WO2014047432A1

WO2014047432A1 - Clinical predictors of weight loss

Info

Publication number: WO2014047432A1
Application number: PCT/US2013/060901
Authority: WO
Inventors: Lee M. Kaplan; Ida HATOUM; Jason L. Harris
Original assignee: General Hospital Corp; Ethicon Endo Surgery Inc
Current assignee: General Hospital Corp; Ethicon Endo Surgery Inc
Priority date: 2012-09-21
Filing date: 2013-09-20
Publication date: 2014-03-27
Anticipated expiration: 2015-03-21
Also published as: US20180066315A1; US11236392B2; EP2898101A4; EP2898101A1; US20230096225A1

Description

CLINICAL PREDICTORS OF WEIGHT LOSS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present invention claims priority to U.S. Patent Application Serial No.

13/828,809, filed on March 14, 2013, which claims priority to U.S. Provisional Application Serial No. 61/704,434, filed on September 21, 2012, to U.S. Provisional Application Serial No. 61/704,077, filed on September 21, 2012, and to U.S. Provisional Application Serial No.

61/740,678, filed on December 21, 2012, all of which are hereby incorporated by reference in their entirety. In the case of any inconsistency, the instant application supersedes the prior applications.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing, which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on March 14, 2013 is named 100873-629_SL.txt and is 1,318,653 bytes in size.

FIELD OF THE INVENTION

[0003] The present invention relates to genetic predictors for treatment of metabolic disorders and diseases, such as obesity.

BACKGROUND OF THE INVENTION

[0004] It is estimated that 66% of adults in the United States are overweight, including 32% who have obesity. The myriad metabolic, inflammatory, degenerative, cognitive, and neoplastic sequealae of obesity together cost more than $168 billion annually and account for nearly 10% of all healthcare expenditures in the United States.

[0005] Behavioral and pharmacotherapeutic treatments for severe obesity have been met with limited long-term success. In contrast, metabolic and bariatric operations such as Roux-en-Y gastric bypass (RYGB) lead to significant and sustained weight loss. Because of its excellent clinical outcomes, RYGB is currently the most commonly used surgical therapy for obesity. Metabolic and bariatric surgical procedures are increasingly being performed laparoscopically. Reduced postoperative recovery time, markedly decreased post operative pain and wound infection, and improved cosmetic outcome are well established benefits of laparoscopic surgery, derived mainly from the ability of laparoscopic surgeons to perform an operation utilizing smaller incisions of the body cavity wall.

[0006] Despite the various metabolic and bariatric surgical procedures each providing chances for weight loss and associated improvements in comorbid conditions, there is wide variability in outcomes (e.g., weight loss, improvements in diabetes and other comorbidities of obesity, adverse sequelae, etc.) among individual patients who receive such surgeries. Several clinical, demographic, psychological, and surgical predictors of weight loss have been reported, but these factors explain only a small fraction of the variation in weight loss after surgery. The

identification of novel predictors of outcomes after metabolic and bariatric surgical procedures can both provide insight into the biological mechanisms of action of these procedures, as well as provide predictive markers that may be used to stratify those patients who may respond best to surgery or alternative treatments. Additionally, because numerous factors can affect a patient's outcomes following metabolic and bariatric surgery, and because some factors may be more relevant for some patients more than others depending on an individual's overall health and other biological characteristics, it can be difficult for medical professionals to consider and balance these factors to arrive at an accurate prediction as to how the surgery will affect a particular patient. It can be even more difficult, and likely impossible, for non-medical professionals, e.g., patients, to consider and balance such factors.

[0007] Accordingly, there remains a need for improved systems and methods for predicting metabolic and bariatric surgery outcomes and treatments that incorporate these systems and methods.

SUMMARY OF THE INVENTION

[0008] The present invention generally provides methods and compositions for treating a subject having a metabolic disorder, e.g., obesity and weight related disorders. In one embodiment, a method of treating a subject having a metabolic disorder can include the steps of (a) obtaining a biological sample from the subject; (b) evaluating the sample for the presence or absence of at least one genetic indicator, and (c) performing a first metabolic procedure on the subject, if the at least one genetic indicator is/are absent, or (d) if the at least one genetic indicator is/are present, performing a second metabolic procedure, wherein the first metabolic procedure is different from a second metabolic procedure. A genetic indicator can include a genetic variation, such as a single nucleotide polymorphism, or a level of gene expression within or without a reference range.

[0009] In one embodiment, a method of treating a weight-related disorder in a subject can include (a) obtaining a sample comprising nucleic acids from the subject; (b) evaluating the nucleic acids for an absence or presence of one or more genetic indicators; and (c) based on if the genetic indicator(s) is absent in (b), performing a first metabolic procedure, or if the genetic indicator(s) is present in (b), performing a second metabolic procedure wherein the second metabolic procedure is different from the first metabolic procedure. The first and second metabolic procedures can be surgical, such as bariatric surgery, or non- surgical. In some aspects, the biological sample includes nucleic acids and evaluating the sample includes evaluating the nucleic acids for the presence or absence of at least one genetic indicator. In an exemplary embodiment, the nucleic acids in the samples can be deoxyribonucleic acids (DNA) or ribonucleic acids (RNA). The nucleic acids can also be positive or negative for the genetic indicators. In some instances, the genetic indicators can be at least one single nucleotide polymorphism (SNP) selected from Appendix A (i.e., SEQ ID NOs 129-837 (SNPs identified as statistically significant for percent weight loss)), Appendix B (additional SNPs identified as statistically significant for percent weight loss) and/or Appendix C (SNPs identified as statistically significant for percent excess body weight loss). The absence or presence of the SNP can correlate with therapeutically effective weight loss of at least 20% weight change after a first metabolic procedure or a second metabolic procedure in the subject. In an exemplary embodiment, the absence or presence of the SNP can correlate with therapeutically effective weight loss of at least 20% weight change after a first metabolic procedure or a second metabolic procedure without a bariatric surgery in the subject.

[0010] In another embodiment, additional clinical measurements can be obtained from the subject. The additional clinical measurements can be obtained prior to evaluating the nucleic acids in the sample or prior to performing a first metabolic procedure or a second metabolic procedure without a bariatric surgery. In an exemplary embodiment, the additional clinical measurements can be obtained prior to evaluating the nucleic acids in the sample or prior to performing a first metabolic procedure or a second metabolic procedure without a bariatric surgery. The clinical measurement can include at least one of a pre-operative body mass index (BMI), anthropometric assessment, body composition, fat distribution, and energy expenditure assessment of the subject, a glucose tolerance or other marker of metabolic homeostasis, a bile acid profile, and a measurement of a biomarker obtained from a fluid, tissue, feces, or other sample obtained from the subject. In an exemplary embodiment, the clinical measurement is a pre-operative body mass index (BMI) of the subject. The clinical measurements can also include weight, gender, age, medical history, weight history, comorbid disease, physical activity, and/or status, BMI, ethnicity, prescription history and/or status, and types and outcomes of treatments previously tried (such as medications or other surgical and non-surgical treatments, etc). In another exemplary embodiment, the nucleic acids can be negative for the genetic indicators and the clinical measurement is a pre-operative BMI of the subject, where the BMI is greater than 25 kg/m².

[0011] In one embodiment, a method of treating a subject having a metabolic disorder can include the steps of (a) obtaining a biological sample from the subject; (b) evaluating expression of at least one gene in the sample, wherein the gene is differentially expressed after bariatric surgery or whose expression correlates with weight loss after a metabolic procedure; and (c) comparing the expression level of the gene(s) evaluated in (b) to a reference range, if expression of the gene(s) is outside the reference range, performing a first metabolic procedure on the subject, or if expression of the gene is inside the reference range, performing a second metabolic procedure on the subject. The first and second metabolic procedures can be surgical, such as bariatric surgery, or non-surgical.

[0012] In one embodiment, the gene(s) can be selected from SEQ ID NOs 1-128. The gene(s) can correlate with therapeutically significant weight loss associated with a metabolic procedure, such as bariatric surgery; improvement, alleviation or amelioration of one or more co- morbid conditions; absence of an adverse metabolic effect; and/or lack of therapeutically significant weight loss, lask of improvement, alleviation or amelioration of one or more co- morbid conditions, or an adverse metabolic event associated with bariatric surgery; or increased risk of obesity, or obesity-related co-morbid conditions in the subject. In one embodiment, expression of the gene can correlate with therapeutically significant weight loss after a metabolic procedure, such as bariatric surgery.

[0013] In some instances, the reference range of gene expression can be determined from multiple patients having undergone a metabolic procedure, such as bariatric surgery. The reference range of gene expression can be an average of gene expression from multiple patients. The reference range of gene expression can be about +30%, +25%, +20%, +15%, +10%, or +5% of an average of gene expression from multiple patients. These may be a group of patients that have experienced therapeutically significant weight loss associated with a metabolic procedure, such as bariatric surgery; improvement, alleviation and/or amelioration of one or more co- morbid conditions, or the absence of an adverse metabolic event. Alternatively, the group of patients may have experienced lack of therapeutically significant weight loss, lack of

improvement, alleviation and/or amelioration of one or more co-morbid conditions, or an adverse metabolic event associated with a metabolic procedure, such as bariatric surgery, or an increased risk of obesity or obesity-related co-morbid conditions.

[0014] In one embodiment, the first and second metabolic procedures can be the same or different procedures. The first metabolic procedure can be a surgical procedure, such as bariatric surgery, including, but not limited to, gastric bypass, Roux-en-Y gastric bypass (RYGB), biliopancreatic diversion, partial gastrectomy procedures such as vertical sleeve gastrectomy, adjustable gastric banding, duodenal switch, duodenojejunal bypass, vertical banded

gastroplasty, intragastric balloon therapy, greater curvature plication, gastric placation (including anterior and anteroposterior plication) and other forms of gastric volume reduction, Magenstrasse and Mill, ileal transposition or interposition, small bowel transposition, biliary diversion, procedures involving anastomotic connections of the gastrointestinal tract, gastric balloon implantation and other gastric or intestinal device implantation, gastric, duodenal or intestinal endoluminal barrier implantation, gastric electrical stimulation, small bowel electrical stimulation, vagal electrical stimulation, and vagal electrical inhibition. Alternatively, the first metabolic procedure can be a non-surgical procedure, such as, but not limited to, administering pharmacological and nutritional therapies, such as hormone and neuropeptide therapy, receptor agonists and antagonists, etc.; providing an alternative medical device based therapy, such as, but not limited to, gastric balloon implantation and other gastric or intestinal device implantation, gastric, duodenal or intestinal endoluminal barrier implantation, etc.; and/or the activation of brown adipose tissue.

[0015] As mentioned above, the second metabolic procedure can be the same as or different from the first metabolic procedure. In one embodiment, the first metabolic procedure can be different from the second metabolic procedure. For example, the first metabolic procedure can be a surgical procedure, such as bariatric surgery, and the second metabolic procedure can be non-surgical. In another example, the first metabolic procedure can be a surgical procedure, and the second metabolic procedure can be a different surgical procedure. In one embodiment, the first metabolic procedure can be a non-surgical procedure and the second metabolic procedure can be a surgical procedure.

[0016] In another embodiment, a clinical measurement can be obtained from the subject. The clinical measurement can be obtained prior to or after obtaining a biological sample from the subject, prior to or after comparing the expression level of the gene(s), or prior to performing a first metabolic procedure or second metabolic procedure. The clinical measurement can include at least one of a pre-operative body mass index (BMI), a glucose tolerance, bile acid profile, and body composition/fat distribution of the subject. In an exemplary embodiment, the clinical measurement is a pre-operative body mass index (BMI) of the subject. The clinical

measurement can also include weight, gender, age, medical history and/or status, ethnicity, medical, prescription history and/or status, and types of treatments previously tried (such as medications or other surgical and non-surgical treatments, etc.). In another exemplary embodiment, the nucleic acids can be negative for the genetic indicators and the clinical measurement is a pre-operative BMI of the subject, where the BMI is greater than 23 kg/m .

[0017] In one aspect, diagnostic kits are disclosed for assessing the presence of a single nucleotide polymorphism (SNP) shown in Appendix A (SEQ ID NOs. 129-837), Appendix B, and/or Appendix C in a sample. The kit can include, but is not limited to, a pair of primers that specifically hybridize to regions proximal to the SNP selected from Appendix A (SEQ ID NOs. 129-837), Appendix B, and/or Appendix C and reagents for polymerase chain reaction (PCR). The kit can also include reagents for preparation, isolation and/or purification of nucleic acids from a sample. The kit can also be used in a method having the steps of (a) obtaining a sample comprising nucleic acids, such as deoxyribonucleic acids (DNA), from the subject; (b) evaluating the nucleic acids for an absence or presence of one or more genetic indicators; and if the genetic indicator(s) is absent in (b), performing a first metabolic procedure, such as a bariatric surgery, or if the genetic indicator(s) is present in (b), performing a second metabolic procedure, wherein the second metabolic procedure is different from the first metabolic procedure. In an exemplary embodiment, the second metabolic procedure can exclude bariatric surgery.

[0018] In another aspect, a method of treating obesity or a weight-related disorder in a subject is disclosed. The method can include (a) obtaining a sample comprising nucleic acids from the subject; (b) evaluating the nucleic acids for an absence or presence of one or more genetic indicators; (c) predicting an outcome of performing a first metabolic procedure based on the absence or presence of the genetic indicator(s); and (d) performing the first metabolic procedure or performing an alternative second metabolic procedure based on the predicted outcome. For example, the method can include (a) obtaining a sample comprising nucleic acids from the subject; (b) evaluating the nucleic acids for an absence or presence of one or more genetic indicators; (c) predicting an outcome of performing a first metabolic procedure, such as a bariatric surgery, based on the absence or presence of the genetic indicator(s); and (d) performing the first metabolic procedure or performing an alternative second metabolic procedure. In an exemplary embodiment, the alternative second metabolic procedure can exclude bariatric surgery.

[0019] In one embodiment, the nucleic acids in the samples can be deoxyribonucleic acids (DNA) or ribonucleic acids (RNA). The nucleic acids can also be positive or negative for the genetic indicators. In some instances, the genetic indicators can be at least one single nucleotide polymorphism (SNP) selected from Appendix A (SEQ ID NOs 129-837), Appendix B, and/or Appendix C that can be absence or presence in the nucleic acids.

[0020] In another embodiment, the outcome predicted from performing the metabolic procedure can be a therapeutically effective weight loss and/or the outcome can be an amelioration of or reduction of at least one weight-related co-morbid condition. In some embodiments when the outcome is a therapeutically effective weight loss, the weight loss can be at least 20% weight change. The outcome can also be a therapeutically effective weight loss when the genetic indicator(s) is absent. The outcome can further be a therapeutically effective weight loss and the metabolic procedure can be performed in the absence of the genetic indicator(s). In some embodiments, the outcome predicted from performing the metabolic procedure can be lack of therapeutically significant weight loss or an adverse metabolic event associated with bariatric surgery, increased risk of obesity, or obesity-related co-morbid conditions in the subject, lack of therapeutically significant weight loss, lask of improvement, alleviation or amelioration of one or more co-morbid conditions, or an adverse metabolic event associated with bariatric surgery; or increased risk of obesity, or obesity-related co-morbid conditions in the subject.

[0021] In yet another embodiment, the outcome predicted from performing the metabolic procedure can be an amelioration of or reduction of at least one weight-related co-morbid condition. The co-morbid condition can be at least one of hypertension, dyslipidemia, triglyceride levels, diabetes, gastroesophageal reflux, fatty liver disease, steatohepatitis, heart or vascular disease, heart failure, cardiovascular risk, sleep apnea, Barrett's esophagus, asthma, osteoarthritis, compression fractures, gallstones, lymphedema, urinary incontinence, stroke, cognitive dysfunction, pseudotumor cerebri, inflammatory diseases, autoimmune diseases, gout, polycystic ovarian syndrome, infertility, depression, anxiety and/or panic disorders, cognitive or other neurological disorders, cancer risk and mortality (cancers including adenocarcinoma of pancreas, esophagus, gallbladder, pancreas, colon, rectum, breast, prostate; cervical carcinoma, endometrial carcinoma, ovarian carcinoma, renal cell carcinoma, non-Hodgkins lymphoma), weight regain, excess weight loss, nutritional deficiency, constipation, diarrhea, marginal ulceration, dumping syndrome, reactive hypoglycemia, beta cell hyperfunction, gastrointestinal stenosis, liver disorders, nausea/vomiting and/or other metabolic syndromes.

[0022] In yet another aspect, predicting the outcome can include inputting the subject's data into a metabolic procedure outcome prediction system. The metabolic procedure outcome prediction system can be an interactive interface for modeling metabolic procedure outcomes, such as bariatric surgery outcomes. Examples of patient data that can be used for predicting outcomes can include the evaluation of the absence or presence of the genetic indicator(s), and at least one clinical measurement including a pre-operative body mass index (BMI), a glucose tolerance, bile acid profile, and body composition/fat distribution of the subject, or another measurement of gene expression in a cell or tissue, measurement of a peptide, protein, metabolite or other compound in blood or in a cell or in a tissue.

[0023] In another aspect, a method of treating a metabolic disorder in a subject is disclosed. The method can include measuring expression of the gene(s) in a sample from the subject;

comparing the expression level of the gene(s) to a reference range of expression of the gene, wherein the reference range is determined from multiple patients having undergone a bariatric surgery; and administering to the subject a composition that modulates expression of the gene(s) to mimic the expression after bariatric surgery, thereby treating the metabolic disorder. The method can result in a therapeutically significant weight loss. The method can also result in a therapeutically significant weight loss that is at least a 20% body weight change or an amelioration of or reduction of at least one weight-related co-morbid condition, where the co- morbid condition can be hypertension, dyslipidemia, triglyceride levels, diabetes,

gastroesophageal reflux, fatty liver disease, steatohepatitis, heart or vascular disease, heart failure, cardiovascular risk, sleep apnea, Barrett's esophagus, asthma, osteoarthritis, compression fractures, gallstones, lymphedema, urinary incontinence, stroke, cognitive dysfunction, pseudotumor cerebri, inflammatory diseases, autoimmune diseases, gout, polycystic ovarian syndrome, infertility, depression, anxiety and/or panic disorders, cognitive or other neurological disorders, cancer risk and mortality (cancers including adenocarcinoma of pancreas, esophagus, gallbladder, pancreas, colon, rectum, breast, prostate; cervical carcinoma, endometrial carcinoma, ovarian carcinoma, renal cell carcinoma, non-Hodgkins lymphoma), weight regain, excess weight loss, nutritional deficiency, constipation, diarrhea, marginal ulceration, dumping syndrome, reactive hypoglycemia, beta cell hyperfunction, gastrointestinal stenosis, liver disorders, nausea/vomiting and/or other metabolic syndromes.

[0024] In yet another aspect, kits are disclosed for assessing expression of at least one gene associated with response to a metabolic procedure in a sample. In yet another aspect, kits are disclosed for assessing the sequence of a gene or other chromosomal DNA. The kit can include, but is not limited to, a pair of primers that specifically hybridize to an expression product of the gene(s) selected from SEQ ID NOs 1-128. The kit can also include reagents for preparation, isolation and/or purification of nucleic acids and/or expression products from a sample. [0025] In another aspect, use of a single nucleotide polymorphism to evaluate a course of treatment for a metabolic disorder is disclosed. In yet another aspect, use of a gene expression level to evaluate a course of treatment for a metabolic disorder is disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The invention will be more fully understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

[0027] Figure 1 is a bar graph showing the excess body weight loss (EBWL) at postoperative weight nadir (achieved after at least 10 months of surgery without coexisting debilitating illness or use of weight lowering medications) after Roux-en-Y gastric bypass (RYGB) in 848 patients with severe obesity;

[0028] Figure 2 is a bar graph showing the mean difference in percent EBWL within patient pairs, according to type of relationship;

[0029] Figure 3 is a bar graph showing no difference on the residuals (deviations of the regressing postoperative BMI on preoperative BMI from the sample mean) from the cohort of 848 patients;

[0030] Figure 4 is a bar graph showing absolute change in weight of the 848 patients after 1 year postoperative with patients in lower BMI groups losing significantly less weight;

[0031] Figure 5 is a bar graph showing final weights obtained at 1 year postoperative of the 848 patients after 1 year postoperative;

[0032] Figure 6 is a bar graph showing the change in body mass index (BMI) of the 848 patients after 1 year postoperative with patients in lower BMI groups having significantly less change in BMI;

[0033] Figure 7 is a bar graph showing final BMI obtained at 1 year postoperative of the 848 patients after 1 year postoperative; [0034] Figure 8 is a bar graph showing percent excess body weight lost ( EBWL) obtained at 1 year postoperative of the 848 patients after 1 year postoperative with patients at a lower pBMI losing more EBWL at both 1 year and weight nadir;

[0035] Figure 9 is a bar graph showing percent weight change (%WC) obtained at 1 year postoperative of the 848 patients after 1 year postoperative with no significant association between pBMI group and WC at one year, and a relatively weak association between pBMI and WC and weight nadir;

[0036] Figure 10 is a bar graph showing percent change in weight in 858 unrelated

Caucasian individuals grouped according to preoperative BMI;

[0037] Figure 11 is a bar graph showing percent change of weight nadir measured in a subgroup consisting of 693 patients (Cohort 1);

[0038] Figure 12 is a bar graph showing percent change of weight nadir measured in an independent group of 349 Caucasian RYGB patients (Cohort 2);

[0039] Figure 13 is a graphical representation of 112 significant (P < 5 x 10^"5) single nucleotide polymorphisms (SNPs) identified in Cohort 2;

[0040] Figure 14 is a flow diagram illustrating direct or indirect association of the SNP with surrounding loci;

[0041] Figure 15 is a graphical representation of chromosome 11 with the SNPs identified as having significant association with percent total weight loss (%WL) at the lowest weight (weight nadir) after RYGB;

[0042] Figure 16 is graph showing the association of carrying the minor allele (MA), rsl7702901, and percent weight change at nadir in patients homozygous null for MA, heterozygous for MA or homozygous for MA;

[0043] Figure 17 is a bar graph showing percent weight change at nadir measured in pooled data from combination of Cohort 1 and Cohort 2 (953 RYGB patients). The shaded area identifies 171 patients having %WL categorized as less than or greater than or equal to 30% at weight nadir;

[0044] Figure 18 is a bar graph showing percent weight loss with patients carrying at least one copy of the MA being 2.54 times more likely to fall below 30% WL (left shaded area) and no patients with this polymorphism lost more than 50% of his or her weight (right shaded area);

[0045] Figure 19 is an area under the receiver operating characteristic curve (AUROC) showing that inclusion of rsl7709201 has a higher probability of being a predictor of weight loss than rsl7709201 as a random positive influence on weight loss;

[0046] Figure 20 is a bar graph of percent weight loss showing association of rs 17702901 with weight loss in RYGB patients; and

[0047] Figure 21 is a bar graph of percent weight loss showing a lack of an association of validated BMI locus, Melanocortin 4 Receptor (MC4R), with weight loss in RYGB patients.

[0048] Figure 22 is a schematic diagram of the anatomy of Roux-en-Y gastric bypass.

Tissues noted were dissected from mice 10 weeks after RYGB or sham operation;

[0049] Figure 23A is a bar graph showing the comparative expression of st8sia2 in RYGB- treated and sham operated, weight matched mice (WMS). Grey bars denote the WMS group, blue bars the RYGB group. Error bars denote the standard error of the mean. * p<0.05, ** p<0.01, *** p<0.001;

[0050] Figure 23B is a bar graph showing the comparative expression of slco3al in RYGB- treated and sham operated, weight matched mice (WMS). Grey bars denote the WMS group, blue bars the RYGB group. Error bars denote the standard error of the mean. * p<0.05, ** p<0.01, *** p<0.001;

[0051] Figure 24A is a regional association plot showing the AQP11 locus. Each SNP is plotted as a diamond based on its chromosomal location (jc-axis) and -logw P value (left _y-axis). Recombination rates are plotted in grey toward the bottom of the graph (right _y-axis). The large upper diamond represents the top SNP in the region (rs7129556) from the genome- wide association study (GWAS), and the large lower diamond represents the p-value from that SNP in the replication cohort;

[0052] Figure 24B is a bar graph showing the relative expression level of aqpll . Light bars denote the WMS group, dark bars the RYGB group. Error bars denote the standard error of the mean. * p<0.05, ** p<0.01, *** p<0.001; and

[0053] Figure 24C is a bar graph showing the relative expression level of clnsla. Light bars denote the WMS group, dark bars the RYGB group. Error bars denote the standard error of the mean * p<0.05, ** p<0.01, *** p<0.001.

DETAILED DESCRIPTION OF THE INVENTION

[0054] Certain exemplary embodiments will now be described to provide an overall understanding of the principles of the structure, function, manufacture, and use of the therapeutics and methods disclosed herein. One or more examples of these embodiments are illustrated in the accompanying drawings. Those skilled in the art will understand that the therapeutics and methods specifically described herein and illustrated in the accompanying drawings are non-limiting exemplary embodiments and that the scope of the present invention is defined solely by the claims. The features illustrated or described in connection with one exemplary embodiment may be combined with the features of other embodiments. Such modifications and variations are intended to be included within the scope of the present invention.

[0055] SEQ ID NOs 1-128 in the present application correspond to SEQ ID NOs 1-128 of U.S. Provisional Application Serial No. 61/740,678, and SEQ ID NOs 129-837 correspond to the sequences from Appendix A of U.S. Provisional Application Serial No 61/704,434 (which is identical to Appendix A of the present application).

Metabolic Disorders (Obesity and Other Weight-Related Disorders)

[0056] Methods and kits are provided to evaluate genetic indicators, such as by identifying genetic indicators, and/or measuring and assessing gene expression for treatment of obesity and/or weight-related disorders. It has been discovered that genetic indicators, such as single nucleotide polymorphisms, can be indicators for weight loss potential after metabolic surgery, such as bariatric surgery. Given the correlation between weight loss and improvements in comorbidities associated with excess weight, these genetic predictors can be indicators or predictors of improvements in comorbid conditions after bariatric surgery. It has also been discovered that certain genes demonstrate differential gene expression after a metabolic procedure, such as bariatric surgery. It has been further discovered that a correlation exists between the weight loss and other improvements in comorbidities and gene expression of certain genes. Therefore, modulating certain gene expression can be therapeutic to improve comorbid conditions after bariatric surgery. Further, the gene expression can serve a surrogate marker for whether a surgical procedure (e.g., bariatric surgery) is likely to lead to a successful outcome, or an alternative procedure is better suited for a certain patient or patient population. Therefore, the invention disclosed is generally directed to therapeutic methods and compositions for treating metabolic disorders, such as obesity and/or other weight-related disorders, in a subject by (1) evaluating genetic indicators, such as by evaluating the subject's deoxyribonucleic acids (DNA) for a presence or absence of one or more genetic indicators, and/or evaluating gene expression in the subject for an overexpression or an underexpression of one or more specific genes associated with metabolic disorders.

[0057] Weight loss can be characterized using a number of different metrics, including the absolute number of pounds or body mass index (BMI) points lost, weight or BMI achieved after weight loss, the percent of baseline weight or BMI lost (% weight change (WC)), and percent excess body weight lost (% EBWL).

[0058] The phrase "weight-related disorder" as used herein, refers to disorders, diseases, and conditions that are caused or characterized by abnormal energy use or consumption leading to excessive weight gain or loss, altered responses to ingested or endogenous nutrients, energy sources, hormones or other signaling molecules within the body or altered metabolism of carbohydrates, lipids, proteins, nucleic acids or a combination thereof. A weight-related disorder can be associated with either a deficiency or excess in a metabolic pathway resulting in an imbalance in metabolism of nucleic acids, proteins, lipids, and/or carbohydrates. Factors affecting metabolism include, and are not limited to, the endocrine (hormonal) control system (e.g., the insulin pathway, the enteroendocrine hormones including GLP-1, PYY or the like), the neural control system (e.g., GLP-1 or other neurotransmitters in the brain, spinal cord, peripheral or enteric nervous systems) or the like. Some non-limiting examples of weight-related disorders can be obesity, diabetes, including type II diabetes, insulin-deficiency, insulin-resistance, insulin- resistance related disorders, glucose intolerance, syndrome X, inflammatory and immune disorders, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver, abnormal lipid metabolism, obstructive sleep apnea, asthma, autoimmune and inflammatory disorders, cancer, cognitive and neurodegenerative disorders, hypertension, high cholesterol, anxiety, congestive heart failure, ischemic heart disease, GERD, atherogenic dyslipidemia, hyperlipidemic conditions such as atherosclerosis, hypercholesterolemia, and other coronary artery diseases in mammals, and other disorders of metabolism.

[0059] As used herein, the term "obesity" or "obese" typically refers to a non-Asian individual having a body mass index (BMI) of >30 kg/m 2 or >27 kg/m 2 in Asian individuals and "overweight" typically refers to a non-Asian individual having a body mass index (BMI) of >25 kg/m 2 or >23 kg/m 2 in Asian individuals. BMI is a measure expressing the relationship (or ratio) of weight-to-height based on a mathematical formula in which a person's body weight in kilograms is divided by the square of his or her height in meters (i.e., wt/(ht) ). Individuals having BMI of >25 kg/m 2 in non-Asians or >23 kg/m 2 in Asians have a substantially increased risk of at least one weight-related co-morbid condition or having a metabolic disorder or syndrome. As used herein, the terms "co-morbidity" or "co-morbid condition" typically refers to, but is not limited to, hypertension, dyslipidemia, triglyceride levels, diabetes,

gastroesophageal reflux, fatty liver disease, steatohepatitis, heart or vascular disease, heart failure, cardiovascular risk, sleep apnea, Barrett's esophagus, asthma, osteoarthritis, compression fractures, gallstones, lymphedema, urinary incontinence, stroke, cognitive dysfunction, pseudotumor cerebri, inflammatory diseases, autoimmune diseases, gout, polycystic ovarian syndrome, infertility, depression, anxiety and/or panic disorders, cognitive or other neurological disorders, cancer risk and mortality (cancers including adenocarcinoma of pancreas, esophagus, gallbladder, pancreas, colon, rectum, breast, prostate; cervical carcinoma, endometrial carcinoma, ovarian carcinoma, renal cell carcinoma, non-Hodgkins lymphoma), weight regain, excess weight loss, nutritional deficiency, constipation, diarrhea, marginal ulceration, dumping syndrome, reactive hypoglycemia, beta cell hyperfunction, gastrointestinal stenosis, liver disorders, nausea/vomiting and/or other metabolic syndromes. As the name suggests, "metabolic disorder or syndrome" is tied to the body's metabolism, and more likely to conditions that influence metabolism, such as insulin resistance. Metabolic disorder or syndrome can also be characterized by excess body fat, atherogenic dyslipidemia, elevated blood pressure and insulin resistance, among others.

[0060] Other weight-related disorders can include conditions that occur or cluster together, and/or increase the risk for heart disease, stroke, diabetes, and obesity. Having just one of these conditions such as increased blood pressure, elevated insulin levels, excess body fat around the waist or abnormal cholesterol levels can increase the risk of the above mentioned diseases. In combination, the risk for coronary heart disease, stroke, insulin-resistance syndrome, and diabetes is even greater.

[0061] The increasing prevalence of obesity in the population has led to a parallel rise in metabolic procedures, like bariatric surgery, as a treatment for obesity and related comorbid conditions. As used herein, the term "metabolic procedures" can include surgical and nonsurgical procedures. Surgical procedures can achieve a sustained weight reduction of up to 70% of excess body weight in the majority of patients, and are often more effective than nonsurgical approaches. Nonlimiting examples of surgical procedures can include bariatric surgery. As used herein, "bariatric surgery" generally refers and can include procedures often referred to as metabolic surgery or therapy, as well as a variety of procedures performed in a subject that leads to a physiologic improvement in energy balance, nutrient utilization, or metabolic disorders. These procedures often, but not always, result in weight loss. Bariatric surgery refers to a surgical procedure to alter gastrointestinal structure or function so as to affect body weight, body composition, or energy balance regulation or otherwise alter metabolic function. Some non-limiting examples of bariatric surgery can be any form of gastric bypass, Roux-en-Y gastric bypass (RYGB), biliopancreatic diversion, partial gastrectomy procedures such as vertical sleeve gastrectomy, adjustable gastric banding, duodenal switch, duodenojejunal bypass, vertical banded gastroplasty, intragastric balloon therapy, greater curvature plication, gastric plication (including anterior and anteroposterior plication) and other forms of gastric volume reduction, Magenstrasse and Mill, ileal transposition or interposition, small bowel transposition, biliary diversion, procedures involving anastomotic connections of the gastrointestinal tract (e.g., jejunoileostomy, etc.), gastric electrical stimulation, small bowel electrical stimulation, vagal electrical stimulation, vagal electrical inhibition, and variations of the procedures above as well as other methods known by those skilled in the art. Metabolic procedures can also include non- surgical procedures including, by way of non-limiting examples, administering pharmacological and nutritional therapies, such as hormone and neuropeptide therapy, receptor agonists and antagonists, etc.; providing an alternative medical device based therapy, such as, but not limited to, gastric balloon implantation and other gastric or intestinal device implantation, gastric, duodenal or intestinal endoluminal barrier implantation, etc.; and/or the activation of brown adipose tissue. Each of the surgical and non-surgical procedures may be performed alone or in addition to other treatments.

[0062] It has been discovered that subjects with certain diagnostic markers respond to therapeutic interventions, such as gastric bypass surgery. Therefore, in an exemplary

embodiment, a method of treating a metabolic disorder, such as obesity, in a subject can include obtaining a sample with DNA from the subject, evaluating the DNA for the presence or absence of one or more genetic indicators and performing a first metabolic procedure, such as bariatric surgery or a second metabolic procedure, excluding bariatric surgery, depending on the absence or presence of one or more genetic indicators.

[0063] It has also been discovered that expression of certain genes is associated with a better response to therapeutic interventions, such as gastric bypass surgery. Therefore, in an exemplary embodiment, a method of treating a metabolic disorder in a subject can include obtaining a sample from the subject, evaluating the sample for expression of at least one gene (wherein the gene is shown to be differentially expressed after bariatric surgery or wherein expression of the gene correlates with weight loss after a metabolic procedure), and performing a first metabolic procedure or a second metabolic procedure excluding a bariatric surgery depending on the expression of gene(s).

Differentially Expressed Genes and Genetic Indicators

[0064] Identification of specific genetic indicators, such as SNPs associated with weight loss after RYGB, or expression patterns, such as expression of genes associated with weight loss after RYGB, may both enhance the understanding of the mechanisms of weight loss as well as help identify those patients for whom bariatric surgery procedures are most effective.

[0065] As used herein, "polymorphism" refers to a variation in the sequence of a gene in the genome amongst a population, such as allelic variations and other variations that arise or are observed. "Genetic polymorphisms" refers to the variant forms of DNA sequences that can arise as a result of nucleotide alteration or substitution, deletion, insertion, rearrangement or duplication, for example. Thus, a polymorphism refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population. These polymorphisms can occur in coding and non-coding portions of the genome, and can be manifested or detected as differences in nucleic acid sequences, gene expression, and/or other differences in mRNA structure and function, including, for example transcription, processing, translation, transport, protein processing, trafficking, DNA synthesis, expressed proteins, other gene products or products of biochemical pathways or in post-translational modifications and any other differences manifested among members of a population. A "single nucleotide polymorphism" or "SNP" refers to a polymorphism that arises as the result of a single base change, such as an insertion, deletion or change in a base.

[0066] A polymorphic marker or site is the locus at which divergence occurs. Such a site may be as small as one base pair (an SNP). Polymorphic markers include, but are not limited to, restriction fragment length polymorphisms, copy number variations, variable number of tandem repeats (VNTR's), hypervariable regions, minisatellites, dinucleotide repeats, trinucleotide repeats, tetranucleotide repeats and other repeating patterns, simple sequence repeats and insertional elements, such as Alu.

[0067] Polymorphic forms also are manifested as different mendelian alleles for a gene. The genomes of all organisms undergo spontaneous mutation in the course of their continuing evolution, generating variant forms of progenitor genetic sequences. A variant form may confer differences in proteins, protein modifications, RNA expression, RNA modification, DNA and RNA methylation, regulatory factors that alter gene expression and DNA replication, and any other manifestation of alterations in genomic nucleic acid or organelle nucleic acids. [0068] As used herein, an "isolated" nucleic acid molecule, such as a nucleic acid molecule containing a SNP genetic indicator or an expression product of a gene or other transcript (e.g., messenger RNA, microRNA or other non-coding RNA), can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. A nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered "isolated." Nucleic acid molecules present in non-human transgenic animals, which do not naturally occur in the animal, are also considered "isolated." For example, recombinant DNA molecules contained in a vector are considered "isolated." Further examples of "isolated" DNA molecules include recombinant DNA molecules maintained in heterologous host cells, and purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the isolated SNP-containing DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.

[0069] A nucleic acid molecule can include one or more SNPs with flanking nucleotide sequences on either side of the SNPs. A flanking sequence can include nucleotide residues that are naturally associated with the SNP site and/or heterologous nucleotide sequences. Preferably the flanking sequence can be up to about 500, 300, 100, 60, 50, 30, 25, 20, 15, 10, 8, or 4 nucleotides (or any other length in between) on either side of a SNP.

[0070] As used herein, an "isolated protein," once expressed, can be isolated by lysing cells and applying standard protein isolation techniques to the lysates or the pellets. Monitoring the purification process can be accomplished by using Western blot techniques or radioimmunoassay or other standard immunoassay techniques.

[0071] As used herein, an "amplified polynucleotide" can include a nucleic acid molecule containing one or more SNPs or a gene that can be replicated by at least two fold through any nucleic acid amplification method performed in vitro. In one embodiment, an amplified polynucleotide is the result of at least a ten fold, fifty fold, one hundred fold, one thousand fold, or even ten thousand fold increase as compared to its starting amount in a test sample. In a typical PCR amplification, a polynucleotide of interest is often amplified at least fifty thousand fold in amount over the unamplified DNA template, but the precise amount of amplification needed for an assay depends on the sensitivity of the subsequent detection method used.

[0072] A subject or patient may be homozygous or heterozygous for an allele at each SNP position. A SNP can, in some instances, be referred to as a "cSNP" to denote that the nucleotide sequence containing the SNP is an amino acid coding sequence. While SNPs can be bi-, tri-, or tetra-allelic, the vast majority of the SNPs are bi-allelic, and are thus often referred to as "bi- allelic markers," or "di-allelic markers."

[0073] As used herein, references to "SNPs" and SNP genotypes include individual SNPs and/or haplotypes, which are groups of SNPs that are generally inherited together. Haplotypes can have stronger correlations with diseases or other phenotypic effects compared with individual SNPs, and therefore may provide increased diagnostic accuracy in some cases. An "allele" is an alternative form or variation in a DNA sequence. Many SNPs have only two alleles: minor and major alleles. SNPs are routinely used in SNP-based genetic linkage analysis to map a disease to a particular locus, the position of a gene (or SNP) on a chromosome.

[0074] Methods and kits are provided to identify genetic indicators and/or evaluate and assess expression of genes associated with response to a metabolic procedure, such as bariatric surgery, for treatment of metabolic disorders. It has been discovered that genetic indicators, such as single nucleotide polymorphisms, and gene expression, such as genes associated with response to a metabolic procedure, can indicate weight loss potential after the metabolic procedure, such as bariatric surgery. By obtaining a sample from a subject and extracting nucleic acids from or analyzing gene expression in the sample, response to the metabolic procedure, e.g. weight loss potential after the bariatric surgery, can be predicted and/or assessed.

[0075] The term "subject" as used herein refers to any living organism, including, but not limited to, humans, nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats, rabbits and guinea pigs, and the like. The term does not denote a particular age or sex. In a specific embodiment, the subject is human. In an exemplary embodiment, the subject is a patient. [0076] The term "sample" is intended to include tissues, cells, fluids and biological samples isolated from a subject, as well as tissues, cells and fluids present within a subject. The sample can be a tissue sample, such as from an organ, or fluid, ascites, and any other sample that is used by those familiar with the art. The sample can be derived from any source which contains proteins or expression products and/or nucleic acids, DNA (e.g., chromosomal nucleic acids) or RNA, such as a blood sample, body excrements such as semen, saliva, stool, urine, amniotic fluid and so forth, sample of cerebrospinal fluid, or tissue sample from skin, muscle, buccal or conjunctival mucosa, placenta, gastrointestinal tract or other organs. A sample of proteins and/or nucleic acid from fetal cells or tissue can be obtained by appropriate methods, such as by amniocentesis or chorionic villus sampling (direct or cultured). In one aspect, the sample can be a biopsy sample or a small number of cells or a tissue sample removed for processing. Common examples of biopsy methods can include, but are not limited to, oral swab, brush cytology, core needle biopsy, surgical biopsy, punch biopsy, shave biopsy, incisional/excisional biopsy and curettage biopsy.

[0077] In some embodiments, the sample of cells or tissue sample can be obtained from the subject by biopsy or surgical resection. A sample of cells, tissue, or fluid can be removed by needle aspiration biopsy. For this, a fine needle attached to a syringe is inserted through the skin and into the organ or tissue of interest. The needle is typically guided to the region of interest using ultrasound or computed tomography (CT) imaging. Once the needle is inserted into the tissue, a vacuum is created with the syringe such that cells or fluid may be sucked through the needle and collected in the syringe. A sample of cells or tissue may also be removed by incisional or core biopsy. For this, a cone, a cylinder, or a tiny bit of tissue can be removed from the region of interest. CT imaging, ultrasound, or endoscopy can be used to guide this type of biopsy.

[0078] Once a sample of cells or sample of tissue is removed from the subject, it may be processed for the isolation of RNA or protein using techniques well known in the art and disclosed in standard molecular biology reference books. A sample of tissue may also be stored in RNAlater (Ambion; Austin, TX) or flash frozen and stored at -80 °C for later use. The tissue sample may also be fixed with a fixative, such as formaldehyde, paraformaldehyde, or acetic acid/ethanol. The fixed tissue sample may be embedded in wax (paraffin) or a plastic resin. The embedded tissue sample (or frozen tissue sample) may be cut into thin sections. RNA or protein may also be extracted from a fixed or wax-embedded tissue sample.

[0079] Direct assessment for the presence of the genetic identifiers or for gene expression can be performed on a sample without processing to isolate nucleic acids or gene expression products. Alternatively, a sample can be processed to enhance access to gene expression products, nucleic acids, or copies of nucleic acids (e.g., amplification of nucleic acids), and the processed sample can then be used to assess for the presence of the genetic identifiers or for gene expression. For example, in one embodiment, cDNA is prepared from a sample comprising mRNA, for use in the methods. The mRNA can be isolated from the sample and converted into cDNA. Alternatively or in addition, if desired, an amplification method can be used to amplify nucleic acids for use as the test sample in the assessment for the presence or absence of a genetic identifier(s) or for gene expression. The nucleic acids can be isolated from the samples or can be processed and analyzed within the sample.

[0080] Nucleic acids, including RNA, DNA, or cDNA, proteins or other expression products can be analyzed for the genetic indicator(s) or measured to determine gene expression from a sample. The presence of genetic indicator(s) or gene expression can be evaluated in nucleic acids or proteins in vitro, in situ, as well as in vivo. For example, in vitro techniques for detection of genetic indicator(s) in mRNA or for measuring expression can include assays such as ELISA assay and Western blot analysis, immunocytochemical assays, assessment of mRNA in PCR, q-PCR, northern hybridizations and in situ hybridizations, assessment of cDNA in Southern hybridizations, PCR, quantitative PCR (qPCR), and introduction of labeled nucleic acids for incorporation into the nucleic acids, for example, the radiolabeled nucleic acids whose presence and location in a subject can be detected by standard imaging techniques.

[0081] Another embodiment for identifying genetic indicators in RNA or DNA or for measuring gene expression can include the use of a labeled nucleic acid probe capable of hybridizing to a mRNA or cDNA. A wide variety of conventional techniques are available, including mass spectrometry, chromatographic separations, 2-D gel separations, microarrays, binding assays (e.g., immunoassays), competitive inhibition assays, one- and two-dimensional gels and sandwiched ELISA. Typical methodologies for RNA detection include RNA extraction from a cell or tissue sample, followed by hybridization of a labeled probe, (e.g., a

complementary polynucleotide) specific for the target RNA to the extracted RNA, and detection of the probe (e.g., Northern blotting), direct sequencing, gel electrophoresis, column

chromatography, and quantitative PCR.

[0082] Primers based on a nucleotide sequence specific for one or more of the genetic indicators or genes can be used to analyze the presence or absence in or to measure expression of the corresponding gene(s) or genetic indicator(s). In some embodiments, a primer pair can be designed by utilizing primer design software, such as GenScript, Primer3, PRIDE and Primer Express. Commercial primers are also available for purchase corresponding to multiple locations throughout the gene. The primers can be at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, or 40 basepairs in length. In an exemplary embodiment, the primers can be at least 10 basepairs in length. The primers can also hybridize to a region of nucleic acids (mRNA, cDNA or genomic DNA) proximal or in the vicinity of the genetic indicator or the gene.

[0083] The primer can be similar (sufficiently similar or identical to hybridize to the sequence) or complementary (sufficiently similar or identical to hybridize to the complement sequence) to a nucleic acid sequence upstream or downstream from the genetic indicator. The primer can hybridize to a sequence that is at least 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 850, 900, 950, 1000 basepairs or more or any number of basepairs in between from the genetic indicator. In one embodiment, the primers can specifically hybridize to a region proximal to one or more genetic indicators (such as a single nucleotide polymorphism (SNP) as shown in Appendix A (SEQ ID NOs 129-837), Appendix B, or Appendix C). In an exemplary embodiment, the primers can be complementary to a nucleic acid sequence of at least 10 bases found at least 200 basepairs or more from the genetic indicator.

[0084] The primer can be similar (sufficiently similar or identical to hybridize to the sequence) or complementary (sufficiently similar or identical to hybridize to the complement sequence) to a nucleic acid sequence of the gene. In one embodiment, the primers can be specific for at least one of SEQ ID NOs 1-128. See Table 10 for correspondence of SEQ ID NO to gene

name/description and accession number. [0085] Diagnostic kits and/or devices are also included. The diagnostic kit and/or device can include, but is not limited to, sample collection materials (storage solutions and collection apparatus such as swab, biopsy needle, blood/body fluid needle, brush, etc), protein, DNA or RNA extraction and isolation materials (solutions and enzymes for performing such procedures); nucleic acid amplification materials (solutions, enzymes and primers specific for the genetic indicator(s) or primers for performing such procedures); and sequencing materials (solutions, enzymes and gene specific primers for performing such procedures). The diagnostic kit and/or device can include any of the above and exclude any materials from the above. In an exemplary embodiment, the diagnostic kit and/or device can include DNA polymerase chain reaction amplification solutions and/or enzymes and at least one set of primers specific for a genetic indicator. In another exemplary embodiment, the diagnostic kit and/or device can include DNA polymerase chain reaction amplification solutions and/or enzymes and at least one set of gene specific primers.

[0086] In one aspect, a method of treating obesity or weight-related disorders includes evaluating DNA for the absence or presence of one or more genetic indicators, such as SNPs. The DNA can be positive or negative for one or more indicators. A DNA sample from a subject can be evaluated for the presence or absence of the genetic indicator(s). In some instances, the DNA can be negative for the indicator(s) and the pre-operative BMI can be greater than 20 kg/m².

[0087] Also, the presence or absence of the genetic indicator(s) can correlate with therapeutically effective weight loss associated with bariatric surgery; improvement, alleviation or amelioration of one or more co-morbid conditions; and/or lack of therapeutically effective weight loss associated with bariatric surgery, increased risk of obesity or obesity-related co- morbid conditions in the subject.

[0088] In one embodiment, the presence or absence of the genetic indicator(s) can correlate with therapeutically effective weight loss after bariatric surgery in the subject. Therapeutically effective weight loss can be characterized by loss of at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85% or more of excess body weight. Therapeutically effective weight loss can also be characterized as at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, or more weight change. Alternatively or in addition to, therapeutically effective weight loss can be characterized by change of at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85% or more of body mass index. Excess body weight, weight change, and/or body mass index measurements can be determined by taking the measurement of the subject prior to treatment or preoperatively and compare the

measurement to another measurement taken at a time point after treatment or surgery. The time point for taking the measurement can be 1, 3, 6, 9, 12, 18, 24, 36, 48, 72, 84, 96, 108, 120 months post treatment or postoperative or any number of months in between. In an exemplary embodiment, the therapeutically effective weight loss is at least 20% weight change after bariatric surgery or alternative treatment in the subject.

[0089] Also, the presence or absence of one or more genetic indicator(s) can correlate with improvement, alleviation or amelioration of one or more co-morbid conditions in the subject. The presence or absence of the genetic indicator(s) can correlate with, for example, reduced hypertension, reduced dyslipidemia, improvement or alleviation of diabetes, reduced acid reflux, alleviation of fatty liver or steatohepatitis, reduced risk of heart disease, alleviation of depression, alleviation of sleep apnea, alleviation of asthmatic symptoms, alleviation of arthritis, reduced risk of compression fractures, reduced occurrence of gallstones, lymphoedema, alleviation of urinary incontinence, reduced risk of stroke, reduced risk of cancer and/or reduced risk of other metabolic syndromes.

[0090] Alternatively, the presence or absence of one or more genetic indicator(s) can correlate with lack of therapeutically effective weight loss associated with bariatric surgery or increased risk of obesity, or obesity-related co-morbid conditions in the subject. The presence or absence of the genetic indicator(s) can correlate with, for example, lack of weight loss after bariatric surgery, increased hypertension, risk of dyslipidemia, development of diabetes, acid reflux, fatty liver disease or steatohepatitis, heart disease, depression, sleep apnea, asthmatic symptoms, arthritis, compression fractures, gallstones, lymphoedema, urinary incontinence, stroke, cancer and/or risk of other metabolic syndromes.

[0091] The genetic indicators can be single nucleotide polymorphisms that occur in coding and non-coding portions of the genome, and can be manifested or detected as differences in nucleic acid sequences (DNA), gene expression products (RNA and proteins), including, for example transcripts (mRNA, miRNA, and others), proteins, other gene products or products of biochemical pathways or in post-translational modifications and any other differences manifested among members of a population. In one embodiment, one or more genetic indicators are absent from the subject's nucleic acid sample, such as DNA, where at least one genetic indicator is a single nucleotide polymorphism (SNP) shown in Appendix A (SEQ ID NOs 129-837), Appendix

B, and/or Appendix C. In another embodiment, one or more genetic indicators are present in the subject's nucleic acid sample, such as DNA, where at least one genetic indicator is a single nucleotide polymorphism (SNP) shown in Appendix A (i.e, SEQ ID NOs 129-837 (SNPs identified as statistically significant for percent weight loss)), Appendix B (additional SNPs identified as statistically significant for percent weight loss) or Appendix C (SNPs identified as statistically significant for percent excess body weight loss). Each SNP sequence is associated with a unique accession number (e.g., rs number) that is available in the Single Nucleotide Polymophism Database hosted by the National Center for Biotechnology Information (NCBI) to identify the genetic variation and sequence information. In yet another embodiment, one or more genetic indicators are absent from the subject's nucleic acid sample, such as DNA, and one or more different genetic indicators are present in the subject's DNA sample, where at least one genetic indicator is a single nucleotide polymorphism (SNP) shown in Appendix A (SEQ ID NOs 129-837), Appendix B, or Appendix C.

[0092] For example, the genetic indicator(s) can be at least one single nucleotide

polymorphism (SNP) shown in Appendix A (SEQ ID NOs 129-837), Appendix B, or Appendix

C. Selected genetic indicators, such as the SNPs shown in Appendix A (SEQ ID NOs 129-837), Appendix B, or Appendix C, can be positive or negative indicators for successful obesity or weight-related disorder treatment. In one embodiment, the genetic indicator can be one or more SNPs shown in Appendix A (SEQ ID NOs 129-837), Appendix B, or Appendix C, such as SEQ ID NO 129-SEQ ID NO 138, SEQ ID NO 129-SEQ ID NO 148, SEQ ID NO 129-SEQ ID NO 158, SEQ ID NO 129-SEQ ID NO 168, SEQ ID NO 129-SEQ ID NO 178, SEQ ID NO 129- SEQ ID NO 188, SEQ ID NO 129-SEQ ID NO 198, SEQ ID NO 129-SEQ ID NO 208, SEQ ID NO 129-SEQ ID NO 218, SEQ ID NO 129-SEQ ID NO 228, SEQ ID NO 129-SEQ ID NO 328, SEQ ID NO 129-SEQ ID NO 428, SEQ ID NO 129-SEQ ID NO 528, SEQ ID NO 129-SEQ ID NO 628, SEQ ID NO 129-SEQ ID NO 728, SEQ ID NO 129-SEQ ID NO 828, SEQ ID NO 129- 837, SEQ ID NO 129-SEQ ID NO 928, SEQ ID NO 129-SEQ ID NO 1028, SEQ ID NO 129- SEQ ID NO 1128, SEQ ID NO 129-SEQ ID NO 2128, SEQ ID NO 129-SEQ ID NO 3128, SEQ ID NO 129-SEQ ID NO 4128, SEQ ID NO 129-SEQ ID NO 5128, etc. In another embodiment, the genetic indicator can be one or more SNPs shown in Appendix A (SEQ ID NOs 129-837), Appendix B, or Appendix C, as identified by the unique SNP identifier (e.g. rs number). In another embodiment, the genetic indicator can be one or more SNPs located on chromosome 6, chromosome 11, and/or chromosome 15. In yet another embodiment, the genetic indicators are one or more SNPs selected from rs7158359, rs7129556, rsl0899387, rs934760, rsl 104959, rsl7702901, rs588217 and rs9357419. In an exemplary embodiment, at least one genetic indicator can be located on chromosome 15. In another exemplary embodiment, the at least one genetic indicator can be located within a cluster or cloud of SNPs within a region of a chromosome that may be in linkage disequilibrium with one another. The genetic indicator(s) can include one or more SNPs within the cloud and/or all the SNPs within the cloud. The genetic indicator(s) can also include one or more SNPs in linkage disequilibrium. In yet another exemplary embodiment, at least one genetic indicator is rs 17702901. At least one genetic indicator can be rsl7702901 and the DNA can be negative for rsl7702901. In another embodiment, the DNA is negative for the rs 17702901 and the pre-operative BMI of the subject can be greater than 25 kg/m .

[0093] In one aspect, a method of treating metabolic or weight-related disorders includes evaluating expression of one or more genes associated with response to a metabolic procedure. The gene can also be shown to be differentially expressed in patients before or after bariatric surgery. The expression level of the gene(s) can be compared to a reference range of expression of the gene and if expression of the gene(s) is outside the reference range, a first metabolic procedure can be performed, or if expression of the gene is inside the reference range, an alternative second metabolic procedure can be performed. For example, the expression level of the gene(s) can be compared to a reference range of expression of the gene and if expression of the gene(s) is outside the reference range, a first metabolic procedure can be performed, or if expression of the gene is inside the reference range, an alternative second metabolic procedure without a bariatric surgery can be performed. In some instances, the reference range of gene expression can be determined from multiple patients having undergone a metabolic procedure, such as bariatric surgery. The reference range of gene expression can be an average of gene expression from multiple patients. The reference range of gene expression can be about +30%, +25%, +20%, +15%, +10%, or +5% of an average of gene expression from multiple patients. The multiple patients may be a group of patients that have experienced therapeutically significant weight loss associated with a metabolic procedure, such as bariatric surgery;

improvement, alleviation and/or amelioration of one or more co-morbid conditions.

Alternatively, the group of patients may have experienced lack of therapeutically significant weight loss or an adverse metabolic event associated with a metabolic procedure, such as bariatric surgery, increased risk of obesity or obesity-related co-morbid conditions.

[0094] The gene can be at least one of SEQ ID NOs 1-128, also shown in Table 10. The gene can correlate with therapeutically significant weight loss associated with a metabolic procedure, such as bariatric surgery; improvement, alleviation or amelioration of one or more co- morbid conditions; and/or lack of therapeutically significant weight loss or an adverse metabolic event associated with bariatric surgery, increased risk of obesity or obesity-related co-morbid conditions in the subject. In one embodiment, gene expression can correlate with therapeutically significant weight loss after a metabolic procedure, such as bariatric surgery. Therapeutically significant weight loss can be characterized by loss of at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85% or more of excess body weight. Therapeutically significant weight loss can also be characterized as at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, or more weight change. Alternatively or in addition to, therapeutically significant weight loss can be characterized by change of at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85% or more of body mass index.

[0095] Also, gene expression can correlate with improvement, alleviation or amelioration of one or more co-morbid conditions in the subject. Gene expression can correlate with, for example, reduced hypertension, reduced dyslipidemia, improvement or alleviation of diabetes, reduced acid reflux, alleviation of fatty liver or steatohepatitis, reduced risk of heart disease, alleviation of depression, alleviation of sleep apnea, alleviation of asthmatic symptoms, alleviation of arthritis, reduced risk of compression fractures, reduced occurrence of gallstones, lymphedema, alleviation of urinary incontinence, reduced risk of stroke, reduced risk of cancer and/or reduced risk of other metabolic syndromes.

[0096] Alternatively, gene expression can correlate with lack of therapeutically significant weight loss or an adverse metabolic event associated with bariatric surgery or increased risk of obesity, or obesity-related co-morbid conditions in the subject. Gene expression can correlate with, for example, lack of weight loss after bariatric surgery, increased hypertension, risk of dyslipidemia, development of diabetes, acid reflux, fatty liver disease or steatohepatitis, heart disease, depression, sleep apnea, asthmatic symptoms, arthritis, compression fractures, gallstones, lymphedema, urinary incontinence, stroke, cancer and/or risk of other metabolic syndromes.

[0097] Gene expression can be measured prior to any metabolic procedure or preoperative procedure. Gene expression can be also be measured and compared to one or more

measurements taken at different time points, such as after a metabolic procedure or post-surgery. Gene expression can be measured at 1, 3, 6, 9, 12, 18, 24, 36, 48, 72, 84, 96, 108, 120 months post metabolic procedure or postoperative or any number of months in between. In an exemplary embodiment, gene expression is measured prior to a metabolic procedure.

Modulating Gene Expression

[0098] Methods and compositions for modulating expression of at least one gene associated with response to a metabolic procedure in a target tissue to treat a subject having a metabolic disorder are also disclosed. Methods and pharmaceutical compositions to modulate gene expression can include delivering regulatory proteins, ligands, agonists and antagonists of expression of the gene to a target tissue. Gene therapy can be used to modulate gene expression and can also be accomplished by methods known to those skilled in the art. For example, one approach is to use an inducible promoter to drive expression of the gene delivered. In return, the in vivo steady state level of the gene can be increased, through augmented expression of the gene.

The terms "modulate" or "modulating" are used herein to refer to an increase or or change in expression of at least one target protein or gene. [00100] In one aspect, methods and composition are disclosed to modulate gene expression by providing a full-length, a portion or fragment of, or variant of the gene or its encoded protein and expressing the full-length, a portion or fragment of, or variant of the gene or its encoded protein in the target tissue. The term "full-length" refers to the entire open reading frame, capable of expressing a full-length encoded protein.

[00101] A "portion" or "fragment" of the gene or encoded protein refers to any sequence that has fewer nucleic acids or amino acids than the entire sequence of the gene or its encoded protein. Sizes of nucleic acid fragments can be 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater of the full-length gene. Sizes of peptide fragments can be about 500 amino acids, about 400 amino acids, about 300 amino acids, about 200 amino acids, about 100 amino acids, about 80 amino acids, about 60 amino acids, about 40 amino acids, about 20 amino acids, about 10 amino acids or any fragment in between of the full-length protein.

[00102] "Variant" as the term is used herein, can be a polynucleotide or polypeptide that differs from a reference nucleic acid or protein (i.e., SEQ ID NOs 1-128), but may retain essential properties (i.e., biological activity or conserved domains). A typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the gene or sequence including or affected by the reference

polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. Variant polynucleotides can include polynucleotides having at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity or at least 99% identity to the reference nucleotide sequence of the gene.

[00103] Variant polypeptides can include any polypeptide having an amino acid residue sequence substantially identical to a sequence specifically shown herein in which one or more residues have been conservatively substituted with a functionally similar residue, and which displays the ability to mimic essential properties of the reference protein. Variant polypeptides can include polypeptides having at least 70% homology, at least 80% homology, at least 90% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology or at least 99% homology to the reference protein sequence.

[00104] In another embodiment, the compositions can include vectors to modulate expression of the gene. In an exemplary embodiment, the vector can include a full-length, a portion or fragment of, or variant of at least one of the nucleic acid sequences found in SEQ ID NOs 1-128. The vector can also be a viral vector, such as adenoviral vectors, adeno-associated viral vectors, retroviral vectors (including lentiviral vectors), alphaviral vectors (e.g., Sindbis vectors), and herpes virus vectors. The vector can also include an inducible promoter. The inducible promoter can be inducible through response to a regulator, such as cellular conditions, inducer molecules or stimuli. Regulatable promoters include inducible promoters, which are usually "off," but which may be induced to turn "on," and "repressible" promoters, which are usually "on," but may be turned off. Many different regulators are known to effect control over the activity of regulatable promoters, including temperature, hormones, growth factors, cytokines, heavy metals, and regulatory proteins. In one embodiment, the promoter can be inducible through exposure to an energy source. In another embodiment, the promoter can be inducible through exposure to light.

[00105] The compositions include a therapeutic agent that may be administered in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. An exemplary form will depend on the intended mode of delivery and therapeutic application. Typical therapeutic agents are in the form of injectable or infusible solutions, such as therapeutic agents similar to those used for passive immunization of humans. Another mode of delivery is parenteral (e.g. , intravenous, subcutaneous,

intraperitoneal, intramuscular). In one embodiment, the therapeutic agent is delivered by intravenous infusion or injection. In another embodiment, the therapeutic agent is delivered by intramuscular or subcutaneous injection. In another embodiment, the therapeutic agent is delivered perorally. In yet another embodiment, the therapeutic agent is delivered to a specific location using stereotactic delivery. In an exemplary embodiment, the therapeutic agent is formulated for delivery to the target tissue selected from the group consisting of a brain, a spinal cord, a sympathetic nervous system, a parasympathetic nervous system, an enteric nervous system, a gastrointestinal tract and a pancreas.

[00106] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the vector in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filter sterilization.

[00107] The composition can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. In certain embodiments, the therapeutic agent may be prepared with a carrier that will protect the agent against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. The carrier may also target delivery to at least one of a brain, a spinal cord, a sympathetic nervous system, a parasympathetic nervous system, an enteric nervous system, a gastrointestinal tract and a pancreas.

[00108] Carriers can be made of biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. The compositions may include a "therapeutically effective amount" to modulate expression of the gene(s). A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as gene expression modulation. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the treatment to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the treatment are outweighed by the therapeutically beneficial effects. [00109] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially

advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

Bariatric Surgery and Alternative Metabolic Procedures

[00110] After analyzing for the presence or absence of genetic indicators or measuring expression of the gene(s), a determination of whether to perform a first metabolic procedure or a second metabolic procedure, which is different than the first metabolic procedure, can be made. For example, after analyzing for the presence or absence of genetic indicators or measuring expression of the gene(s), a determination of whether to perform a first metabolic procedure, such as a bariatric surgery, or a second metabolic procedure excluding bariatric surgery can be made.

[00111] Bariatric surgery includes procedures often referred to as metabolic surgery or therapy, as well as a variety of procedures performed in a subject that leads to a physiologic improvement in energy balance, nutrient utilization, or metabolic disorders. Surgical procedures to treat severe obesity or obesity-related conditions have included various forms of bariatric surgery, such as but not limited to, gastric bypass, Roux-en-Y gastric bypass (RYGB), biliopancreatic diversion, partial gastrectomy procedures such as vertical sleeve gastrectomy, adjustable gastric banding, duodenal switch, duodenojejunal bypass, vertical banded

gastroplasty, intragastric balloon therapy, greater curvature plication, gastric plication (including anterior and anteroposterior plication) and other forms of gastric volume reduction, Magenstrasse and Mill, ileal transposition or interposition, small bowel transposition, biliary diversion, procedures involving anastomotic connections of the gastrointestinal tract (e.g., jejunoileostomy, etc.), gastric balloon implantation and other gastric or intestinal device implantation, gastric, duodenal or intestinal endoluminal barrier implantation, gastric electrical stimulation, small bowel electrical stimulation, vagal electrical stimulation, vagal electrical inhibition, and variations of the procedures above as well as other methods known by those skilled in the art. Such surgical procedures have increasingly been performed laparoscopically. Reduced postoperative recovery time, markedly decreased post-operative pain and wound infection, and improved cosmetic outcome are well established benefits of laparoscopic surgery, derived mainly from the ability of laparoscopic surgeons to perform an operation utilizing smaller incisions of the body cavity wall. Non-surgical procedures can include, but are not limited to, pharmacological and nutritional therapies, such as hormone and neuropeptide therapy, receptor agonists and antagonists, etc.; procedures including a device, such as gastric balloon

implantation and other gastric or intestinal device implantation, gastric, duodenal or intestinal endoluminal barrier implantation, etc.; and/or the activation of brown adipose tissue. In one embodiment, the first metabolic procedure is a bariatric surgery. In one embodiment, the first metabolic procedure is a non-surgical procedure. In another embodiment, the second metabolic procedure is a non- surgical procedure. In another embodiment, the second metabolic procedure is a procedure different from the first metabolic procedure.

[00112] Different metabolic procedures, like bariatric surgery, can also produce similar end results, such as weight loss or amelioration of co-morbid conditions, by acting through similar mechanisms. Studies done by Chambers et al. in Gastroenterology 2011; 141(3):950-958, which is incorporated herein by reference in its entirety, have shown similar effects of vertical sleeve gastrectomy and gastric bypass on weight loss, food intake, insulin sensitivity, glucose tolerance, insulin secretion, endogenous glucose production, and glucagon-like peptide- 1 secretion. In addition, studies performed by Peterli et al. in Ann Surg 2009; 250(2):234-41, which is incorporated herein by reference in its entirety, have reproduced similar studies in humans showing that vertical sleeve gastrectomies and RYGB have similar effects on glucose

production, and glucagon-like peptide- 1 secretion. In particular, gastric bypass, biliopancreatic diversion, sleeve gastrectomy and endoluminal sleeve have shown similar effects on weight loss, food intake, insulin sensitivity, glucose tolerance, insulin secretion, and endogenous glucose production. Moreover, bariatric surgical procedures have also shown similar effects on the gastrointestinal endocrine system. Levels of ghrelin, glucagon-like peptide- 1, peptide YY, amylin and gastric inhibitory polypeptide have shown similar changes in post-prandial secretion levels in individuals who have undergone gastric bypass, biliopancreatic diversion, sleeve gastrectomy, ileal interposition or duodenal endoluminal sleeve.

[00113] Some non-surgical examples of alternative metabolic procedures to bariatric surgery can include, but are not limited to, administering pharmacological and nutritional therapies, such as hormone and neuropeptide therapy, receptor agonists and antagonists, etc.; providing an alternative medical device based therapy, such as, but not limited to, gastric balloon implantation and other gastric or intestinal device implantation, gastric, duodenal or intestinal endoluminal barrier implantation, etc.; and/or the activation of brown adipose tissue. (See US Pat. Pub. No. 2011/0263490 entitled "Diagnostic Methods And Combination Therapies Involving MC4R" filed December 29.2010, which is hereby incorporated by reference in its entirety.) The treatments can be temporary. By temporarily performing the treatments, assessment of the efficacy of the treatment can be made. Moreover, as the treatment can be temporary, and possibly reversible, evaluating the efficacy of the treatment can influence whether additional treatments need to be performed or if the treatment alone is sufficient to attain the desired weight loss and other clinical results.

[00114] Hormone and neuropeptide therapy can also be used to regulate or suppress appetite, increase body energy expenditure, and/ or decrease fat mass accumulation (McMinn, J. E., Baskin, D. G. & Schwartz, M. W., Obes. Rev. 2000; 1:37-46; Drazen, D. L. & Woods, S. C, Curr. Opin. Clin. Nutr. Metab. Care 2003; 6:621-629).

[00115] Activation of brown adipose tissue (BAT) can further lead to mobilization of fat stores within brown adipocytes to increase fat metabolism. The controlled activation of BAT can be optimized, leading to weight loss by reducing the stores of triglycerides in white adipose tissue (WAT). [00116] BAT activation can occur either directly or transcutaneously. Either can stimulate the sympathetic nervous system to physiologically activate BAT. Whether BAT is activated directly and/or transcutaneously, target areas for BAT stimulation can include areas in the vicinity of BAT depots, e.g., the nape of the neck, over the scapula, alongside the spinal cord, and around the kidneys. Any BAT depot can be selected for activation. In the course of treating a patient, BAT nerves can be stimulated at any one or more BAT depots and can be stimulated

simultaneously, e.g., two or more BAT depots being concurrently stimulated, or stimulated sequentially, e.g., different BAT depots being stimulated at different times. Simultaneous stimulation of BAT can help encourage more and/or faster energy expenditure. Sequential stimulation of BAT can help prevent the "burning out" of target nerves and can help stimulate the creation of new BAT cells. Sequential nerve stimulation can include stimulating the same BAT depot more than once, with at least one other BAT depot being activated before activating a previously activated BAT depot.

[00117] Generally, direct activation of BAT can include implanting a device below the skin surface proximate to a BAT depot, e.g., within a BAT depot, and activating the device to deliver an electrical signal to the nerves innervating the BAT depot and/or to brown adipocytes directly. BAT itself is densely innervated, with each brown adipocyte being associated with its own nerve ending, which suggests that stimulating the BAT directly can target many if not all brown adipocytes and depolarize the nerves, leading to activation of BAT. The sympathetic nerves that innervate BAT can be accessed directly through standard surgical techniques, as will be appreciated by a person skilled in the art.

[00118] The electrical signal, whether transcutaneously or directly delivered to BAT, can be configured in a variety of ways. The stimulation "on" time amplitude can be higher for shorter periods and increased or decreased for longer periods of application. The electrical signal can have any "geometry" of the applied voltage, e.g., square waves, ramp waves, sine waves, triangular waves, and waveforms that contain multiple geometries. A transcutaneous device can be used to transcutaneously activate BAT through a variety of sizes, shapes, and configurations. Generally, the device can be configured to generate and/or deliver an electrical signal to tissue at predetermined intervals, in response to a manual trigger by the patient or other human, in response to a predetermined trigger event, or any combination thereof. In an exemplary embodiment, the transcutaneous device can include an electrical stimulation patch configured to be applied to an external skin surface and to deliver an electrical signal to tissue below the skin surface, e.g., to underlying BAT.

[00119] Stimulation of BAT using an electrical signal is described in further detail in US Pat. Pub. No. 2011/0270360 entitled "Methods And Devices For Activating Brown Adipose Tissue Using Electrical Energy" filed December 29, 2010, and stimulation of BAT using other exemplary modes of stimulation are described in further detail in PCT Pat. App. No.

PCT/US11/66399 entitled "Methods And Devices For Activating Brown Adipose Tissue With Targeted Substance Delivery" filed December 21, 2011, PCT Pat. App. No. PCT/US 11/66358 entitled "Brown Adipocyte Modification" filed December 21, 2011, PCT Pat. App. No.

PCT/US 11/66409 entitled "Methods And Devices For Activating Brown Adipose Tissue With Light" filed December 21, 2011, and PCT Pat. App. No. PCT/US 11/66415 entitled "Methods And Devices For Activating Brown Adipose Tissue With Cooling" filed December 21, 2011.

[00120] The electrical signal, whether transcutaneously or directly delivered, can be configured in a variety of ways. The stimulation "on" time amplitude can be higher for shorter periods and increased or decreased for longer periods of application. The electrical signal can have any "geometry" of the applied voltage, e.g., square waves, ramp waves, sine waves, triangular waves, and waveforms that contain multiple geometries. A transcutaneous device can be used to transcutaneously activate BAT through a variety of sizes, shapes, and configurations. Generally, the device can be configured to generate and/or deliver an electrical signal to tissue at predetermined intervals, in response to a manual trigger by the patient or other human, in response to a predetermined trigger event, or any combination thereof. In an exemplary embodiment, the transcutaneous device can include an electrical stimulation patch configured to be applied to an external skin surface and to deliver an electrical signal to tissue below the skin surface, e.g., to underlying BAT.

Procedure Outcome Indicators

[00121] Indicators that predict outcomes after a metabolic procedure can also be measured and used in the methods disclosed herein. Such indicators can include genetic indicators and/or clinical measurements obtained from the patient. Examples of genetic indicators are described in more detail in U.S. Prov. Pat. App. No. 61/704,434 entitled "Clinical Predictors of Weight Loss" filed on September 21, 2012, Atty. Dkt. No. 100873-572 (END7094USPSP), which is incorporated herein by reference in its entirety. Examples of clinical measurements can include, but are not limited to, pre-operative BMI, a glucose tolerance, bile acid profile, and body composition/fat distribution of the subject. Pre-operative BMI can be greater than 23 kg/m in non-Asians or greater than 21 kg/m in Asians. Individuals at a lower than "overweight" BMI may also be at risk of at least one weight-related comorbidity and therefore be applicable subjects to be treated or assessed by methods of this invention (e.g., Caucasians having a BMI of greater than 23 and Asians, a BMI of greater than 21). In some embodiments, pre-operative BMI can be greater than 25 kg/m in non-Asians or greater than 23 kg/m in Asians.

[00122] Additional non-limiting examples of indicators include, but are not limited to height, weight, gender, age, medical history and/or status, ethnicity, medical prescription history and/or status, types of previously received medical treatments for obesity (e.g., medications, BAT stimulation, gastric banding, gastric bypass, sleeve gastrectomy, etc), types of medical treatments previously received for health issues other than obesity (e.g., medications, surgical treatments, and non- surgical treatments), insurance information, diet information for the patient, and psychological history of the patient.

Predicting Bariatric Surgery Outcomes

[00123] Various systems and methods are provided for predicting metabolic therapy, e.g., metabolic and bariatric surgery, outcomes, i.e., a composite predictive model. The systems and methods can also provide predictions for non-surgical metabolic and bariatric treatments. In general, a user, e.g., a patient, a medical professional involved with treating a patient, a medical student, a hospital administrator, a health insurance administrator, etc., can receive predictive outcomes of multiple metabolic therapies that could be performed on a patient. The collection of therapies for the treatment of obesity and metabolic disease (e.g., diet and exercise,

pharmaceutical therapy, medically supervised therapy), metabolic surgery (open, laparoscopic, natural orifice, etc.), bariatric surgery (open, laparoscopic, natural orifice, etc.) are collectively defined herein as metabolic therapies. In one embodiment, a user can electronically access a metabolic therapy outcome prediction system, e.g., using one or more web pages. The system can provide predictive outcomes of one or more different metabolic therapies, such as bariatric surgeries, for the patient based on data gathered from the user and on historical data regarding outcomes of the different bariatric surgeries. The system can additionally provide predictive outcomes for not having any treatment and/or a comparison of the predictive outcomes of the one or more different bariatric surgeries to the predictive outcomes for not having any treatment or for having non-surgical treatment. Generally, the predictive outcomes provided by the system can include a potential clinical metabolic outcome of each of the different bariatric surgeries, e.g., a predicted amount of weight loss, a predicted amount of body mass index (BMI) reduction, an improvement in a health condition associated with a metabolic disease, an associated risk of complications from the treatment, and/or an associated cost of the surgery and post-operative care. The predictive outcomes can be based on a plurality of patient- specific characteristics, e.g., age, weight, height, BMI, ethnicity, medical prescription history and/or status, genetic data (e.g., a genetic indicator), gene expression profiles (expression of one or more genes, expression over a time period and/or in different tissues), types of previously received medical treatments for obesity (e.g., gastric banding, gastric bypass, sleeve gastrectomy, etc), medical history and/or status, gender, etc. The predictive outcomes can also be based on historic results of the different types of bariatric surgeries on other patients. The predictive outcomes can thus be based at least in part on data specific to the patient and not just on historical data, e.g., data gathered by the user from previous personal experience, friends or colleagues, journal articles, internet research, clinical data, etc. The outputs can thus be personalized to the patient. The system can help the user be more informed about which of the bariatric surgeries would be most effective if performed on the patient, help specifically compare and contrast the different bariatric surgeries, and help the user decide which of the different bariatric surgeries, if any, to pursue for the patient. The system can therefore help maximize effectiveness of treatment for the patient by allowing a most effective option to be identified and pursued by the patient and/or by medical practitioner(s) treating the patient. The system can also help inform the user about bariatric surgery options that they might not have been aware of at all, e.g., new procedures, and/or deepen understanding of bariatric surgery procedures previously known to the user. The system can be configured to allow the user to save the predictive outcomes, which can then be accessed at a later date/time by the user and/or one or more other users, e.g., the user's surgeon, the user's endocrinologist, the user's primary care physician, or other healthcare providers, etc., to which the user grants access to the saved data.

[00124] Methods and systems for providing predictive outcomes are described in more detail in U.S. Prov. Pat. App. No. 61/704,077 entitled "Systems and Methods for Predicting Bariatric Surgery Outcomes" filed on September 21, 2012, Atty. Dkt. No. 100873-610

(END7094USPSP1)), which is incorporated herein by reference in its entirety.

[00125] Output results can be reported in multiple ways, either individually or simultaneously via the system. In one embodiment, output results can be reported in parameters such as, but are not limited to, target weight loss in pounds or kilograms, target weight, percent excess body weight loss, percent weight change, percent change in BMI. In another embodiment, output results can be reported as continuous or at various cutoff points ranging from 1-100%. An example of various cutoff may include achieving at least or at most 50% excess weight loss. Another example of various cutoff may include achieving at least or at most 70% excess weight loss. In yet another embodiment, the output results can be reported as results obtained at various timepoints post metabolic procedure such as bariatric surgery or alternative metabolic procedure without bariatric surgery. Timepoints can include 1, 3, 6, 9, 12, 18, 24, 36, 48, 72, 84, 96, 108, 120 months post treatment or any number of months in between. In another embodiment, the output results can be reported as nadir weight. "Nadir weight" as used herein is defined as the lowest weight achieved at least 10 months after surgery without coexisting debilitating illness, with or without use of weight lowering medications.

[00126] The input parameters can be analyzed via the interface in a model algorithm. The algorithm can apply univariate analyses, multivariable regression analyses, advanced regression analyses, and other data mining techniques on multiple data sets to build, train and prospectively model predicted results after metabolic procedure such as bariatric surgery or alternative metabolic procedure without bariatric surgery.

[00127] The interface can also be used to output varying levels of confidence of the prediction on the results after metabolic procedure such as bariatric surgery or alternative metabolic procedure without bariatric surgery. Such examples can include the predicted result based on variable changes in weight, e.g., 20% chance of 40 lb change in weight, 40% chance of 201b change in weight, etc. The output can further include predictions based on complications associated with a metabolic procedure such as bariatric surgery or an alternative metabolic procedure without bariatric surgery.

[00128] The output results can be collected and used by patients, primary care providers, and/or other referring physicians or other healthcare providers. The information can be provided in the patient's home, during a health care provider seminar, during a physician's office visit, and/or prior to treatment. Moreover, the interface can be accessed through various routes. In one embodiment, the interface can be accessed via the internet to record and model the patient information. In another embodiment, the interface can be accessed via a mobile device or "app," software application designed specifically for mobile or handheld devices. In yet another embodiment, the interface can be accessed via application software that can be installed on a computer or other device.

[00129] In addition to providing predictive outcomes, the system can optionally provide educational information regarding each of the different bariatric surgeries and/or other types of information related to bariatric surgery such as estimated patient monetary cost (based on one or more factors such as the patient' s insurance carrier, similar procedures performed in the patient' s geographic location, etc.), estimated insurance reimbursement (based on one or more factors such as the patient's insurance carrier, similar procedures performed in the patient's geographic location, etc.), estimated length of post-surgery hospital stay (based on one or more factors such as similar procedures performed in the patient's geographic location, the patient's age, the patient's other health conditions or disorders, etc.). The system can therefore help the user be more fully informed about the various risks and benefits of the various bariatric surgeries before deciding which of the bariatric surgeries to pursue, if any. Applying similar modeling techniques, personalized predictions can be provided for use of one or more of the preceding educational and/or other information.

[00130] One skilled in the art will appreciate further features and advantages of the invention based on the above-described embodiments. Accordingly, the invention is not to be limited by what has been particularly shown and described in the examples or figures, except as indicated by the appended claims. All publications and references cited herein are expressly incorporated herein by reference in their entirety.

EXAMPLES

Example 1: Genetic Analysis Materials and Methods

[00131] Study Population: To identify genetic factors contributing to weight loss after RYGB, an exploratory genome- wide association study of individuals of European descent undergoing RYGB was performed. The study was approved by and performed in accordance with the guidelines of the Human Studies Committee at the Massachusetts General Hospital. From February 2000 until April 2007, consent was obtained from 1018 (97%) of MGH Weight Center patients undergoing RYGB to collect and extract DNA from tissue samples removed at the time of surgery. Intraoperative liver, subcutaneous fat, omental fat, and stomach tissues were collected in RNAlater (Amnion/ Applied Biosystems) and stored at -80°C. Operations were either open (41%) or laparoscopic (59%) RYGB. For the open procedure, the stomach was partitioned but not divided, and for the laparoscopic procedure the pouch was partitioned and divided from the remaining stomach. Otherwise, the techniques were the same, with an approximately 30 ml pouch, a 100-120 cm Roux (alimentary) limb fashioned in a retrocolic, retrogastric configuration, and a pancreaticobiliary limb extending approximately 75 cm beyond the ligament of Treitz.

[00132] Demographic and clinical information was extracted from review of the electronic medical records. Weight nadir was defined as the lowest weight achieved at least 10 months after surgery. Chart-derived nadir weight was validated through telephone interviews in a subset of patients (n=306); there was a 97% correlation between these two sources. Percent weight loss (%WL) at weight nadir was calculated by subtracting the patient's weight at nadir from his or her presurgical weight, and then dividing by the patient' s presurgical weight.

[00133] The following materials and methods apply to Examples 2 through 8:

[00134] Surgical procedures: For the open procedure, the stomach was partitioned but not divided, and for laparoscopic procedure the pouch was partitioned and divided from the remaining stomach. Otherwise, the techniques were the same, with an -30 ml pouch and a 100- 120 cm Roux limb fashioned in a retrocolic, retrogastric configuration, and the pancreaticobiliary limb extending -75 cm from the ligament of Treitz.

[00135] DNA analysis: Genomic DNA was extracted from collected liver samples, and genotyping was performed using the Illumina HumanHap 650Y BeadChip array (Illumina Inc., San Diego, CA). Race was genetically determined by principal component analysis using EIGENSTRAT; there was 97% concordance with self -reported race. Patients were excluded if they were on weight-lowering medications after surgery (1.7%), had cancer or other severe illness (including severe postoperative complications or reoperations; 2.4%), their DNA was not available (4.7%), or their postoperative body mass index (BMI) at least 10 months after RYGB could not be determined (7.9%). Using identity-by-descent methods (PLINK software), we identified 13 first-degree relative pairs, defined as pairs of individuals who share approximately 50% of their genetic variation, six second-degree relative pairs (-25% of genetic variation shared) and four third-degree relative pairs (-12.5% of genetic variation shared). An additional eight patients who were genetically related to established first-degree pairs were excluded, leading to a final sample of 848 (83.3%). Then the samples were matched on genetically identified race, randomly paired 794 unrelated individuals from the cohort, and compared for the similarity in their weight loss outcomes after surgery. Also identified were 20 cohabitating individuals by review of the medical records of all patients who had undergone RYGB at this center. No cohabitating individuals were genetically related.

[00136] Endpoint and covariate assessment: Demographic and clinical information was extracted from the medical record. A patient's weight nadir was identified, defined as the lowest weight achieved at least 10 months after surgery without coexisting debilitating illness or use of weight lowering medications. The percent excess body weight lost (EBWL) at weight nadir was calculated by subtracting the patient's nadir BMI from his or her preoperative BMI and dividing this difference by the difference between the patient's initial BMI and the upper normal BMI of 25 kg/m . Chart-derived weights were validated via telephone interviews in a subset of patients (n = 306); there was a 97% concordance between these two sources.

[00137] Statistical analyses: The average difference in outcome was calculated by pair, and analyses based on these mean differences were based on one entry per pair. Wilcoxon rank sum tests were used to test differences in mean response between groups. Linear mixed effects models were constructed to determine the intraclass correlation coefficients (ICC) by type of relationship. All nongenetic analyses were performed using SAS statistical software (SAS Institute, Cary, NC).

[00138] The following materials and methods apply to Example 9:

[00139] Genotyping and Data Cleaning of Study Population: Samples were shipped to the Rosetta Inpharmatics Gene Expression Laboratory (Seattle, WA) where genomic DNA was extracted from liver samples. Nine hundred fifty samples were successfully genotyped using the Illumina HumanHap 650 Y BeadChip array (Illumina Corp, San Diego, CA). Data were converted to PLINK format (Hatoum, I. J., Stein, H. K., Merrifield, B. F. & Kaplan, L. M., Capacity For Physical Activity Predicts Weight Loss After Roux-En-Y Gastric Bypass, Obesity (Silver Spring) 17, 92-99 (2009)), and all genetic analyses were performed using this software. Using identity-by-descent (IBD) coefficients for all pairs of individuals, 36 related individuals were identified, defined as an IBD coefficient >0.125. One person per family was included for analysis, based on completeness of phenotypic and genetic information. In addition, one person was removed from the analysis because >10 of the person's genetic information was missing. Of the remaining 933 individuals, 806 self-identified as European. To address population structure not captured through self-identification, EIGENSTRAT (Hatoum, I. J. et al. Heritability Of The Weight Loss Response To Gastric Bypass Surgery, / Clin Endocrinol Metab 96, E1630- 3 (2011)) was used to calculate principal components of ancestry, and identified 25 outlying samples (greater than six standard deviations) for a sample of 781 patients. Of these 781 samples, 693 had a weight nadir value, were not on weight-lowering medications after surgery, did not have cancer or other severe illness, and were thus included in the final data set.

Imputation of 1,674,205 additional SNPs was performed using MACH software. (Ochner, C. N. et al,. Selective Reduction In Neural Responses To High Calorie Foods Following Gastric Bypass Surgery, Ann. Surg. 253, 502-507 (2011).) A SNP was excluded from analyses if it was missing in >10 of the samples, if it had less than 1% minor allele frequency, or if it was not in Hardy- Weinberg equilibrium, resulting in a SNP set of 1,943,373 SNPs. A genomic control inflation factor of 1.01 was observed, indicating minimal inflation of test statistics due to population stratification (Figure 1). [00140] Gene Expression Profiling: Total RNA was extracted from liver, subcutaneous fat, and omental fat tissues. Liver, subcutaneous fat and omental fat RNA was amplified and converted to fluorescently labeled cRNA that was hybridized to custom 44K DNA

oligonucleotide microarrays from Agilent Technologies (Santa Clara, CA, USA). A detailed description of the normalization and data cleaning methods has been described previously. (Shin, A. C, Zheng, H., Pistell, P. J. & Berthoud, H.-R., Roux-En-Y Gastric Bypass Surgery Changes Food Reward In Rats, Int J Obes (Lond) 35, 642-651 (2011).) 707, 870, and 916 samples were profiled from liver, subcutaneous fat, and omental fat, respectively.

[00141] Replication Cohort: From May 2007 until October 2009, we obtained consent from 369 Caucasian MGH Weight Center patients undergoing RYGB to collect and extract DNA from tissue samples removed at the time of surgery. Intraoperative liver tissue samples were collected in RNAlater (Amnion/ Applied Biosystems) and stored at -80°. Operations were as described for the GWAS cohort. Clinical traits were extracted from the electronic medical records, as described for the GWAS cohort.

[00142] Genotyping and Data Cleaning of Replication Cohort: Samples were shipped to the Eli and Edythe Broad Institute (Cambridge, MA) where genomic DNA was extracted from liver samples. Three hundred sixty-nine samples were successfully genotyped using Sequenom MassARRAY (Sequenom Inc., San Diego, CA). Of these, 327 had a recorded weight nadir value, were not on weight-lowering medications after surgery, did not have cancer or other severe illness, and were thus included in the final data set.

[00143] Endpoint and Covariate Assessment: Demographic and clinical information was extracted from review of electronic medical records. Weight nadir was defined as the lowest weight achieved at least 10 months after surgery. Chart-derived nadir weight was validated through telephone interviews in a subset of patients (n=306); there was a 97% correlation between these two sources. Percent weight loss (%WL) at weight nadir was calculated by subtracting the patient's weight at nadir from his or her presurgical weight, and then dividing by the patient's presurgical weight. Percent excess body weight loss (%EBWL) was calculated by subtracting the patient's current weight from the patient's presurgical weight, and dividing this difference by the difference between the patient's presurgical weight and the patient's ideal or target weight.

[00144] Animal Studies: All experiments in mice were performed in compliance with and were approved by the Institutional Animal Care and Use Committee of the Massachusetts General Hospital. We have developed a mouse model of RYGB that closely mimics the procedure in humans. At 12 weeks of age, male, diet-induced obese C57BL/6 mice on a high fat diet from weaning (Jackson Laboratories, Bar Harbor, ME) were randomized to RYGB, sham operation with post-operative ad libitum food intake, or sham operation with food restriction to match the weights of the RYGB mice weekly. In the RYGB procedure, the stomach was divided into a gastric pouch and distal stomach using a vascular clip (Ethicon Endo-Surgery, Inc., Cincinnati, OH). For each of the Roux (alimentary) and biliopancreatic limbs, the length of the intestine was 6 cm, approximating the 12-15% instestinal bypass used in the human operation. The alimentary limb was then secured to the gastric fundus by a gastrojejunal anastamosis. Sham operations consisted of a laparotomy and repair. Mice were maintained on a high-fat diet (D12492 diet; Research Diets, New Brunswick, NJ) except during the 7-14 days after surgery, when all mice were maintained on a postoperative protocol that progressed from water only to liquid diet to solid diet. Animals were individually housed in a 12-hour light, 12-hour dark cycle under controlled temperature and humidity conditions.

[00145] Animals were euthanized by carbon dioxide inhalation followed by cervical dislocation at 10 weeks after surgery. All tissues were harvested immediately, flash frozen and stored at -80°C until further processing. For gene expression studies, total RNA was extracted using Superscript^® III First-Strand Synthesis System for RT-PCR kit (Invitrogen, Carlsbad CA), according to the manufacturer's instructions. One μg of total RNA was used as template for cDNA synthesis using TaqMan^® Gene Expression Master Mix kit (Applied Biosciences, Carlsbad CA), according to the manufacturer's instructions. Relative expression level was determined by qPCR using pre- optimized, gene-specific primer probe sets purchased from Applied Biosciences for AQP11 (Mm00613023_ml; Cat# 4331182), SLC03A1

(Mm00452449_ml; Cat# 4331182), CLNS1A (Mm00445821_ml; Cat# 4331182) and ST8SIA2 (Mm01311039_ml; Cat# 4331182) and a CFX96™ Real-Time PCR Detection System

(BioRad:, Hercules, CA). All expression level data was presented relative to actin. [00146] Tissue Samples: Intraoperative liver, subcutaneous fat, omental fat, and stomach tissues were collected in RNAlater (Amnion/ Applied Biosystems) and stored at -80 °C.

Operations were either open (41%) or laparoscopic (59%) RYGB. For the open procedure, the stomach was partitioned but not divided, and for the laparoscopic procedure the pouch was partitioned and divided from the remaining stomach. Otherwise, the techniques were the same, with an approximately 30 ml pouch, a 100-120 cm Roux limb fashioned in a retrocolic, retrogastric configuration, and a pancreaticobiliary limb extending approximately 75 cm beyond the ligament of Treitz.

[00147] Gene Expression Profiling: Total RNA was extracted from liver, subcutaneous fat, and omental fat tissues. Liver, subcutaneous fat and omental fat RNA was amplified and converted to fluorescently labeled cRNA that was hybridized to custom 44K DNA

oligonucleotide microarrays from Agilent Technologies (Santa Clara, CA). A detailed description of the normalization and data cleaning methods has been described previously. Successful profiling of 707, 870, and 916 samples from liver, subcutaneous fat, and omental fat, respectively, was performed.

Example 2: Genetic Analysis

[00148] Preoperatively, patients in this cohort had an average BMI of 50.2+8.6kg/m2, an average age of 44.7+11.3 yr, and were 74.8% female and 86% Caucasian. These characteristics were similar among the different groups studied (see Table 1).

Table 1: Patient demographics at baseline by type of relationship.

HISPANIC 7.8 7.7 10.0

BLACK 4.4 7.7 0

[00149] After RYGB, patients lost an average of 119.2 + 41.7 pounds at weight nadir, corresponding to an EBWL of 79.7+21.8%; the population pattern of percent excess weight loss follows the wide and normal distribution observed previously (Figure 1).

[00150] To determine whether there is a genetic component to the variation in weight loss after surgery, weight loss after RYGB within pairs of genetically related was compared to genetically unrelated individuals. Unrelated individuals demonstrated far less similar weight loss after surgery than first-degree relatives (Figure 2), with an average difference in EBWL of 25.4% in unrelated individuals and 9.9% in first-degree relatives (P = 0.001). Because the observed similarity in weight loss could result primarily from shared environmental influences, weight loss within pairs of individuals who were living together but who are genetically unrelated was compared. Pairs of these environmentally related controls had a shared response similar to completely unrelated individuals (mean 26.1% difference in EBWL; P = 0.60), a response that was substantially different from that of first-degree relatives (P = 0.005). The small number of second- and third-degree relative pairs in this cohort precludes statistical analysis of these groups.

[00151] The ICC represents the portion of total variation in outcome explained by the pair relationship. Using mixed-effects models adjusted for age, sex, year of surgery, and preoperative BMI, the ICC were 70.4% for first-degree relatives (P = 0.02), 14.3% for environmentally related controls (P = 0.67), and 0.9% for randomly paired individuals (P = 0.48). Because preoperative BMI is strongly associated with postoperative percent EBWL, we additionally matched the unrelated controls based on 5-kg/m2-wide BMI groups to mimic the distribution of differences in preoperative BMI between first-degree relatives. After this adjustment, first degree relative pairs still demonstrated significantly less difference in weight loss compared with the unrelated controls (difference in EBWL for unrelated pairs 22.3+17.9%; P = 0.01 vs. first- degree relatives). Thus, first-degree relatives have weight loss outcomes after surgery that closely and significantly resemble each other, a characteristic not shared by environmentally related (cohabitating) controls or randomly paired individuals. Similar results were seen when men and women were examined separately (data not shown).

Example 3: Reporting Methods Materials and Methods

[00152] Study Population: Participants were recruited from the population of patients undergoing RYGB at a single academic center that is part of a larger 13-hospital network in the Boston metropolitan area. From February 2000 until April 2007, we obtained consent from 1018 (97%) of the patients undergoing RYGB at this center. Operations included both open and laparoscopic RYGB performed by one of two surgeons using the same operative techniques; the surgical methods have been described previously. This study was approved by the institutional review board of the Massachusetts General Hospital.

[00153] Endpoint and Covariate Assessment: Demographic and clinical information was extracted from the medical record. We indentified a patient' s weight nadir, defined as the lowest weight achieved at least 10 months after surgery without coexisting debilitating illness or use of weight-lowering medications. One-year weight was defined as the weight closest to 12 months from surgery, within the range of 10-14 months after surgery. Post- operative weights were available for 848 patients (83.3%). Chart-derived nadir weights were validated by telephone interviews in a subset of patients (n=306); there was a 94% concordance between these two sources. Diabetes diagnosis was extracted from patient charts and defined as the documentation of diabetes, a fasting glucose measurement > 126 mg/dL, or the use of diabetes medication (insulin or metformin).

[00154] Weight loss was characterized at one year and at weight nadir using seven different metrics (Table 2). Residuals were calculated by regressing postoperative BMI (the dependent variable) on preoperative BMI (the independent variable) and outputting the residuals from this model. Because residuals derived from regressing postoperative BMI on preoperative BMI represent, by definition, postoperative BMI independent of preoperative BMI, these residuals were used as the benchmark of independence from preoperative BMI. Weight loss characterized by number of pounds lost was calculated by subtracting the patient's final weight from his or her baseline weight. As BMI is a function of weight and height, and height is almost always stable over the course of a weight loss study, BMI and pounds lost are closely similar methods for measuring weight loss. Percent weight change was calculated by dividing the absolute pounds lost by the patient's initial weight, and is statistically interchangeable with percent BMI change. Percent EBWL was calculated by dividing the difference between initial BMI and final BMI by the difference between initial BMI and a "normal" BMI. A BMI of 25 kg/m is commonly used to represent the target, or upper limit of a "normal" BMI, but other standards, including race- specific BMI standards or other "ideal weights" according to the Metropolitan Life Insurance Company (MLIC) life tables, may also be used to represent "normal." In this study, EBWL was calculated as described above, using a reference BMI of 25 kg/m .

[00155] Statistical Analyses: Patients were divided into seven preoperative BMI (pBMI) groups (35-39.9, 40-44.9, 45-49.9, 50-54.9, 55-59.9, 60-64.9, 65+). Means for each weight loss metric were calculated for each pBMI group, and linear trends across the groups were assessed using a test for trend of the median value within each group. Correlations between pBMI and each continuous metric were assessed using Spearman correlations, and r measures were derived from linear regressions. All analyses were performed using SAS statistical software (SAS Institute, Cary, NC).

Example 4: Reporting Methods of Weight Loss

[00156] At baseline, participants had an average BMI of 50.0 (SD ± 8.3) kg/m , an average age of 44.7 (± 11.5) years, 74.3% were female and 26.2% had diabetes. One year after RYGB, patients lost an average of 17.1 kg/m , 34.2 % of baseline weight, and 71.7 % of excess body weight (Table 2). By weight nadir, which on average occurred 28.5 months after surgery, patients lost an average of 19.4 kg/m , 38.7 % of baseline weight, and 81.2% of excess body weight (Table 3).

Table 2: Different Parameterizations of Weight Loss.

Metric Abbreviation Formula

e = Observed Final BMI— Predicted Final BMI

Residuals Predicted BMI from the equation:

Final BMI = Initial BMI

Weight lost (in pounds or kg) Δ pounds, Δ kg Initial Weight(lbs or kgs) - Final Weight(lbs or kgs) Weight achieved (in pounds

Final Weight (lbs or kgs) or kg)

BMI units lost Δ BMI Initial BMI - Final BMI

BMI achieved Final BMI

Percent excess body weight Initial BMI - Final BMI

% EBWL * 100

Initial BMI - Ideal BMI

lost

Initial Weight - Final Weight

Percent weight change % WC * 100

Initial Weight Attorney uocKei ΓΝΟ.: IUU»/J-03J ( i u vwtw

Table 3: Weight loss parameterizations by preoperative BMI group.

[00157] The residuals derived from regressing postoperative BMI on preoperative BMI showed no difference across pBMI groups (r=0, p=0.9 at both one year and weight nadir; Table 3, Figure 3). In contrast, there was a strong positive association between absolute change in weight (pounds lost or gained) and pBMI, with patients in lower BMI groups losing significantly less weight (r_ly = 0.52, p_ly = 3.4 * 10^"53; r_nadir = 0.54, p_nadir = 6.5 * 10^~84; Table 3, Figure 4).

[00158] The same pattern is observed when change in BMI, final attained weight, or final attained BMI is used (Table 3, Figures 3-7). When weight loss was characterized as EBWL, the opposite pattern was observed, with patients at a lower pBMI lose more EBWL at both 1 year and weight nadir (r_ly = -0.51, p_ly = 1.0 * 10^"40; r_nadir = -0.43, p_nadi_r = 6.9 * 10^~38; Table 3, Figure 8). In contrast, there was no association (r_ly = 0.04, p_ly = 0.52) between pBMI group and WC at one year, and a relatively weak association between pBMI and WC and weight nadir (rnadir = 0.13, p_nadir = 0.003; Figure 9).

[00159] Similar patterns were seen when a continuous characterization of pBMI was used (Table 3). The number of pounds lost was strongly and positively correlated with pBMI at both one year (r_Spearman=0.53, p = 4.3 * 10^"46) and at weight nadir (r_Spearman=0.55, p = 5.1 * 10^"69); BMI units lost showed a similar pattern (Table 3). EBWL was strongly negatively correlated with pBMI at both one year

-0.45, p =

7.2 * 10 ). In contrast, WC was not associated with pBMI at one year (r_S S_pPee_aa_rr_mm_aa_nn=" 0.04, p =

0.33) and was only weakly associated with pBMI at weight nadir (rs_pearman= 0.13, p = 0.0002). While pBMI explains a substantial proportion of the variability in number of pounds lost (r _ly =

2 2 2

0.36, r _nadi_r = 0.39) and EBWL (r _ly = 0.25, r _na(ji_r = 0.18), it explains only a small percentage of the variability in WC (r² _iy = 0.002, r² _nadir = 0.02).

[00160] The findings reflect the biology of weight loss after RYGB - to the extent that a higher pBMI represents a more severe form of obesity, severe obesity may "normalize" less completely after RYGB. A parallel can be drawn to other metabolic conditions, such as systolic blood pressure (SBP), where patients with more extreme levels of SBP are less likely to achieve a normal SBP and are more likely to need aggressive treatment with multiple antihypertensive treatments. [00161] Conversely, in this study patients with a lower pBMI lost less absolute weight relative to those patients with a higher pBMI, thus appearing less "successful" if absolute pounds are chosen as the weight loss metric. Whether the association between the weight loss metric and preoperative BMI is observed for biological or artificial reasons, the results indicate a potential for confounding by preoperative BMI (hidden variables associated with BMI) when searching for novel predictors. While it may be possible to partly account for the effects of preoperative BMI through adjustment for preoperative BMI using statistical models, if a relationship between the potential predictor and preoperative BMI exists there will be the potential for collinearity, which can result in incorrect estimation of the effect size and standard error of the novel predictor. Thus, it is advantageous to utilize a weight loss metric that both minimizes the association with pBMI (unlike pounds lost or EBWL) and that is clinically interpretable (unlike the use of residuals).

Example 5: Genetic Factors Materials and Methods

[00162] Cohort 1: To identify genetic factors contributing to weight loss after RYGB, an exploratory genome-wide association study of Caucasian individuals undergoing RYGB was performed. From February 2000 until April 2007, consent was obtained from 1018 (97%) of Massachusetts General Hospital (MGH) Weight Center patients undergoing RYGB to collect and extract DNA from tissue samples removed at the time of surgery. Cohort 1 may also be described herein as the original cohort, the GWAS cohort, the training cohort, or the training set.

[00163] In addition, one person was removed from analyses due to >10% of genetic information missing. Of the remaining 933 individuals, 806 self-identified as Caucasian. To address population structure not captured through self-identification, EIGENSTRAT (Price, A. L. et ah, Nat. Genet. 38, 904-909 (2006)) was used to calculate principal components of ancestry, and identified 25 outlying samples (greater than six standard deviations) for a sample of 781 patients. Of these 781 samples, 693 had a weight nadir value, were not on weight- lowering medications after surgery, did not have cancer or other severe illness, and were thus included in the final data set. Imputation of 1,674,205 SNPs was performed using MACH software (Li, Y., Wilier, C. J., Ding, J., Scheet, P. & Abecasis, G. R., Genet. Epidemiol. 34, 816- 834 (2010)). A SNP was excluded from analyses if it was missing in >10% of samples, if it has less than 1% minor allele frequency, or if it was not in Hardy- Weinberg equilibrium, resulting in a SNP set of 1,943,373 SNPs.

[00164] Genotyping and Data Cleaning of Cohort 1: Samples were shipped to Rosetta Inpharmatics Gene Expression Laboratory (Seattle, WA) where genomic DNA was extracted from liver samples. Nine hundred fifty samples were successfully genotyped using the Illumina HumanHap 650Y BeadChip array (Illumina Corp, San Diego, CA). Data were converted to PLINK format (Purcell, S. et al. PLINK: a tool set for whole-genome association and population-based linkage analyses. Am. J. Hum. Genet. 81, 559-575 (2007)) and all genetic analyses were performed using this software. Using identity-by-descent (IBD) coefficients for all pairs of individuals, 36 related individuals were identified, defined as an IBD coefficient >0.125. One person per family was included for analysis, based on completeness of phenotypic and genetic information.

[00165] Cohort 2: From May 2007 until October 2009, consent was obtained from 369 Caucasian MGH Weight Center patients undergoing RYGB to collect and extract DNA from tissue samples removed at the time of surgery. Intraoperative liver was collected in RNAlater (Amion/ Applied Biosystems) and stored at -80°. Operations were as described for the Cohort 1. Clinical traits were extracted from the electronic medical records, as described for the GWAS cohort. Cohort 2 may also be described herein as the replication cohort, the test cohort, or the test set.

[00166] Genotyping and Data Cleaning of Cohort 2: Samples were shipped to the Eli and Edyth Broad Institute (Cambridge, MA) where genomic DNA was extracted from liver samples. Three hundred sixty-nine samples were successfully genotyped using Sequenom MassARRAY (Sequenom Inc., San Diego, CA). Of these, 327 had a weight nadir value, were not on weight- lowering medications after surgery, did not have cancer or other severe illness, and were thus included in the final data set.

[00167] Tissue Samples: Intraoperative liver, subcutaneous fat, omental fat, and stomach tissues were collected in RNAlater (Amion/ Applied Biosystems) and stored at -80 °C.

Operations were either open (41%) or laparoscopic (59%) RYGB. For the open procedure, the stomach was partitioned but not divided, and for the laparoscopic procedure the pouch was partitioned and divided from the remaining stomach. Otherwise, the techniques were the same, with an approximately 30ml pouch, a 100-120 cm Roux limb fashioned in a retrocolic, retrogastric configuration, and a pancreaticobiliary limb extending approximately 75 cm beyond the ligament of Treitz.

[00168] Endpoint and Covariate Assessment: Demographic and clinical information was extracted from review of the electronic medical charts. Weight nadir was defined as the lowest weight achieved after surgery. Chart-derived nadir weight was validated through telephone interviews in a subset of patients (n=306); there was a 94% correlation between these two sources. Percent weight loss (%WL) at weight nadir was calculated by subtracting the patient's weight at nadir from his or her presurgical weight, and then dividing by the patient's presurgical weight. Percent excess body weight loss (%EBWL) was calculated by subtracting the the patient's current weight from the patient's presurgical weight, and dividing this difference by the difference between the patient's presurgical weight and the patient's ideal weight.

[00169] Gene Expression Profiling: Total RNA was extracted from liver, subcutaneous fat, and omental fat tissues. Liver, subcutaneous fat and omental fat RNA was amplified and converted to fluorescently labeled cRNA that was hybridized to custom 44K DNA

oligonucleotide microarrays from Agilent Technologies (Santa Clara, CA, USA). A detailed description of the normalization and data cleaning methods has been described previously.⁴ Successful profiling of 707, 870, and 916 samples from liver, subcutaneous fat, and omental fat, respectively, was performed.

[00170] Statistical Analysis: Each SNP was compared to %WL using linear regression in

PLINK. SNPs within a 250 kb window of each other with a r > 0.5 were not considered independent; only the strongest associated SNP for each block was considered for replication and follow-up analyses. Association results from the GWAS and replication cohorts were meta- analzyed. The association between rsl7702901 and gene expression in liver, subcutaneous fat, and omental fat was determined using Kruskal-Wallace tests with adjustment for the effect of surgery year, age, race, and gender using a principle components analysis. All genetic analyses were performed using PLINK. All non-genetic analyses were performed using SAS statistical software (SAS Institute, Cary, NC). [00171] Animal Studies: All experiments in mice were performed in compliance with and were approved by the Institutional Animal Care and Use Committee of the Massachusetts General Hospital. We have developed a mouse model of RYGB that closely mimics the procedure in humans. At 12 weeks of age, mice with a C57BL/6 background (Jackson

Laboratories, Bar Harbor, ME) were randomized to RYGB, sham operation with post-operative ad libitum food intake, or sham operation with food restriction to match the weights of the RYGB mice weekly. In the RYGB procedure, the stomach was divided into a gastric pouch and distal stomach using a vascular clip (Ethicon Endo-Surgery, Inc., Cincinnati, OH). For each of the Roux and biliopancreatic limbs, the length of the intestine was 6cm, approximating the 12- 15% instestinal bypass in human RYGB. The Roux limb was then secured to the gastric fundus by a gastrojejunal anastamosis. Sham operations consisted of a laparotomy and repair. Mice were maintained on a high-fat diet (D 12492 diet; Research Diets, New Brunswick, NJ) except during the 7-14 days after surgery, when all mice were maintained on a postoperative protocol that progressed from water only to liquid diet to solid diet. Animals were individually housed in a 12 hour light, 12 hour dark cycle under controlled temperature and humidity.

[00172] At 10 weeks post-surgery, mice were sacrificed in a carbon monoxide chamber followed by cervical dislocation. Tissues were harvested immediately, flash frozen, and stored at -80°. Samples were shipped to Rosetta Inpharmatics Gene Expression Laboratory (Seattle, WA) where mRNA was extracted and converted to cDNA. Quantitative PCR was performed on genes of interest (to be expanded with primer and QC information as it becomes available).

Example 6: Genetic Factors Contributing to Weight Loss

[00173] To identify genetic factors contributing to weight loss after RYGB, an exploratory genome-wide association study (GWAS) of cohorts of 858 unrelated Caucasian individuals undergoing RYGB was performed. The individuals were grouped according to preoperative BMI (>40, 40-45, 45-50, 50-55, 55-60, 60-65, 65+) and percent change in weight can be found in Figure 10 and demographic information is shown in Table 4. Table 4. Pre- and post-operative characteristics of the RYGB original and replication cohorts

Original Cohort Replication p-value

Cohort

Age (years; ± SD) 45.8 ± 11.2 47.1 ± 11.1 0.07

Preoperative BMI (mean kg/m ; ± SD) 50.3 ± 8.4 48.1 ± 8.5 0.0002

Sex (% female) 73.2 71.7 0.65

Diabetes (%) 40.2 41.5 0.19

BMI at weight nadir (mean kg/m ; ± SD) 30.6 ± 6.4 29.9 ± 6.0 0.32

%WL at nadir (%; ± SD) 39.0 ± 9.1 37.5 0.01

[00174] Single nucleotide polymorphisms (SNPs) in this cohort were genotyped using the Illumina HumanHap 650Y array. After implementing stringent quality control measures, 524,284 SNPs were available for analysis. To increase the coverage of genetic variants additional missing genotypes were imputed and applied to these imputed SNPs. The same quality control measures were performed on the genotyped SNPs. A total of 1,943,373 genetic markers were analyzed.

[00175] To minimize heterogeneity due to underlying population structure, the patients who self-identified as Caucasian using principle components analysis were analyzed. There was 97% concordance between self-identified and genetically-identified race, and only patients genetically determined to be Caucasian were included in analyses. Additionally, patients who were related, patients who had complete follow-up information, patients who were on weight- altering medications or had severe illness after surgery were excluded, leaving 693 patients (Cohort 1) for analysis. Figure 11 shows the percent change of weight nadir measured in Cohort 1.

[00176] Figure 12 shows the percent change of weight nadir measured in an independent cohort of 327 Caucasian RYGB patients (Cohort 2). One hundred and three marginally significant (P < 5 x 10^"5) SNPs were identified (Figure 13; Table 5), representing 26 independent loci (pairwise r < 0.5) from Cohort 1. The top SNP per region was carried forward for validation in Cohort 2, 327 Caucasian RYGB patients (Table 5). Table 5. Association results for 103 SNPs with p < 5.0x10 for nadir WL in the Cohort 1.

Distance from R2 with index

Index SNP Chr Position Other SNPs P-value index SNP (kb) SNP

rsl 051508 159753509 8.21e-08 rs7158359 88664048 3.36e-07 rs4904510 -1.28 1.0 5.4e-07 rs7129556 11 76977696 4.28e-07 rsl 1605638 -61 0.986 5.79e-06 rs 11823651 -46 0.997 1.6e-06 rs7481282 -36 1.0 8.64e-07 rsl 1237220 -31 0.997 5.41e-07 rsl0899387 -23 0.993 4.87e-07 rs20323951 14 1.0 6.51e-07 rs6592738 23 0.967 3.18e-06 rsl 0899394 24 0.961 4.26e-06 rsl 1237249 41 0.957 1.91e-06 rsl 1604207 67 0.71 2.5e-06 rsl 2286317 89 0.711 6.48e-06 rs7950873 113 0.709 4.29e-06 r_s4344516 138 0.703 3.61e-05 rsl 0899404 163 0.696 2.95e-05 rs537811 168 0.696 2.95e-05 rs684813 195 0.691 3.86e-05 rs650171 198 0.696 2.34e-05 rs7123080 199 0.696 2.34e-05 rs4945220 228 0.695 2.29e-05 rs7951033 234 0.695 2.29e-05 rs7185923 16 49802988 8.08e-07 rs2994537 1 44650917 1.17e-06 rs934760 2 122002672 4.6e-06 rs6712661 -183 0.907 3.19e-05 rsl 2622908 -182 0.906 4.58e-05 rs735483 -181 0.908 3.19e-05 rsl 2615396 -176 0.908 3.19e-05 rsl 2615068 -171 0.907 4.58e-05 rs7601226 -171 0.908 3.19e-05 rsl 7006394 -168 0.907 4.58e-05 rsl3414513 -167 0.908 3.19e-05 rs9653407 -166 0.908 3.19e-05 rs7564564 -152 0.898 4.68e-05 rsl2711551 -123 0.917 3.22e-05 rsl 7006485 -79 0.902 3.1e-05 rs2164796 -67 0.918 3.35e-05 rs2118387 -67 0.918 3.35e-05 rsl 3000440 -49 0.918 3.35e-05 rs724403 -48 0.918 3.5e-05 rsl 3413270 -46 0.918 3.35e-05 rs6711921 -13 0.918 3.35e-05 rs9678017 2 0.918 3.35e-05 rs 10177322 4 0.918 3.47e-05 rs7580531 19 0.909 4.11e-05 rsl2711559 120 0.859 3.7e-05 rs 1104959 5 149722429 5.83e-06 rs9403832 6 147296219 9.77e-06 rs7383179 -6.69 1 9.77e-06 rs9322082 -2.17 0.985 1.37e-05 rsl 1155492 -0.453 0.985 1.37e-05 rs9403834 5 1 1.35e-05 rsl 1155494 5.34 1 1.35e-05 rs 1408577 12.6 0.98 1.93e-05 rs7742886 13.8 0.995 1.38e-05 rs4267966 17.7 0.956 4.74e-05 rs4452660 21.9 0.972 2.23e-05 rs4323329 22.3 0.972 2.23e-05 rs9377038 27.4 0.957 3.05e-05 rs7775644 47.8 0.667 2.11e-05 rs9322085 58.6 0.632 4.17e-05 rs6570781 58.8 0.6 3.13e-05 rs6902235 60.8 0.611 2.31e-05 rs7745042 66.9 0.6 2.18e-05 rs8032450 15 87573927 1.09e-05 rs 17702901 15 90731415 1.19e-05 rsl 7646351 -3.58 1 3.17e-05 rsl 7646434 0.93 1 2.38e-05 rsl 7702960 2.12 1 2.38e-05 rsl 7646492 3.45 1 3.21e-05 rs588217 11 77261024 1.36e-05 rs621456 -30.3 0.805 3.93e-05 rs4085813 -15.8 0.985 2.24e-05 rs648601 3.53 0.985 1.86e-05 rs6554217 4 55672161 1.49e-05 rs4450992 0.861 0.979 3.72e-05 rs9357419 6 12690973 1.79e-05 r_s7744769 -1.12 1 4.29e-05 rs 11260025 19 7687753 2.21e-05 rs8111760 0.267 1 2.35e-05 rs7749399 6 96086077 2.39e-05 rs443673 5 3265963 2.62e-05 rs 12803675 11 49858702 2.74e-05 rsl3380914 18 69312767 2.94e-05 rsl954888 -8.71 1 4.08e-05 rsl 2968184 44.7 0.626 3.31e-05 rsl0518316 4 120241167 2.98e-05 rs6911409 6 73910091 3.52e-05 rs6911751 0.198 1 4.87e-05 rs4703388 5 34834358 3.92e-05 rsl6867581 1.58 1 3.92e-05 rsl 2659689 1.75 1 3.92e-05 rs 1952291 14 45410504 4.3e-05 rs 1289666 1 117350312 4.35e-05 rs 10242229 7 92996322 4.37e-05 rs2157814 1.37 1 4.73e-05 rsl 1788785 9 96111183 4.53e-05 rsl883264 22 41834344 4.59e-05 rsl 2696123 3 163099074 4.71e-05 rs2029600 -2.47 1 4.71e-05

[00177] The top SNP per region underwent validation in Cohort 2. Twenty-three mutations were determined using Sequenom MassARRAY, see Figure 14. The association between SNPs and percent total weight loss (%WL) at the lowest weight (weight nadir) after RYGB were analyzed using linear regression models. A genomic control inflation factor of 1.00 was observed, indicating there was no inflation of test statistics due to population stratification.

Results of the original and validation cohorts were meta- analyzed using fixed effects models.

[00178] Multiple regions on several chromosomes, such as chromosome 11, were found to have numerous SNPs or "clouds" of SNPs with signification association to percent total weight loss (%WL) at the lowest weight (weight nadir) after RYGB (Figure 15). Additionally, a SNP at 15q26.1, rsl7702901 was identified as being significantly associated with WL after RYGB

-8

(^replication = 0.002; Pmeta-anaiyzed = 7.4 x 10^" ). The magnitude of effect was strikingly similar across the two cohorts, with betas of -6.70 and -6.52, respectively (Table 6).

Table 6: SNPs Associated with WC at Nadir.

Cohort 1 Cohort 2 Meta-Analyzed

SNP Chr p-value β p-value β p-value β rs7158359 14 3.361E-07 -3.029 0.6593 -0.4075 0.00000456 2.2731 rs7129556 11 4.276E-07 -2.803 0.441 -0.6013 0.000003866 2.073 rsl0899387 11 4.873E-07 -2.876 0.5991 4.336 rs934760 2 0.000004595 -4.276 0.68 0.5647 rsl 104959 5 0.000005829 7.748 0.6605 0.8592 0.0001832 4.7901 rsl 7702901 15 0.00001188 -6.703 0.002259 -6.524 7.439E-08 6.6422 rs588217 11 0.00001363 -2.547 0.4659 -0.5852 0.00006972 1.8713 rs9357419 6 0.0000179 -2.95 0.5694 0.5581 0.001292 1.8029

[00179] Because the physiological mechanisms of weight gain to generate obesity and weight loss after RYGB may be related, previously reported and validated BMI loci associated with weight loss after RYGB in humans were assessed. None of the 32 previously reported BMI loci was associated with weight loss after surgery, nor did loci previously reported to be associated with diabetes (Table 8).

Attorney uocKei ΓΝΟ.: IUU»/J-03J ( i u vwtw

Table 7. Association results for SNPs previously identified as associated with obesity.

Attorney uocKei ΓΝΟ.: IUU»/J-03J ( i u vwtw

[00180] After pooling data from the two cohorts (n=953), patients lacking the minor allele (MA) of rsl7702901 lost an average of 38.7% of their body weight, while patients carrying a single copy of this mutation (n=52, 5.0% of the population) lost an average of 33.5%. The sole patient with two copies of the MA had a percent weight loss of 28.8% (Figure 16).

[00181] To examine the potential predictive utility of this SNP for discriminating amongst patients, %WL was categorized as less than or greater than or equal to 30% at weight nadir (n=171, 17.9%), Figure 17. Patients with at least one copy of the MA were 2.54 times more likely to fall below 30% WL, than patients with no copies of the MA (P < 0.001)(left shaded area in Figure 18). Notably, no patients with this polymorphism lost more than 50% of his or her weight (corresponding to the upper 10% of the weight loss distribution) (right shaded area in Figure 18).

Example 7: Genetic Models

[00182] The predictive ability of this SNP was further tested by adding this marker to a clinical model for predicting weight loss after RYGB, Table 8. The model was constructed by selecting one or more SNPs that met specific p-value criteria and then using backward and stepwise regression. The coefficients for each variable were determined using the training population (Cohort 1) and then the genetic information was entered into a regression model to determine the improvement genetics has on weight loss predictions. In a multivariable model that included age, sex, preoperative BMI (pBMI), and diabetes as variables, the area under the receiver operating characteristic curve (AUROC) was calculated as 0.620. After inclusion of rsl7709201 in the model, the AUROC improved to 0.633 (Figure 19), showing that inclusion of rsl7709201 has a higher probability of being a predictor of weight loss than rsl7709201 as a random positive influence on weight loss.

Table 8: Model Performance.

[00183] To further demonstrate the predictive ability of incorporating genetic information with clinical data, multivariable models were tested by adding multiple SNPs to a clinical model for predicting weight loss after RYGB, Table 9. Variables for the clinical model included age, sex, diabetes, and preoperative BMI (pBMI). Variables for the genetic model included all of the variables in the clinical model with the addition of the following genetic variables: rsl 108723, rsl 1739371, rsl 1942914, rsl2425125, rsl7710780, rs2383289, rs3734399, rs4325727, rs4603757, rs6737079, rs6911751, rs6925786, rs9474779. Similar to the model with one SNP, the multi-SNP model was constructed by selecting multiple SNPs that met specific p-value criteria and then using backward and stepwise regression. For each model, coefficients for each variable were determined using the training population (Cohort 1). Validation that these coefficients are applicable to other data sets was assessed using Cohort 2 as a test set of data. Data from Cohort 2 was not used in establishing the coefficients and therefore serves as a valid test case. For the clinical multivariable model that included age, sex, diabetes, and preoperative BMI (pBMI) as variables, the AUROC score was calculated to be 0.665 in Cohort 1. The AUROC score in the test population, Cohort 2, showed significant similarity (0.699) and affirmed the performance of the model. For the genetic multivariable model that included clinical and genetic factors, the AUROC was significantly better (0.971) after determining the optimal coefficients to this model in the training set (Cohort 1). Using the genetic multivariable model with the coefficients derived from Cohort 1, the AUROC in the test set (Cohort 2) was also improved (0.795) over the respective AUROC scores based on clinical data alone, verifying that inclusion of multiple SNPs (rsl 108723, rsl 1739371, rsl 1942914, rsl2425125, rsl7710780, rs2383289, rs3734399, rs4325727, rs4603757, rs6737079, rs6911751, rs6925786, rs9474779) significantly improves weight loss predictions.

Table 9: Model Performance.

[00184] To determine the potential biological function of this SNP, the presence of the MA associated with expression of any of -44,000 gene expression transcripts in liver, omental fat tissue, and subcutaneous fat tissue was examined. No obvious association between rs 17702901 and expression of other transcripts was found upon initial inspection, including expression of two genes located closest to rsl7702901 - ST8SIA2 (-6.7 kilobases (kb) downstream of rsl7702901) and SLC03A1 (~223kb upstream of rsl7702901).

[00185] Because expression of transcripts may be influenced by the patient's physiological state, expression of these genes in a controlled environment was analyzed. Expression of ST8SIA2 and SLC03A1 were previously analyzed in a mouse model of RYGB (Hatoum, I.J. et al., / Clin Endocrinol Metab, vol 97, pp E1023-E1031, 2012). Age- and sex-matched animals were randomized to RYGB, sham operation with ad libitum food intake (SO-AL), or sham operation with food restriction to match the weight of the RYGB animals (SO-WM). After ten weeks, animals were sacrificed and expression was determined in upper bypassed limb (BL), the upper Roux limb (RL), the upper common limb (CL), the colon, the liver, the muscle, the epididymal fat and the subcutaneous fat. [00186] Expression of ST8SIA2 was not significantly altered in any tissue after RYGB relative to SO-AL and SO-WM. In contrast, expression of SLC03A1 was significantly altered in the RL, CL, and subcutaneous fat after RYGB, relative to ad libitum-fed shams. It appeared that effects in RL and CL were weight loss-independent, as these remained significant in the RYGB compared to SO-WM animals. The effects in the BL and colon were also significant in Roux animals compared to weight matched animals. The biological relationship between rsl7702901 and regulation of ST8SIA2 or SLC03A1 does not appear to be in strong linkage disequilibrium.

[00187] Because the mechanisms of weight gain and weight loss may be shared, previously reported and validated BMI loci (Speliotes, E.K. et al., Nat Genet, vol 42, pp 937-48, 2010) were analyzed for association with weight loss after surgery. Unlike the association of rsl7702901 with weight loss after RYGB (Figure 20), deep sequencing of the MC4R locus did not indicate any association between variants in this gene and weight loss after surgery (Figure 21).

Example 8: Different Bariatric Surgeries Act Through Similar Mechanisms

[00188] To determine if different metabolic procedures produce similar results, possibly using similar mechanisms of action, patients that have undergone gastric bypass, biliopancreatic diversion, sleeve gastrectomy and duodenal endoluminal sleeve were analyzed for effects of weight loss, food intake, insulin sensitivity, glucose tolerance, insulin secretion, and endogenous glucose production. Despite the disparity in the type of procedure performed, gastric bypass, biliopancreatic diversion, sleeve gastrectomy and duodenal endoluminal sleeve surprisingly had similar effects, increased weight loss, decreased food intake, increased energy expenditure, greater insulin sensitivity, increased glucose tolerance and enhanced insulin secretion. (Data not shown.)

[00189] Moreover, the different metabolic procedures also had similar effects on the gastrointestinal endocrine system. Levels of ghrelin, glucagon-like peptide- 1, peptide YY and gastric inhibitory polypeptide demonstrated similar changes in post-prandial secretion levels in individuals that have undergone gastric bypass, biliopancreatic diversion, sleeve gastrectomy, ileal interposition or duodenal endoluminal sleeve. Example 9: Genetic Factors Contributing to Weight Loss

[00190] The effect of RYGB on expression of genes in a previously described (McAuley, E. Z. et ah, Identification Of Sialyltransferase 8B As A Generalized Susceptibility Gene For Psychotic And Mood Disorders On Chromosome 15q25-26, PLoS ONE 7, e38172 (2012)) mouse model was determined. Mice were randomized to RYGB or sham operation with food restriction to match the weight of the RYGB animals (WMS). Animals were sacrificed after 10 weeks, and gene expression was measured in the alimentary limb, liver, and epididymal fat (Figure 22, Table 10).

Table 10: Differentially expressed genes.

cdhl l cadherin 11, osteoblast NM_009866

slcl2a4 solute carrier family 12 NM_009195

(potassium/chloride transporters),

member 4

eraf NM_133245

hoxal homeobox Al NM_010449

satll spermidine/spermine Nl -acetyl ENSMUST00000026601 transferase-like 1

adiporl adiponectin receptor 1 AK182949

adiporl adiponectin receptor 1 NM_028320

adiporl adiponectin receptor 1 AK143680

abcf2 ATP-binding cassette, sub-family F NM_013853

(GCN20), member 2

abcf2 ATP-binding cassette, sub-family F CA751126

(GCN20), member 2

mier3 mesoderm induction early response 1, NM_172593

family member 3

ddr2 discoidin domain receptor tyrosine BB613073

kinase 2

ddr2 discoidin domain receptor tyrosine AK028767

kinase 2

ddr2 discoidin domain receptor tyrosine NM_022563

kinase 2

nphsl nephrosis 1, congenital, Finnish type AK141081

(nephrin)

zranb3 zinc finger, RAN-binding domain ENSMUST00000097598 containing 3

zranb3 zinc finger, RAN-binding domain NM_172642

containing 3

cldnl6 NM_053241

dhrs3 dehydrogenase/reductase (SDR family) AF061743

member 3

dhrs3 dehydrogenase/reductase (SDR family) NM_011303

member 3

adnp2 ADNP homeobox 2 ENSMUST00000066743 scrtl NM_130893

trappc5 trafficking protein particle complex 5 NM_025701

trappc5 trafficking protein particle complex 5 AK003633

polk polymerase (DNA directed) kappa ENSMUST00000091386 polk polymerase (DNA directed) kappa NM_012048

clnsla chloride channel, nucleotide- sensitive, NM_023671

1A

col4a3bp collagen, type IV, alpha 3 AK020301

(Goodpasture antigen) binding protein

104 akap8 A kinase (PRKA) anchor protein 8 ENSMUST00000002699

105 med21 mediator complex subunit 21 NM_025315

106 aplm2 adaptor-related protein complex 1, mu NM_009678

2 subunit

107 ulk4 unc-51-like kinase 4 NM_177589

108 ulk4 unc-51-like kinase 4 ENSMUST00000098284

109 ulk4 unc-51-like kinase 4 BU946109

110 ift57 intraflagellar transport protein 57 NM_028680

111 ift57 intraflagellar transport protein 57 AK014731

112 lca51 NM_001001492

113 armc9 armadillo repeat containing 9 NM_030184

114 armc9 armadillo repeat containing 9 NM_027456

115 armc9 armadillo repeat containing 9 AK019600

116 dio3 deiodinase, iodothyronine, type III NM_172119

117 wfdc8 NM_001080550

118 cfb complement factor B NM_008198

119 eif4e eukaryotic translation initiation factor NM_007917

4e

120 eif4e eukaryotic translation initiation factor M61731

4e

121 eif4e eukaryotic translation initiation factor AK146757

4e

122 igj immunoglobulin J polypeptide, linker NM_152839

protein for immunoglobulin alpha and

mu polypeptides

123 ctnnd2 catenin (cadherin-associated protein), ENSMUST00000081728 delta 2 (neural plakophilin-related arm- repeat protein)

124 ampd2 adenosine monophosphate deaminase 2 NM_028779

(isoform L)

125 rtkn rhotekin NM_009106

126 rslldl ribosomal LI domain containing 1 NM_025546

127 elk4 ELK4, ETS-domain protein AK156537

128 elk4 ELK4, ETS-domain protein ENSMUST00000086556

[00191] To identify genetic factors contributing to weight loss after RYGB, an exploratory genome wide association study (GWAS) of 858 genetically unrelated individuals was performed (Table 2). After stringent quality control measures, 1,943,373 single nucleotide polymorphisms (SNPs) were analyzed. To minimize heterogeneity from the underlying population structure, we limited the analyses to 693 patients genetically determined to be Caucasian. The association between SNPs and percent total weight loss ( WL) at the lowest weight (nadir) after RYGB using additive models of quantitative trait associations was assessed. Table 11: Pre- and post-operative characteristics of the RYGB study population and replication cohort.

[00192] 103 marginally significant (P<5xl0^~5) SNPs were identified (Table 12), representing

26 independent loci (pairwise r <0.5). The most highly associated SNP per region was carried forward for validation in an independent cohort of 327 Caucasian RYGB patients. 23 of the SNPs were successfully genotyped, and associations with percent weight loss were analyzed using linear regression models. Results of the original and validation cohorts were then meta- analyzed using fixed effects models.

Table 12: Top 50 expression transcripts associated with rsl7702901.

SLC12A4 0.000119327 liver

Contig34964 0.000125475 liver

BC009871 0.000139822 liver hCT1646793 0.000169095 liver

HSS00381099 0.000178477 liver

FAM10A4 0.000193237 subcutaneous fat

Contigl2033 0.000200709 omental fat

CNTNAP3B 0.000212491 omental fat

AK094533 0.00022344 liver

ERAF 0.000249779 omental fat

HOXA1 0.000251027 liver

SATL1 0.000251598 subcutaneous fat

U84510 0.000259264 liver

ADIPOR1 0.000260775 liver

ABCF2 0.000289326 liver

HCT1958096 0.000291375 liver

MIER3 0.000305798 liver

DDR2 0.000308075 liver

NPHS1 0.000315369 liver

HSS00053942 0.000327271 liver

ZRANB3 0.000339101 subcutaneous fat

CLDN16 0.000362854 liver

DHRS3 0.000398518 liver

OR2A2 0.000430517 liver

BC022568 0.000463747 liver Contig34719 0.000468504 liver

HCT1845647 0.000470327 liver

ADNP2 0.000470332 liver

HCT2297022 0.000487732 liver

HSS00299143 0.000491702 liver

AF400502 0.000519205 omental fat

SCRT1 0.000521866 liver

Contig43708 0.000537265 liver

HSS00214508 0.000540097 subcutaneous fat

C2orf29 0.000543099 liver

HCT1956088 0.000566656 liver

Contig57822 0.000581398 subcutaneous fat

HCT1641204 0.000589909 liver

CR749513 0.000590453 liver

[00193] SNP rsl7702901 at 15q26.1 was significantly associated with percent weight loss after Roux-en-Y gastric bypass (RYGB)

To determine the potential biological function of rsl7702901, its association with the expression level of -44,000 transcripts in liver, omental fat and subcutaneous fat was examined. No multiple test- corrected, significant associations were detected between rs 17702901 and preoperative expression of any transcripts, including the two nearest genes - ST8SIA2, located -6.7 kilobases (kb) downstream of rsl7702901 and SLC03A1, ~223kb upstream of this SNP (Table 12). The magnitude of effect was similar in the study population and replication cohorts, with betas of - 6.70 and -6.52, respectively (Table 13). Attorney uocKet ΓΝΟ.: IUU» /J-03J ( i u vwtw

Table 13: SNP association results from the genome- wide association analysis.

GWAS Cohort Replication Cohort Combined

Distance from Gene

SNP Closest Gene MA² Chr Position Beta P-value Beta P-value Beta P-val to SNP (bp)¹

rsl0515808 C1QTNF2 23283 A 5 159753509 -4.06 8.2 x 10^"8 -1.17 0.27 -3.08 4.2 x rs7158359 CHES1 28219 G 14 88664048 -3.03 3.4 x 10^"7 -0.41 0.65 -2.27 4.6 x rs7129556 AQP11 631 T 11 76977696 -2.80 4.3 x 10^"7 -0.60 0.44 -2.07 3.9 x rs7185923 SALL1 60335 c 16 49802988 -2.40 8.1 x 10^"7 -0.63 0.40 -1.89 3.5 x rs934760 CLASP1 0 G 2 12202672 -4.28 4.6 x 10^"6 0.56 0.68

rs 1104959 RPS14 79991 T 5 149722429 7.75 5.8 x 10^"6 0.86 0.66 4.79 1.8 x rs9403832 STXBP5 0 c 6 14795969 2.17 9.8 x 10^"6 -0.51 0.47 1.30 O.OC rs 17702901 ST8SIA2 6728 A 15 90731415 -6.70 1.1 x 10^"5 -6.52 0.002 -6.64 7.4 x rs588217 INTS4 6391 A 11 77261024 -2.55 1.4 x 10^"5 -0.59 0.47 -1.87 7.0 x rs6554217 RNU5B 63795 T 4 55672161 2.89 1.5 x 10^"5 -1.43 0.15

rs9357419 PHACTR1 134845 C 6 12690973 -2.95 1.8 x 10^"5 0.56 0.56 -1.80 O.OC rs 11260025 CLEC4G 12090 c 19 7687753 3.33 2.2 x 10^"5 -1.06 0.32 1.82 O.OC rs 12803675 OR4C13 71848 A 11 49858702 3.69 2.7 x 10^"5 0.37 0.79 2.80 1.8 x rsl3380914 FBX015 578813 A 18 69312767 -2.50 2.9 x 10^"5 -0.57 0.51 1.89 1.2 x

Attorney uocKei ΓΝΟ.: IUU»/J-03J ( i u vwtw

rs!0518316 SYNP02 120241167 -2.52 3.0 x 10- 1.37 0.15

rs6911409 KCNQ5 A 73910091 -3.71 3.5 x 10- -1.14 0.32 -2.74 rs 12659689 KAU4 C 34836108 -1.98 3.9 x 10- -0.11 0.87 -1.39 rs 1952291 C14orfl06 618149 A 14 45410504 -4.32 4.3 X 10- 1.27 0.42 -2.65 rs 1289666 TTF2 C 117350312 2.73 4.3 X 10- -0.24 0.80

rsl 1788785 HSD17B3 A 96111183 -2.79 4.5 X 10- -0.68 0.50

rsl 883264 BIK C 22 41834344 2.85 4.6 X 10- 0.76 0.46

rsl 2696123 MIRN135A2 66339 C 163099074 -2.36 4.7 x 10- 0.24 0.77

Absolute value of the distance from the start or stop site of the closest gene. A distance of 0 indicates that the SNP is located within the gene.

MA=Minor allele

[00194] Expression of sta8sia2, the mouse orthologue of the gene closest to rsl7702901, was significantly lower in the epididymal fat and liver of RYGB-treated than WMS mice (Figure 23A). In addition, intestinal alimentary limb expression of slco3al was significantly greater in the RYGB group than WMS controls (Figure 23B).

[00195] Results in humans using a gene-based association test that integrates SNP

associations with linkage disequilibrium patterns within each gene was analyzed and identified a marginally significant association (p=8.0xl0^~ ) for the aquaporin 11 gene (AQPll ; Table 14). While there were 27 SNPs in this region with a P-value<0.001, there was no statistically significant association between the top SNP in this region, rs7129556, and percent weight loss in the replication cohort (Figure 24A). No genome- wide, multiple test-corrected, significant associations between rs7129556 and the expression of any transcripts in humans was detected, but this SNP was marginally associated with expression of AQPll itself (P_omentai=9 xlO^'5 , Pu_ver =1.6xl0^"4; Table 15).

[00196] In the mouse models, aqpll expression in the alimentary limb and liver was significantly lower after RYGB than in WMS mice (Figure 24B). In contrast, expression of clnsla, the gene closest to aqpll, was not significantly changed after RYGB (Figure 24C).

Table 14: Top 50 association results for gene-based association tests.

POLK 5 82 0.0007 rsl 0942739 0.0001566

CLEC4M 19 46 0.00071 rsl 1260025 2.21E-05

CLNS1A 11 39 0.000747 rs7129556 4.276E-07

COL4A3BP 5 82 0.000873 rs6453134 0.0001267

GCNT4 5 61 0.001 rs4632781 0.0002618

INTS4 11 115 0.00102 rs588217 1.363E-05

CKAP2 13 26 0.001241 rsl l618716 0.0007868

SPAG6 10 4 0.001533 rs9665348 0.03949

C3orf39 3 86 0.00167 rs536119 6.329E-05

RPS27A 2 56 0.001706 rsl561231 0.0009679

Clorf87 1 165 0.001751 rs686119 0.0004618

VPS36 13 46 0.00176 _rs4884452 0.0003805

CALML4 15 41 0.00197 rs8025947 0.0005017

THSD1 13 43 0.002 _rs4884452 0.0003805

TBC1D3B 17 2 0.00204 rs4500794 0.0004894

HMGCR 5 66 0.00214 rs7700468 0.000152

CLN6 15 43 0.00219 rs8025947 0.0005017

KLK8 19 81 0.00223 rsl 701948 0.0005329

RSF1 11 104 0.002362 rsl 1237249 1.906E-06

KRT7 12 121 0.002513 rs7953684 0.0001044

ZNF223 19 80 0.00261 rs8105003 0.0003053

LMAN2L 2 25 0.00276 rs2872633 0.001663

LVRN 5 243 0.00276 rsl593872 0.0003657

CNNM4 2 34 0.0028 rsl7119562 0.003587

SLC06A1 5 157 0.002973 rsl0041015 0.0006075

GJA4 1 28 0.00298 rs4653105 0.001588

OPALIN 10 135 0.00298 rsl 1188734 0.0001968 KLK11 19 104 0.00301 rs 1701948 0.0005329

HOXA4 7 60 0.00304 rs983184 0.0005409

RETN 19 33 0.00305 rs 11260025 2.21E-05

KLK9 19 84 0.00307 rs 1701948 0.0005329

ZNF284 19 74 0.003167 rs8105003 0.0003053

KRT81 12 150 0.00317 rs7953684 0.0001044

SIRPG 20 123 0.00318 rs4254565 0.0006302

KLK10 19 95 0.00323 rs 1701948 0.0005329

Clor 212 1 46 0.00329 rs4653105 0.001588

KLK12 19 110 0.00334 rs 1701948 0.0005329

PHF3 6 53 0.00334 rs4710437 0.0001252

GRPEL1 4 74 0.00338 rs 12648134 0.0005674

HOXA5 7 58 0.00349 rs983184 0.0005409

Table 15: Top 50 expression transcripts associated with rsl7702901.

AP1M2 0.000198556 subcutaneous fat

ULK4 0.000234395 liver

Contig36748_RC 0.000248422 omental fat

ZNF711 0.000258753 omental fat

IFT57 0.000266345 liver

IGLV2J4 0.000267441 subcutaneous fat

LCA5L 0.000271869 liver

ADAM3A 0.000276415 liver

AL833696 0.000303924 liver

ARMC9 0.000311108 omental fat

BC040305 0.000315233 liver

DI03 0.000324871 liver

WFDC8 0.000338595 omental fat

CFB 0.000339373 subcutaneous fat

EIF4E 0.000381093 liver

IGJ 0.000381533 liver

ZNF509 0.000394645 liver

PI4KAP2 0.000415647 liver

CTNND2 0.00042246 subcutaneous fat

C3orf38 0.000423274 liver

AMPD2 0.000424693 liver

XM_173060 0.000429279 liver

XM_065192 0.00045425 omental fat

RTKN 0.000455737 liver Contig41220_RC 0.000481428 omen

RSL1D1 0.00048159 subq

AK097908 0.000496614 omental fat

XM_210217 0.000505951 liver

HSS00095153 0.000506833 subq

Contig45100_RC 0.000518199 liver

AK074192 0.000560538 omental fat

CFB 0.000563012 subq hCT1955907_l 0.000617333 liver

XM 73120 0.000620047 liver

AK022383 0.000638859 omental fat

AL833662 0.000639129 omental fat

Contigl5012_RC 0.000641473 omental fat

Contig31661_RC 0.000646011 subcutaneous fat

PEA15 0.000647577 subcutaneous fat

ELK4 0.000650268 liver

[00197] Because the physiological mechanisms of weight gain to generate obesity and weight loss after RYGB may be related, previously reported and validated BMI-associated loci were assessed for association with weight loss after RYGB in humans. None of the 32 previously- reported BMI-associated or 28 diabetes-associated loci was associated with weight loss after surgery (Tables 16 and 17). Deep sequencing of the MC4R locus showed no evidence of an association between variants in this gene and weight loss after RYGB. Attorney uocKei ΓΝΟ.: IUU»/J-03J ( i u vwtw

Table 16: Association results for SNPs previously identified as associated with obesity.

GWAS Cohort Validation Cohort Combined

SNP Closest Genes Chr Position Beta P-value Beta P-value Beta P-value rs3810291 TMEM160, ZC3H4 19 52260843 0.8816 0.09116 1.627 0.03188 1.1222 0.008874 rs9816226 ETV5 3 187317193 1.18 0.08907 0.04788 0.9598 rs713586 RBJ, ADCY3, POMC 2 25011512 0.2023 0.678 1.117 0.1079 rs206936 NUDT3, HMGA1 6 34410847 0.8864 0.1432 -1.133 0.1898 0.2204 0.6562 _rs 12444979 GPRC5B, IQCK 16 19841101 0.4432 0.5179 0.1076 0.9165 0.3396 0.551 rs3817334 MTCH2, NDUFS3, 11 47607569 0.7219 0.1428 0.2568 0.7194 0.5722 0.1579

CUGBP1

rs2890652 LRP1B 2 142676401 1.07 0.2291 rs4929949 RPL27A, TUB 11 8561169 -1.146 0.1109 rs2112347 FLJ35779, HMGCR 5 75050998 -0.252 0.6206 -1.337 0.08203 0.5839 0.1684 rsl558902 FTO 16 52361075 -0.1002 0.8373 0.7299 0.322

Attorney uocKei ΓΝΟ.: IUU»/J-03J ( i u vwtw

Attorney uocKei ΓΝΟ.: IUU»/J-03J ( i u vwtw

Table 17: Association results for SNPs previously identified as associated with diabetes.

rsl63184 KCNQ1 2803645 -0.7157 0.1421

^{1 1}

rs2237892 KCNQ1 2796327 2.437 0.01667

^{1 1}

rs231362 KCNQ1 2648047 0.169 0.7283

^{1 1}

rs5215 KCNJ11 17365206 -0.2529 0.6285 rs 1552224 CENTD2 region 72110746

^{1 1}

rsl0830963 MTNR1B 92348358 0.9426 0.2734

^{1 1}

rs2943634 IRS1 region 226776324 -0.291 0.5857

^{1 1}

rs7961581 TSPAN8/LGR5 12 69949369 -0.1602 0.7799 rs7957197 HNF1A 12 119945069 -0.2222 0.7284 rsl 1642841 FTO 16 52402988 -0.5849 0.2549 rs4430796 HNF1B 17 33172153 0.1217 0.8362

APPENDIX A

The sequences as provided in Appendix A of U.S. Patent Application Serial No. 13/828,809, filed on March 14, 2013, which is hereby incorporated by reference in its entirety. These sequences directly correspond to (i.e., are identical to) SEQ ID NOs 129-837 provided in the sequence listing of the present application.

2252681.1

Appendi xB . txt

SNP p

rs7481282 8. . 636E-7

rs9322082 0. .00001371

rs648601 0. .00001859

rsl408577 0. .00001929

rs4323329 0. .00002226

rs4452660 0. .00002226

rs4085813 0. .00002238

rs4945220 0. .00002292

rs650171 0. .00002343

rs7123080 0. .00002343

rs8111760 0. .00002345

rsl7646434 0. .00002376

rsl7702960 0. .00002376

rsl0899404 0. .00002945

rsl7006485 0. .00003101

rsl7646351 0. .00003173

rsl3414513 0. .00003186

rs6712661 0. .00003186

rs735483 0. .00003186

rs7601226 0. .00003186

rs9653407 0. .00003186

rsl7646492 0. .00003213

rsl2711551 0. .00003219

rsl2968184 0. .00003313

rsl3000440 0. .00003348

rsl3413270 0. .00003348

rs2118387 0. .00003348

rs2164796 0. .00003348

rs6711921 0. .00003348

rs9678017 0. .00003348

rsl0177322 0. .00003468

rsl7006496 0. .00003502

rs724403 0. .00003502

rs4344516 0. .0000361

rsl2711559 0. .000037

rs4450992 0. .0000372

rs684813 0. .00003859

rsl6867581 0. .00003915

rs4703388 0. .00003915

rs621456 0. .00003926

rsl954888 0. .00004077

rs7580531 0. .00004105

rs7744769 0. .00004291

rsl0242229 0. .00004375

rsl2615068 0. .00004576

rsl2622908 0. .00004576

rsl7006394 0. .00004576

rs7564564 0. .00004681

rs2157814 0. .00004726

rs4267966 0. .00004741

rsl2614155 0. .00005011

rsll707244 0. .00005025

rsl7050208 0. .00005125

rs6913861 0. .00005196

rsl0936257 0. .00005258

rsl529014 0. .00005471

rs9386176 0. .00005646

rs9399592 0. .00005646

rs9403837 0. .00005646

rs2328774 0. .00005782

rs6554218 0. .00005783

rs4551178 0. .00005824

rsl456243 0. .00005826

rs7116127 0. .00005826

rs4134463 0. .0000585

rsl945747 0. .00005922

rsl0137389 0. .00006177

rsl0496569 0. .00006274

rs477594 0. .00006589

Page 1 Appendi xB . txt rs504973 0. .00006589

rs834194 0. .00006589

rsl0179345 0. .00006609

rs6756225 0. .00006609

rsl3302476 0. .00006648

rs9360640 0. .00006802

rs484363 0. .00006837

rsl365832 0. .00006923

rs4445078 0. .00006973

rsl2614379 0. .00007143

rsl2087475 0. .00007264

rsl3403769 0. .00007504

rs643388 0. .0000771

rs663674 0. .00007742

rs415234 0. .00007831

rs687037 0. .00007853

rsl2073831 0. .00008024

rs6023633 0. .00008146

rs6736269 0. .00008288

rs7772526 0. .00008298

rs6441408 0. .00008446

rsll018434 0. .00008495

rs2594536 0. .0000852

rsl0185512 0. .00008828

rs2034137 0. .00008828

rsl7087674 0. .00008992

rsl456244 0. .00009005

rslll22861 0. .00009285

rs6746090 0. .00009285

rs9840287 0. .00009301

rs2048504 0. .00009427

rs6014185 0. .00009427

rsl420582 0. .0000946

rs668701 0. .00009522

rsl0170935 0. .00009539

rs6714671 0. .00009539

rs7595601 0. .00009539

rs3912845 0. .00009655

rsl7764592 0. .0000985

rsl0496570 0. .00009884

rsl2611458 0. .00009884

rsl3395695 0. .00009884

rs2304560 0. .00009884

rs2304561 0. .00009884

rs6704587 0. .00009884

rs7566558 0. .00009884

rs934761 0. .00009884

rs6541782 0. .0001021

rs6760211 0. .0001021

rsl633749 0. .000103

rs7538154 0. .0001038

rsl0255791 0. .0001039

rs9446844 0. .0001046

rsl2614123 0. .0001058

rsl0184008 0. .000106

rs6064117 0. .0001079

rs7760644 0. .000109

rs877191 0. .00011

rs611232 0. .0001113

rs6912526 0. .0001153

rs911196 0. .0001162

rs2181942 0. .0001182

rsl512065 0. .0001192

rsl0179294 0. .0001196

rsl3395277 0. .0001196

rs6023645 0. .0001201

rs594406 0. .0001204

rs595459 0. .0001204

rs4811538 0. .0001212

rsl505006 0. .0001214

Page 2 Appendi xB . txt rs8180176 0. .000123

rs4848712 0. .0001243

rsl551151 0. .0001253

rs4711162 0. .0001253

rs2034136 0. .0001279

rs7558771 0. .0001279

rs7188211 0. .0001294

rsl628613 0. .0001301

rsl666790 0. .0001301

rsl666797 0. .0001301

rsl700572 0. .0001301

rsl2493604 0. .0001308

rsl388702 0. .0001327

rsl492146 0. .0001327

rsl492147 0. .0001327

rsl2190601 0. .0001328

rs6704842 0. .0001361

rsl2622810 0. .0001364

rs7599081 0. .0001364

rs9653406 0. .0001364

rsl2614362 0. .0001376

rs6729454 0. .0001376

rs669738 0. .0001384

rs2289192 0. .0001396

rs7672513 0. .00014

rsl0498676 0. .0001406

rs3778166 0. .0001406

rsl388705 0. .000144

rs675079 0. .0001444

rs2962621 0. .0001458

rs638807 0. .000146

rs7943055 0. .000147

rs7765767 0. .0001483

rs685553 0. .0001488

rs608382 0. .0001491

rs7700468 0. .000152

rsl057530 0. .0001524

rs488824 0. .0001535

rs553727 0. .0001535

rs611536 0. .0001535

rs947841 0. .0001535

rs9322081 0. .0001545

rs3916911 0. .000156

rsl0942739 0. .0001566

rs7709282 0. .0001566

rs4121744 0. .0001568

rs524874 0. .0001585

rsl0765186 0. .0001588

rs4440336 0. .0001599

rs6872314 0. .0001599

rs7356637 0. .0001599

rsl881665 0. .0001601

rs9386177 0. .0001601

rslll55493 0. .0001617

rslll89415 0. .0001621

rsl881663 0. .0001627

rsl7389893 0. .000165

rsl2419587 0. .0001697

rsl6922585 0. .0001697

rs6541784 0. .0001716

rs2092859 0. .0001741

rs2591107 0. .0001755

rs9468304 0. .0001759

rslll9723 0. .0001781

rsl2662634 0. .0001793

rs4811532 0. .00018

rsl3401792 0. .000182

rsl388706 0. .0001823

rs6023649 0. .0001843

rsl0929465 0. .0001848

Page 3 Appendi xB . txt rs4669141 0. .0001848

rs4804226 0. .0001852

rs8009216 0. .000186

rs7001847 0. .0001888

rsl2440999 0. .0001898

rs2200674 0. .0001898

rsl7766596 0. .0001927

rs2280565 0. .0001928

rsl335955 0. .0001932

rsl889102 0. .0001942

rs2784465 0. .0001942

rs4358853 0. .0001942

rs4685652 0. .000195

rs2678801 0. .0001951

rsl0816127 0. .0001972

rs4271025 0. .0001972

rs4279660 0. .0001972

rs4403465 0. .0001972

rs4892107 0. .0001981

rsl0177273 0. .0002003

rslll5356 0. .0002006

rs2167107 0. .0002006

rs7926062 0. .0002024

rsl023329 0. .000203

rs9620115 0. .000203

rsl0457864 0. .0002047

rs7250467 0. .0002048

rs4804788 0. .0002064

rs4685650 0. .0002065

rs4685651 0. .0002065

rs2328777 0. .0002073

rs9322084 0. .0002073

rs9497675 0. .0002073

rs6747693 0. .0002075

rsl0150607 0. .000208

rsl420577 0. .0002091

rs2827489 0. .0002098

rs2786262 0. .0002099

rsl0969252 0. .0002105

rs687781 0. .0002105

rs4988386 0. .0002129

rs4988388 0. .0002129

rs4988389 0. .0002129

rsl912890 0. .0002133

rsl553047 0. .0002145

rs2412616 0. .0002145

rsl2454494 0. .0002153

rs608240 0. .0002155

rs9497679 0. .0002176

rs2786258 0. .000218

rs595106 0. .000219

rs596159 0. .000219

rs618472 0. .000219

rs652539 0. .000219

rs6679824 0. .0002201

rs6688321 0. .0002201

rsl0969267 0. .0002222

rsl7105190 0. .0002249

rsl7105228 0. .000225

rs2186053 0. .000225

rsl0755432 0. .0002282

rsl591906 0. .0002284

rs6921180 0. .0002289

rs2027733 0. .0002297

rsl532212 0. .0002308

rs6532675 0. .0002309

rsll014929 0. .0002316

rsll014931 0. .0002316

rsll014933 0. .0002316

rsll014935 0. .0002316

Page 4 Appendi xB . txt rsll014936 0. .0002316

rsl2784683 0. .0002316

rs7862503 0. .0002316

rs7894301 0. .0002316

rs4445901 0. .0002341

rs9929708 0. .0002341

rsl559604 0. .0002351

rs9377040 0. .0002354

rsll014932 0. .0002361

rs2368120 0. .0002361

rs4703674 0. .0002367

rs767676 0. .0002367

rs2786263 0. .0002368

rsl3018316 0. .000237

rsl2232770 0. .0002384

rsl6982264 0. .0002393

rsl6870906 0. .0002398

rs4045166 0. .0002419

rsl402436 0. .0002421

rs748040 0. .0002422

rs6717225 0. .0002436

rs4711170 0. .0002465

rs2658785 0. .0002489

rsl2205302 0. .00025

rsl3084500 0. .00025

rs2919051 0. .0002524

rs296325 0. .0002541

rs296329 0. .0002541

rs296333 0. .0002541

rs296334 0. .0002541

rs296318 0. .0002561

rs296319 0. .0002561

rs296320 0. .0002561

rsl0147303 0. .0002574

rsl2718626 0. .0002594

rsl2718627 0. .0002594

rsl2718628 0. .0002594

rs3118041 0. .0002608

rs2295599 0. .0002629

rs7740233 0. .000265

rsl412727 0. .0002658

rs3949639 0. .0002658

rs7043400 0. .0002658

rs2153356 0. .0002667

rs870913 0. .0002667

rs870914 0. .0002667

rs870915 0. .0002667

rs2857682 0. .0002672

rs6023640 0. .0002673

rsl0281294 0. .0002674

rs234589 0. .0002689

rs6023625 0. .0002699

rsl0209628 0. .0002718

rsl892159 0. .0002728

rsl892160 0. .0002732

rs7917879 0. .0002749

rs9535895 0. .0002751

rsl2599944 0. .0002759

rs995365 0. .0002773

rsl595288 0. .0002785

rs234586 0. .000279

rsl7671266 0. .0002792

rs4685654 0. .000281

rsl0853651 0. .0002816

rs4685649 0. .000282

rsl0491676 0. .0002832

rsl0491679 0. .0002832

rsl7698884 0. .0002842

rs2492279 0. .0002846

rsl2278691 0. .0002857

Page 5 AppendixB.txt rsl0069744 0. .0002892

rsl0484891 0. .0002892

rs7871542 0. .0002892

rs4988398 0. .0002896

rsl455048 0. .000294

rsl943559 0. .0002946

rs2839314 0. .0002946

rsl0457082 0. .0002953

rs6541794 0. .0002962

rs6023624 0. .0002981

rsl0448170 0. .0002986

rsl0969241 0. .0002986

rsl0969245 0. .0002986

rsl0969247 0. .0002986

rsl0969248 0. .0002986

rs736508 0. .0003

rsl513680 0. .0003002

rsl838408 0. .0003002

rsl849566 0. .0003002

rsl521034 0. .0003006

rs2139795 0. .0003006

rs2251640 0. .0003012

rs2784464 0. .0003012

rs7231534 0. .0003017

rsl0491681 0. .0003052

rsl998199 0. .0003053

rs582351 0. .0003058

rsll811546 0. .0003061

rsl0969272 0. .0003081

rsl2771164 0. .0003098

rsl0205258 0. .0003102

rs6100191 0. .0003127

rsl943553 0. .0003128

rsl0139222 0. .0003129

rsl2615868 0. .0003137

rsl2711546 0. .0003137

rs6735895 0. .0003137

rsll635771 0. .0003143

rslll2833 0. .0003155

rsl0196761 0. .0003161

rsl0496568 0. .0003161

rsl7006463 0. .0003161

rs6725314 0. .0003161

rs7383045 0. .0003161

rs7599933 0. .0003161

rs7770235 0. .0003161

rs7038904 0. .0003163

rs9293920 0. .0003169

rs7851775 0. .0003177

rsl0490196 0. .0003193

rs4873792 0. .0003196

rsl0510271 0. .0003201

rsl0969230 0. .0003203

rs4233172 0. .000323

rsl344506 0. .0003237

rs7864733 0. .000324

rsl0203617 0. .0003251

rsl398112 0. .0003255

rslll55742 0. .0003258

rsl2611456 0. .0003268

rsl2611457 0. .0003268

rsl7039719 0. .0003268

rs6761493 0. .0003268

rsl355827 0. .000327

rs7239670 0. .000327

rsll084821 0. .0003278

rsl6962574 0. .000328

rs2025129 0. .0003285

rs4323568 0. .0003289

rs6070587 0. .000329

Page 6 Appendi xB . txt rs657426 0. .0003301

rs4685648 0. .0003311

rs7482394 0. .0003312

rs2889741 0. .0003316

rsl0214511 0. .0003325

rsl885235 0. .0003327

rsl0756650 0. .0003332

rs4804787 0. .0003338

rsll920714 0. .0003353

rsl580569 0. .0003353

rs4855516 0. .0003353

rs4855517 0. .0003353

rs4855518 0. .0003353

rs6549147 0. .0003353

rs6764924 0. .0003353

rsl0123940 0. .0003357

rs9468414 0. .000336

rsl335957 0. .0003361

rs2786266 0. .0003361

rs2312587 0. .0003365

rs6477537 0. .0003375

rsl513686 0. .0003391

rsl3146442 0. .0003393

rs7866684 0. .0003402

rsll085134 0. .0003422

rs2901113 0. .0003445

rs2901114 0. .0003445

rs4560873 0. .000345

rs610199 0. .000345

rslll29637 0. .0003451

rs7741525 0. .000346

rs9398017 0. .000346

rs6509137 0. .0003481

rsll920741 0. .0003485

rsl602200 0. .0003485

rsl6960857 0. .0003485

rsl0813132 0. .0003501

rs2067951 0. .0003504

rs7747720 0. .0003517

rsl7802700 0. .0003522

rs4490947 0. .0003524

rs2327566 0. .0003534

rs2492274 0. .0003552

rs2492276 0. .0003552

rsl6942896 0. .0003579

rsl2662569 0. .000358

rs4570560 0. .0003585

rsl895320 0. .0003596

rsl3078681 0. .0003605

rsl3065961 0. .0003615

rsl3076600 0. .0003615

rsl474276 0. .0003618

rs6482531 0. .0003633

rsl0969240 0. .0003635

rsl7698983 0. .0003653

rs2327567 0. .0003653

rs6914579 0. .0003656

rsl593872 0. .0003657

rs9482270 0. .0003671

rs902246 0. .0003679

rs902247 0. .0003679

rs7894273 0. .0003693

rs7899880 0. .0003693

rs7475020 0. .0003694

rsl3300699 0. .0003697

rsl885236 0. .0003697

rsl807403 0. .0003706

rsl0830220 0. .0003715

rs4688519 0. .0003733

rs6509134 0. .0003734

Page 7 Appendi xB . txt rsl041752 0. .0003735

rs2828109 0. .0003735

rs2828110 0. .0003735

rs7998892 0. .000375

rs319919 0. .0003754

rsl0510604 0. .0003758

rsl0481554 0. .0003772

rsl556463 0. .0003788

rs6570450 0. .0003794

rs9403262 0. .0003794

rs4884452 0. .0003805

rs9526845 0. .0003815

rs8087567 0. .0003825

rs6888545 0. .0003832

rsl2904291 0. .0003857

rs7193895 0. .0003858

rs9630225 0. .0003862

rs6502586 0. .0003884

rsl0518270 0. .0003892

rsl893692 0. .0003893

rsl0176760 0. .0003912

rsl943579 0. .0003952

rsl943580 0. .0003952

rsl943581 0. .0003952

rs2591098 0. .0003952

rs2591099 0. .0003952

rs2733134 0. .0003952

rsl943582 0. .0003962

rs2212962 0. .0003962

rs6932538 0. .0004003

rs9341408 0. .0004005

rs597462 0. .0004006

rs8129099 0. .000401

rsll970254 0. .0004022

rs2236426 0. .000403

rsl7108636 0. .0004039

rs2417752 0. .0004066

rsll946608 0. .0004076

rs4468712 0. .0004082

rsl387731 0. .0004093

rsl602199 0. .0004093

rs9812027 0. .0004093

rs991807 0. .0004093

rs991808 0. .0004093

rsl965118 0. .0004134

rs8041853 0. .0004134

rs876535 0. .0004141

rs2088403 0. .0004147

rs789900 0. .0004147

rs761937 0. .0004165

rsll886348 0. .0004167

rsll891264 0. .0004167

rs6716674 0. .0004167

rs6745850 0. .0004167

rs2328194 0. .0004175

rs9321803 0. .0004175

rs4609047 0. .0004194

rsl349710 0. .0004197

rs9321804 0. .0004208

rs7095668 0. .0004211

rsl435684 0. .0004226

rs6911913 0. .0004246

rs9390445 0. .0004246

rsll24329 0. .0004265

rsl0977739 0. .0004278

rsl2610472 0. .0004282

rs3826941 0. .0004282

rs4742595 0. .0004283

rsl368299 0. .0004288

rs8109615 0. .0004305

Page 8 AppendixB.txt rs662041 0. .0004335

rsl7403480 0. .000434

rs789887 0. .000436

rs789888 0. .000436

rs9428429 0. .0004363

rs4703645 0. .00044

rslll22898 0. .0004414

rs3860383 0. .0004414

rs7605443 0. .0004414

rs4912173 0. .0004421

rs8028380 0. .0004424

rsl547872 0. .0004433

rs7504838 0. .0004433

rs7765349 0. .0004446

rs9596651 0. .000445

rsll854288 0. .0004461

rs3759846 0. .0004461

rs6570443 0. .0004465

rsl332207 0. .0004476

rsll073855 0. .0004481

rs8036314 0. .0004481

rs8028882 0. .0004486

rs9806190 0. .0004486

rsll016936 0. .0004502

rs4503789 0. .0004521

rs9376586 0. .0004523

rsl344196 0. .0004524

rs6468799 0. .0004528

rs7015311 0. .0004528

rs3757508 0. .0004533

rs3948710 0. .0004533

rsl0204078 0. .0004539

rsl0207199 0. .0004539

rs6945109 0. .0004539

rsl0867221 0. .0004545

rslll37747 0. .0004545

rsl2339181 0. .0004545

rsl452990 0. .000456

rs6570441 0. .000456

rs9403252 0. .000456

rslll37748 0. .0004567

rs3761743 0. .0004571

rs370177 0. .0004578

rs403461 0. .0004578

rsl7112249 0. .0004586

rslll37755 0. .00046

rs769289 0. .00046

rs8109313 0. .0004607

rsl0835824 0. .0004609

rsl2986567 0. .0004619

rsl6831392 0. .0004628

rsll853075 0. .0004629

rsl6960864 0. .0004629

rsl0508710 0. .0004638

rsl2248210 0. .0004638

rsl2252825 0. .0004638

rsl2253015 0. .0004638

rsl2260321 0. .0004638

rsl2263561 0. .0004638

rsl2267071 0. .0004638

rsl2765561 0. .0004638

rsl2766929 0. .0004638

rsl2767114 0. .0004638

rsl2768879 0. .0004638

rsl2770526 0. .0004638

rsl2771414 0. .0004638

rsl2773856 0. .0004638

rsl2776584 0. .0004638

rsl2781790 0. .0004638

rsl521029 0. .0004638

Page 9 Appendi xB . txt rsl521030 0. .0004638

rsl521032 0. .0004638

rsl546702 0. .0004638

rsl7739106 0. .0004638

rs2176937 0. .0004638

rs3758441 0. .0004638

rs3758443 0. .0004638

rs3758444 0. .0004638

rs3817419 0. .0004638

rs4581343 0. .0004638

rs4631780 0. .0004638

rs5012431 0. .0004638

rs7084483 0. .0004638

rs7095982 0. .0004638

rs7096274 0. .0004638

rs7097387 0. .0004638

rs7475023 0. .0004638

rs7475188 0. .0004638

rsl352638 0. .0004646

rs4484195 0. .0004646

rsll049887 0. .0004688

rsl480630 0. .0004688

rsl452989 0. .0004689

rs7872398 0. .0004689

rs7989297 0. .0004704

rsl2243741 0. .0004719

rsl2248769 0. .0004719

rsl2249006 0. .0004719

rsl2263998 0. .0004719

rsl2780744 0. .0004719

rs7893838 0. .0004719

rs7915315 0. .0004719

rs3937845 0. .0004723

rs4771400 0. .0004723

rs4855515 0. .0004723

rs7621668 0. .0004723

rs7635448 0. .0004723

rsl489995 0. .0004733

rs6748226 0. .000474

rsll897514 0. .0004765

rs2170466 0. .0004776

rs7529541 0. .0004783

rsl2676502 0. .0004809

rsl0497107 0. .0004811

rsl6831399 0. .0004811

rs7452495 0. .0004842

rs7500154 0. .0004854

rs399355 0. .0004878

rsl3208784 0. .0004884

rsll073091 0. .0004921

rsll073092 0. .0004921

rsll635913 0. .0004921

rs6495766 0. .0004921

rs8031712 0. .0004921

rs8041062 0. .0004921

rs8041339 0. .0004921

rs4298240 0. .0004923

rs4704170 0. .0004923

rsl0765819 0. .0004936

rs6885454 0. .0004946

rs9390449 0. .0004947

rsl6831410 0. .0004961

rs6560561 0. .0004971

rs9403836 0. .0004984

rsll33174 0. .0004993

rsl0969259 0. .0004994

rs679094 0. .0004994

rsl2289316 0. .0005001

rs2070435 0. .0005002

rsl3264258 0. .0005011

Page 10 Appendi xB . txt rs7042831 0. .0005017

rsl414121 0. .000503

rsl6855165 0. .0005031

rsl873263 0. .0005049

rsl7406994 0. .0005058

rs6468794 0. .0005058

rs6468796 0. .0005058

rsl0093531 0. .0005069

rsl0107399 0. .0005069

rsll987293 0. .0005069

rsll989758 0. .0005069

rsll855819 0. .0005077

rs2356558 0. .000508

rs4544345 0. .000508

rs4140424 0. .0005085

rs6076819 0. .0005085

rs6076820 0. .0005085

rs6076823 0. .0005085

rs6516026 0. .0005085

rs234595 0. .0005095

rsl0152716 0. .0005105

rsl0519999 0. .0005105

rsl0520001 0. .0005105

rsll637062 0. .0005105

rsll853444 0. .0005105

rsll853446 0. .0005105

rsl455047 0. .0005105

rsl6960879 0. .0005105

rsl6960916 0. .0005105

rs8038618 0. .0005105

rs8040254 0. .0005105

rsl0488687 0. .0005112

rs6039529 0. .0005141

rs6056764 0. .0005141

rs341870 0. .0005146

rsl0515405 0. .0005147

rsl349541 0. .0005178

rs4241220 0. .0005178

rs9858963 0. .0005182

rs6936461 0. .0005183

rs9497673 0. .0005183

rs3118045 0. .0005216

rs212293 0. .0005221

rs567990 0. .000524

rsll632343 0. .000525

rsll636113 0. .000525

rsl455045 0. .000525

rs8034582 0. .000525

rs8034766 0. .000525

rs6576603 0. .0005251

rs7165604 0. .0005251

rsl0107160 0. .0005258

rsl2203394 0. .000526

rsl0129671 0. .000528

rsl0483588 0. .000528

rsl2586169 0. .000528

rsl2588980 0. .000528

rsl3075703 0. .0005282

rsl354439 0. .0005282

rs7871453 0. .0005323

rs2850287 0. .0005334

rs534815 0. .0005352

rs2277882 0. .0005383

rs7043954 0. .0005383

rsll629935 0. .0005422

rsl431603 0. .0005461

rs2163996 0. .000547

rs4373347 0. .000547

rsl452988 0. .0005474

rsl489992 0. .0005476

Page 11 Appendi xB . txt rsl7537990 0. .0005476

rsl0491673 0. .0005485

rsl408508 0. .0005485

rsl0767906 0. .0005493

rsl2221893 0. .0005493

rsl0418179 0. .0005497

rs6689022 0. .0005542

rs7539805 0. .0005542

rsl0064203 0. .0005544

rs6495767 0. .0005545

rs684371 0. .0005599

rs2828132 0. .0005603

rs4896533 0. .0005623

rsll676548 0. .0005633

rsl489993 0. .0005669

rsll947379 0. .0005687

rsl746338 0. .0005689

rs3823764 0. .0005695

rsl7154523 0. .0005714

rs6468793 0. .0005725

rsl2176199 0. .0005741

rsl3269411 0. .0005775

rsl405252 0. .0005776

rs6916676 0. .0005796

rsll923978 0. .0005808

rs6711537 0. .0005816

rs6730732 0. .0005816

rsl0969221 0. .0005818

rsl2376718 0. .0005818

rsl6851002 0. .0005824

rs9946863 0. .0005827

rsll678315 0. .0005834

rsl529013 0. .0005852

rsl3257327 0. .0005855

rs4666283 0. .0005865

rsl3377546 0. .0005889

rs6810192 0. .0005952

rsl595286 0. .0005958

rs7646856 0. .0005984

rs2138823 0. .0005987

rs2138824 0. .0005987

rs2176431 0. .0005987

rs8104694 0. .0005987

rs663651 0. .0005994

rs4131293 0. .0005996

rs4131455 0. .0006005

rsll31497 0. .0006007

rs6570784 0. .000601

rs4284720 0. .0006015

rsl70347 0. .000603

rs296323 0. .0006035

rs296328 0. .0006035

rs296335 0. .0006035

rs4740981 0. .0006042

rsl943545 0. .0006062

rsl954972 0. .0006062

rs2591104 0. .0006062

rs2733113 0. .0006062

rs2850276 0. .0006062

rsl943555 0. .0006064

rsl332204 0. .0006065

rsl332206 0. .0006065

rs3902451 0. .0006065

rs6914395 0. .0006067

rsl901515 0. .0006075

rsll237338 0. .0006098

rsl540023 0. .0006098

rsl7429236 0. .0006098

rsl7501198 0. .0006098

rsl943546 0. .0006098

Page 12 Appendi xB . txt rs2097049 0. .0006098

rs2591102 0. .0006098

rs9990383 0. .0006098

rsl0106459 0. .0006138

rs2278155 0. .0006145

rs967444 0. .0006151

rs414509 0. .0006181

rs4839651 0. .0006194

rs6440181 0. .0006194

rsl3258631 0. .0006196

rs890343 0. .000621

rs4326011 0. .0006213

rsl481110 0. .0006225

rsll607512 0. .000624

rsl567819 0. .0006251

rs4734595 0. .0006254

rs7667878 0. .0006261

rslll07212 0. .0006279

rs9490520 0. .0006285

rs8086418 0. .0006292

rsl0781390 0. .0006298

rs9749801 0. .0006324

rs4452425 0. .0006327

rs944559 0. .0006327

rsl0813114 0. .0006347

rs3918322 0. .000635

rs7319214 0. .0006355

rsll56491 0. .000637

rs2504906 0. .0006382

rsll097562 0. .0006383

rsl0813115 0. .0006391

rsl7709654 0. .0006391

rs7745871 0. .0006407

rs6468797 0. .0006434

rs6468798 0. .0006434

rs7925289 0. .0006499

rs6663538 0. .0006506

rsl575054 0. .0006533

rsl7090631 0. .0006562

rs6449263 0. .0006564

rsl2279880 0. .0006572

rsl823507 0. .0006572

rs7115801 0. .0006572

rsl7671496 0. .0006632

rs6453116 0. .0006632

rs6890143 0. .0006632

rs6890320 0. .0006632

rs6893216 0. .0006632

rs717587 0. .0006632

rs6668923 0. .0006636

rsl408507 0. .0006639

rs319924 0. .000664

rs319925 0. .000664

rs6100193 0. .000664

rs2121777 0. .0006647

rs7681041 0. .0006666

rsl0876268 0. .0006675

rsl532763 0. .0006693

rs2209168 0. .0006702

rs2588844 0. .0006712

rs8122333 0. .0006718

rsl0497108 0. .0006725

rsl0497111 0. .0006725

rsl6823807 0. .0006725

rsl0961885 0. .000673

rsl407978 0. .000673

rs427608 0. .0006739

rs451388 0. .0006739

rs3754205 0. .0006745

rs9965748 0. .0006781

Page 13 Appendi xB . txt rs875429 0. .0006792

rs7871676 0. .0006819

rsl7752458 0. .0006822

rs7745473 0. .0006822

rsl0464926 0. .0006833

rsl0464928 0. .0006833

rsll787096 0. .0006833

rsl2679840 0. .0006833

rsl2680350 0. .0006833

rsl543010 0. .0006833

rs9293488 0. .000684

rsl2711699 0. .0006843

rs2421357 0. .0006843

rsl974868 0. .0006848

rs2305317 0. .0006858

rsl2066808 0. .0006862

rsl6840486 0. .0006874

rs292209 0. .0006876

rs9376605 0. .0006901

rs756274 0. .0006926

rs7336398 0. .0006934

rs7039335 0. .0006974

rs627387 0. .0006978

rsll261299 0. .0006982

rs7534106 0. .0006986

rs493412 0. .0006989

rsl7054953 0. .0006996

rs403668 0. .0006996

rs4368235 0. .0007

rs7106157 0. .0007007

rsl862245 0. .0007025

rs986861 0. .0007037

rs7452823 0. .0007051

rsl2441919 0. .0007053

rs6688496 0. .0007057

rslll06529 0. .0007058

rs6934306 0. .000709

rs978960 0. .000709

rs598741 0. .0007093

rs622020 0. .0007093

rs7046871 0. .0007102

rs9536632 0. .0007103

rsl3243392 0. .0007121

rsl2319366 0. .0007123

rs601519 0. .0007132

rsl0790449 0. .0007143

rs4729093 0. .0007143

rs6468795 0. .0007144

rsl6831413 0. .0007156

rsl2368136 0. .0007159

rsl480639 0. .0007159

rsl0876257 0. .000718

rsl2149168 0. .0007181

rs2854516 0. .0007225

rs4236869 0. .0007253

rs7345130 0. .0007269

rs3935754 0. .0007284

rsll892222 0. .0007302

rs4803668 0. .0007322

rs4242641 0. .0007326

rs4895591 0. .0007326

rs6570456 0. .0007326

rsl0786282 0. .000733

rsl0736107 0. .0007335

rs6721161 0. .0007336

rsl2308231 0. .0007337

rsl013507 0. .0007338

rsl225740 0. .0007341

rsl0206342 0. .0007383

rs4517379 0. .0007383

Page 14 Appendi xB . txt rs632496 0. .0007383

rs7760482 0. .0007387

rsl342404 0. .0007392

rs7420862 0. .0007428

rs275737 0. .0007437

rs2588849 0. .0007438

rs7029062 0. .0007456

rsl0501696 0. .0007463

rs6686154 0. .0007466

rs7537518 0. .0007466

rs873982 0. .0007486

rsl0455430 0. .00075

rsl333201 0. .00075

rs7996728 0. .0007502

rsl949914 0. .0007516

rs2063034 0. .000753

rs2016496 0. .0007532

rs7431080 0. .0007557

rsl573515 0. .0007565

rsl945295 0. .0007565

rs226478 0. .0007591

rs438666 0. .0007592

rsl3092390 0. .0007605

rs9937891 0. .000761

rs4603646 0. .0007621

rsl925558 0. .0007634

rs7914124 0. .0007634

rsl7061059 0. .0007639

rs6560559 0. .0007642

rs440980 0. .0007662

rsl406538 0. .0007681

rs9526913 0. .0007699

rsl513681 0. .0007704

rs9952567 0. .0007704

rs7835485 0. .0007712

rsl7704557 0. .0007715

rs8036702 0. .0007715

rs8037124 0. .0007715

rsl2681932 0. .000774

rsll682773 0. .0007748

rs9399593 0. .000775

rsl393914 0. .0007753

rsll698603 0. .0007779

rsl2625034 0. .0007779

rsl2625431 0. .0007779

rsl2625628 0. .0007779

rsl2625665 0. .0007779

rsl438106 0. .0007779

rsl550266 0. .0007779

rs4254564 0. .0007779

rs4353709 0. .0007779

rs6034368 0. .0007779

rs6034370 0. .0007779

rs6043726 0. .0007779

rs7509079 0. .0007779

rs585721 0. .0007782

rs7222072 0. .0007793

rs4880981 0. .0007826

rsl2063812 0. .000783

rs4839649 0. .0007863

rsll618716 0. .0007868

rsl448822 0. .000787

rs7087620 0. .000788

rs4749914 0. .0007883

rsll245624 0. .0007902

rs3096735 0. .0007905

rs2023256 0. .0007907

rs4710440 0. .0007935

rs6936487 0. .0007936

rs7656857 0. .000794

Page 15 AppendixB.txt rs6914649 0. .0007951

rsl0876266 0. .0007952

rsl0876267 0. .0007952

rsl3121574 0. .0007987

rs7067673 0. .0007992

rs3735530 0. .0008004

rsll845831 0. .0008031

rsl7278718 0. .000804

rsl2005037 0. .0008041

rsl2454081 0. .0008046

rsl2149202 0. .000806

rsl943530 0. .0008061

rslll67538 0. .0008066

rsl2519170 0. .0008066

rs4814547 0. .0008091

rsl513679 0. .0008093

rsl3277059 0. .0008098

rsl0748673 0. .00081

rs2015439 0. .00081

rsl0027730 0. .0008101

rs268899 0. .0008104

rs2225595 0. .0008127

rs7136664 0. .0008146

rs985652 0. .0008156

rsl2510520 0. .0008166

rsl2510610 0. .0008166

rsl3338089 0. .000817

rsl7483533 0. .0008171

rs402617 0. .0008206

rs569420 0. .0008214

rs4319886 0. .0008219

rsl0818876 0. .0008236

rs2895621 0. .0008248

rs7600875 0. .0008261

rsl3123933 0. .0008268

rs7601126 0. .0008273

rsll25335 0. .0008274

rsl2297679 0. .0008288

rsl2303248 0. .0008288

rsl2309427 0. .0008288

rsl388688 0. .0008288

rsl2352641 0. .0008295

rsl889054 0. .0008314

rs2303021 0. .0008315

rs2303022 0. .0008315

rslll27237 0. .0008317

rsl073373 0. .000832

rs2892324 0. .000832

rslll48252 0. .0008322

rsll620062 0. .0008322

rs4886018 0. .0008322

rs7166935 0. .0008325

rsl3105679 0. .000833

rsl6836521 0. .0008337

rs4371301 0. .0008384

rs7616888 0. .0008394

rs2186392 0. .0008397

rs9946213 0. .0008399

rsl924036 0. .0008422

rsl2625207 0. .0008429

rs6043688 0. .0008429

rs4485297 0. .000843

rsl009359 0. .0008444

rsll782123 0. .0008448

rsllll886 0. .0008465

rsl497482 0. .0008473

rsl0257555 0. .0008487

rs980608 0. .0008491

rs949391 0. .0008498

rsl6940979 0. .00085

Page 16 Appendi xB . txt rs6730351 0. .0008508

rslll07218 0. .0008509

rsl7280309 0. .0008524

rs4921281 0. .0008532

rs7664389 0. .0008544

rs2123451 0. .0008552

rs4896534 0. .0008552

rs6905756 0. .0008552

rs9376601 0. .0008552

rs9403280 0. .0008552

rs7338409 0. .0008561

rsl7478082 0. .0008573

rsl2619716 0. .0008574

rsl335307 0. .0008576

rsl722545 0. .0008578

rsl722546 0. .0008578

rs6848514 0. .0008578

rsl2466324 0. .0008581

rsll07436 0. .0008615

rsl2677525 0. .0008626

rs2609115 0. .0008649

rs2301721 0. .0008656

rsll040444 0. .0008659

rs605323 0. .0008672

rs607160 0. .0008672

rsl6950049 0. .0008694

rs6557545 0. .0008695

rs9327842 0. .0008696

rs2248674 0. .0008704

rs4671750 0. .0008714

rs912507 0. .0008723

rslll88735 0. .0008737

rs317221 0. .0008738

rsl2292560 0. .0008766

rs2159106 0. .0008769

rsl557336 0. .0008787

rsl893305 0. .0008788

rsl3360698 0. .0008789

rsl037293 0. .0008799

rsl2149210 0. .00088

rsl907153 0. .00088

rsl701916 0. .0008816

rsl2472272 0. .0008822

rs7418932 0. .0008822

rsll639204 0. .0008825

rs7179938 0. .0008825

rs7179940 0. .0008825

rs6689433 0. .0008838

rs6123426 0. .0008852

rs912512 0. .0008852

rs2387616 0. .0008854

rsl2205984 0. .000886

rs450855 0. .000886

rs2081725 0. .0008887

rsl350072 0. .0008922

rs4949165 0. .0008926

rs6934166 0. .0008931

rs6911292 0. .0008934

rs9657649 0. .000894

rsl2292039 0. .0008947

rs7179747 0. .0008966

rsll668398 0. .0008968

rs4848983 0. .0008984

rsl2797590 0. .0008991

rsl0786281 0. .0009004

rs319920 0. .0009027

rs3792775 0. .000903

rs7999587 0. .0009054

rsl370500 0. .0009058

rs7164397 0. .0009065

Page 17 Appendi xB . txt rsl2643660 0. .0009071

rs6468792 0. .0009071

rs6987380 0. .0009071

rs7769835 0. .0009082

rslll90728 0. .0009084

rs906190 0. .0009101

rsl345170 0. .0009108

rs4839650 0. .0009108

rsl7764682 0. .0009125

rs2636723 0. .000916

rs3742297 0. .0009183

rsl2049174 0. .0009201

rs7829940 0. .0009232

rsl0209582 0. .0009239

rsl2052554 0. .0009239

rsl2053338 0. .0009239

rsl3419898 0. .0009239

rsl3429785 0. .0009239

rs2045041 0. .0009239

rs6746493 0. .0009239

rsl64734 0. .0009241

rsl64735 0. .0009241

rs351689 0. .0009241

rs351693 0. .0009241

rs6838150 0. .0009247

rs914804 0. .0009255

rsl971167 0. .0009265

rs2013356 0. .0009265

rs9965899 0. .0009265

rs2414594 0. .000927

rs2414597 0. .000927

rs2414598 0. .000927

rs2414599 0. .000927

rsl539948 0. .0009274

rsll46931 0. .000928

rsl0076638 0. .0009285

rs7742698 0. .0009289

rsl6962582 0. .0009298

rs7131359 0. .0009319

rs2764609 0. .0009358

rs4483986 0. .0009381

rs4591380 0. .0009382

rs6750501 0. .0009382

rs2256121 0. .0009393

rsl0091905 0. .0009414

rs9482272 0. .0009415

rs7764184 0. .0009423

rsl533172 0. .0009442

rs7096228 0. .0009443

rs4659424 0. .0009459

rs2120054 0. .0009466

rs771620 0. .0009467

rs7608301 0. .0009486

rs608539 0. .0009512

rs611671 0. .0009512

rs9388315 0. .0009517

rs3767999 0. .0009525

rsl0079948 0. .0009534

rsl2995362 0. .0009537

rs2835027 0. .0009553

rsl0876271 0. .0009555

rsl0876272 0. .0009555

rs6545476 0. .0009566

rs4769474 0. .0009571

rs4769475 0. .0009571

rs5022638 0. .0009583

rslll37756 0. .0009589

rslll37757 0. .0009589

rsl049868 0. .0009592

rsl654559 0. .0009616

Page 18 Appendi xB . txt rsl701921 0. .0009616

rs996609 0. .0009628

rs9371462 0. .0009667

rs6921058 0. .0009668

rsl0795234 0. .000967

rsl561231 0. .0009679

rs2118283 0. .0009679

rs2164715 0. .0009679

rs6723870 0. .0009679

rsl0049352 0. .0009692

rsl2476575 0. .0009699

rsl335304 0. .000972

rs6678949 0. .0009722

rsl7593128 0. .0009731

rsl0876270 0. .0009755

rsl7627505 0. .0009756

rs319923 0. .0009756

rs9313918 0. .0009761

rs761228 0. .0009773

rs9977734 0. .0009774

rs7993748 0. .000978

rs9946774 0. .0009786

rsl0882777 0. .0009788

rs4282632 0. .0009797

rs4744313 0. .0009797

rs7239336 0. .0009806

rs4395124 0. .0009808

rsll694900 0. .0009822

rs4799513 0. .0009828

rs6854195 0. .0009836

rsl015669 0. .0009842

rs7714352 0. .0009842

rs7714503 0. .0009842

rs2154652 0. .0009843

rs3864151 0. .0009844

rs7585690 0. .0009844

rs7586111 0. .0009844

rs4767012 0. .0009847

rsl749689 0. .0009848

rsl0765183 0. .0009851

rs7929619 0. .0009851

rs7932640 0. .0009851

rsl613720 0. .0009858

rslll01266 0. .0009871

rsl0737329 0. .0009919

rs4142492 0. .0009919

rs867541 0. .0009919

rs477424 0. .0009937

rs2788872 0. .0009947

rs667213 0. .0009953

rsll747342 0. .0009956

rsl0183953 0. .0009965

rs6545479 0. .0009965

rs6715694 0. .0009965

rs7588591 0. .0009965

rsl2189092 0. .0009975

rs7709412 0. .0009975

rsl0505012 0. .0009983

rs4777023 0. .0009993

rsl981815 0. .0009999

rs834194

rs473467

rs4085813

rs24356

rs6502586

rs4924773

rs6532675

rs4482769

rs6888545

rs420828

Page 19 Appendi xB . txt rs7116127

rs2658787

rs7383045

rs4895590

rs7475020

rs4378287

rs7475023

rs4313469

rs7475188

rs4581344

rs7481282

rs4509797

rs7482394

rs4517513

rs7504838

rs2850277

rs7509079

rs4254566

rs7926062

rs2605589

rsl0936257

rs9810082

rsll084821

rs7249031

rsll635913

rs9788669

rsl2176199

rs9371463

rsl2493604

rs9857513

Page 20 Appendi xB . txt

SNP p

rs7481282 8. . 636E-7

rs9322082 0. .00001371

rs648601 0. .00001859

rsl408577 0. .00001929

rs4323329 0. .00002226

rs4452660 0. .00002226

rs4085813 0. .00002238

rs4945220 0. .00002292

rs650171 0. .00002343

rs7123080 0. .00002343

rs8111760 0. .00002345

rsl7646434 0. .00002376

rsl7702960 0. .00002376

rsl0899404 0. .00002945

rsl7006485 0. .00003101

rsl7646351 0. .00003173

rsl3414513 0. .00003186

rs6712661 0. .00003186

rs735483 0. .00003186

rs7601226 0. .00003186

rs9653407 0. .00003186

rsl7646492 0. .00003213

rsl2711551 0. .00003219

rsl2968184 0. .00003313

rsl3000440 0. .00003348

rsl3413270 0. .00003348

rs2118387 0. .00003348

rs2164796 0. .00003348

rs6711921 0. .00003348

rs9678017 0. .00003348

rsl0177322 0. .00003468

rsl7006496 0. .00003502

rs724403 0. .00003502

rs4344516 0. .0000361

rsl2711559 0. .000037

rs4450992 0. .0000372

rs684813 0. .00003859

rsl6867581 0. .00003915

rs4703388 0. .00003915

rs621456 0. .00003926

rsl954888 0. .00004077

rs7580531 0. .00004105

rs7744769 0. .00004291

rsl0242229 0. .00004375

rsl2615068 0. .00004576

rsl2622908 0. .00004576

rsl7006394 0. .00004576

rs7564564 0. .00004681

rs2157814 0. .00004726

rs4267966 0. .00004741

rsl2614155 0. .00005011

rsll707244 0. .00005025

rsl7050208 0. .00005125

rs6913861 0. .00005196

rsl0936257 0. .00005258

rsl529014 0. .00005471

rs9386176 0. .00005646

rs9399592 0. .00005646

rs9403837 0. .00005646

rs2328774 0. .00005782

rs6554218 0. .00005783

rs4551178 0. .00005824

rsl456243 0. .00005826

rs7116127 0. .00005826

rs4134463 0. .0000585

rsl945747 0. .00005922

rsl0137389 0. .00006177

rsl0496569 0. .00006274

rs477594 0. .00006589

Page 1 Appendi xB . txt rs504973 0. .00006589

rs834194 0. .00006589

rsl0179345 0. .00006609

rs6756225 0. .00006609

rsl3302476 0. .00006648

rs9360640 0. .00006802

rs484363 0. .00006837

rsl365832 0. .00006923

rs4445078 0. .00006973

rsl2614379 0. .00007143

rsl2087475 0. .00007264

rsl3403769 0. .00007504

rs643388 0. .0000771

rs663674 0. .00007742

rs415234 0. .00007831

rs687037 0. .00007853

rsl2073831 0. .00008024

rs6023633 0. .00008146

rs6736269 0. .00008288

rs7772526 0. .00008298

rs6441408 0. .00008446

rsll018434 0. .00008495

rs2594536 0. .0000852

rsl0185512 0. .00008828

rs2034137 0. .00008828

rsl7087674 0. .00008992

rsl456244 0. .00009005

rslll22861 0. .00009285

rs6746090 0. .00009285

rs9840287 0. .00009301

rs2048504 0. .00009427

rs6014185 0. .00009427

rsl420582 0. .0000946

rs668701 0. .00009522

rsl0170935 0. .00009539

rs6714671 0. .00009539

rs7595601 0. .00009539

rs3912845 0. .00009655

rsl7764592 0. .0000985

rsl0496570 0. .00009884

rsl2611458 0. .00009884

rsl3395695 0. .00009884

rs2304560 0. .00009884

rs2304561 0. .00009884

rs6704587 0. .00009884

rs7566558 0. .00009884

rs934761 0. .00009884

rs6541782 0. .0001021

rs6760211 0. .0001021

rsl633749 0. .000103

rs7538154 0. .0001038

rsl0255791 0. .0001039

rs9446844 0. .0001046

rsl2614123 0. .0001058

rsl0184008 0. .000106

rs6064117 0. .0001079

rs7760644 0. .000109

rs877191 0. .00011

rs611232 0. .0001113

rs6912526 0. .0001153

rs911196 0. .0001162

rs2181942 0. .0001182

rsl512065 0. .0001192

rsl0179294 0. .0001196

rsl3395277 0. .0001196

rs6023645 0. .0001201

rs594406 0. .0001204

rs595459 0. .0001204

rs4811538 0. .0001212

rsl505006 0. .0001214

Page 2 Appendi xB . txt rs8180176 0. .000123

rs4848712 0. .0001243

rsl551151 0. .0001253

rs4711162 0. .0001253

rs2034136 0. .0001279

rs7558771 0. .0001279

rs7188211 0. .0001294

rsl628613 0. .0001301

rsl666790 0. .0001301

rsl666797 0. .0001301

rsl700572 0. .0001301

rsl2493604 0. .0001308

rsl388702 0. .0001327

rsl492146 0. .0001327

rsl492147 0. .0001327

rsl2190601 0. .0001328

rs6704842 0. .0001361

rsl2622810 0. .0001364

rs7599081 0. .0001364

rs9653406 0. .0001364

rsl2614362 0. .0001376

rs6729454 0. .0001376

rs669738 0. .0001384

rs2289192 0. .0001396

rs7672513 0. .00014

rsl0498676 0. .0001406

rs3778166 0. .0001406

rsl388705 0. .000144

rs675079 0. .0001444

rs2962621 0. .0001458

rs638807 0. .000146

rs7943055 0. .000147

rs7765767 0. .0001483

rs685553 0. .0001488

rs608382 0. .0001491

rs7700468 0. .000152

rsl057530 0. .0001524

rs488824 0. .0001535

rs553727 0. .0001535

rs611536 0. .0001535

rs947841 0. .0001535

rs9322081 0. .0001545

rs3916911 0. .000156

rsl0942739 0. .0001566

rs7709282 0. .0001566

rs4121744 0. .0001568

rs524874 0. .0001585

rsl0765186 0. .0001588

rs4440336 0. .0001599

rs6872314 0. .0001599

rs7356637 0. .0001599

rsl881665 0. .0001601

rs9386177 0. .0001601

rslll55493 0. .0001617

rslll89415 0. .0001621

rsl881663 0. .0001627

rsl7389893 0. .000165

rsl2419587 0. .0001697

rsl6922585 0. .0001697

rs6541784 0. .0001716

rs2092859 0. .0001741

rs2591107 0. .0001755

rs9468304 0. .0001759

rslll9723 0. .0001781

rsl2662634 0. .0001793

rs4811532 0. .00018

rsl3401792 0. .000182

rsl388706 0. .0001823

rs6023649 0. .0001843

rsl0929465 0. .0001848

Page 3 Appendi xB . txt rs4669141 0. .0001848

rs4804226 0. .0001852

rs8009216 0. .000186

rs7001847 0. .0001888

rsl2440999 0. .0001898

rs2200674 0. .0001898

rsl7766596 0. .0001927

rs2280565 0. .0001928

rsl335955 0. .0001932

rsl889102 0. .0001942

rs2784465 0. .0001942

rs4358853 0. .0001942

rs4685652 0. .000195

rs2678801 0. .0001951

rsl0816127 0. .0001972

rs4271025 0. .0001972

rs4279660 0. .0001972

rs4403465 0. .0001972

rs4892107 0. .0001981

rsl0177273 0. .0002003

rslll5356 0. .0002006

rs2167107 0. .0002006

rs7926062 0. .0002024

rsl023329 0. .000203

rs9620115 0. .000203

rsl0457864 0. .0002047

rs7250467 0. .0002048

rs4804788 0. .0002064

rs4685650 0. .0002065

rs4685651 0. .0002065

rs2328777 0. .0002073

rs9322084 0. .0002073

rs9497675 0. .0002073

rs6747693 0. .0002075

rsl0150607 0. .000208

rsl420577 0. .0002091

rs2827489 0. .0002098

rs2786262 0. .0002099

rsl0969252 0. .0002105

rs687781 0. .0002105

rs4988386 0. .0002129

rs4988388 0. .0002129

rs4988389 0. .0002129

rsl912890 0. .0002133

rsl553047 0. .0002145

rs2412616 0. .0002145

rsl2454494 0. .0002153

rs608240 0. .0002155

rs9497679 0. .0002176

rs2786258 0. .000218

rs595106 0. .000219

rs596159 0. .000219

rs618472 0. .000219

rs652539 0. .000219

rs6679824 0. .0002201

rs6688321 0. .0002201

rsl0969267 0. .0002222

rsl7105190 0. .0002249

rsl7105228 0. .000225

rs2186053 0. .000225

rsl0755432 0. .0002282

rsl591906 0. .0002284

rs6921180 0. .0002289

rs2027733 0. .0002297

rsl532212 0. .0002308

rs6532675 0. .0002309

rsll014929 0. .0002316

rsll014931 0. .0002316

rsll014933 0. .0002316

rsll014935 0. .0002316

Page 4 Appendi xB . txt rsll014936 0. .0002316

rsl2784683 0. .0002316

rs7862503 0. .0002316

rs7894301 0. .0002316

rs4445901 0. .0002341

rs9929708 0. .0002341

rsl559604 0. .0002351

rs9377040 0. .0002354

rsll014932 0. .0002361

rs2368120 0. .0002361

rs4703674 0. .0002367

rs767676 0. .0002367

rs2786263 0. .0002368

rsl3018316 0. .000237

rsl2232770 0. .0002384

rsl6982264 0. .0002393

rsl6870906 0. .0002398

rs4045166 0. .0002419

rsl402436 0. .0002421

rs748040 0. .0002422

rs6717225 0. .0002436

rs4711170 0. .0002465

rs2658785 0. .0002489

rsl2205302 0. .00025

rsl3084500 0. .00025

rs2919051 0. .0002524

rs296325 0. .0002541

rs296329 0. .0002541

rs296333 0. .0002541

rs296334 0. .0002541

rs296318 0. .0002561

rs296319 0. .0002561

rs296320 0. .0002561

rsl0147303 0. .0002574

rsl2718626 0. .0002594

rsl2718627 0. .0002594

rsl2718628 0. .0002594

rs3118041 0. .0002608

rs2295599 0. .0002629

rs7740233 0. .000265

rsl412727 0. .0002658

rs3949639 0. .0002658

rs7043400 0. .0002658

rs2153356 0. .0002667

rs870913 0. .0002667

rs870914 0. .0002667

rs870915 0. .0002667

rs2857682 0. .0002672

rs6023640 0. .0002673

rsl0281294 0. .0002674

rs234589 0. .0002689

rs6023625 0. .0002699

rsl0209628 0. .0002718

rsl892159 0. .0002728

rsl892160 0. .0002732

rs7917879 0. .0002749

rs9535895 0. .0002751

rsl2599944 0. .0002759

rs995365 0. .0002773

rsl595288 0. .0002785

rs234586 0. .000279

rsl7671266 0. .0002792

rs4685654 0. .000281

rsl0853651 0. .0002816

rs4685649 0. .000282

rsl0491676 0. .0002832

rsl0491679 0. .0002832

rsl7698884 0. .0002842

rs2492279 0. .0002846

rsl2278691 0. .0002857

Page 5 AppendixB.txt rsl0069744 0. .0002892

rsl0484891 0. .0002892

rs7871542 0. .0002892

rs4988398 0. .0002896

rsl455048 0. .000294

rsl943559 0. .0002946

rs2839314 0. .0002946

rsl0457082 0. .0002953

rs6541794 0. .0002962

rs6023624 0. .0002981

rsl0448170 0. .0002986

rsl0969241 0. .0002986

rsl0969245 0. .0002986

rsl0969247 0. .0002986

rsl0969248 0. .0002986

rs736508 0. .0003

rsl513680 0. .0003002

rsl838408 0. .0003002

rsl849566 0. .0003002

rsl521034 0. .0003006

rs2139795 0. .0003006

rs2251640 0. .0003012

rs2784464 0. .0003012

rs7231534 0. .0003017

rsl0491681 0. .0003052

rsl998199 0. .0003053

rs582351 0. .0003058

rsll811546 0. .0003061

rsl0969272 0. .0003081

rsl2771164 0. .0003098

rsl0205258 0. .0003102

rs6100191 0. .0003127

rsl943553 0. .0003128

rsl0139222 0. .0003129

rsl2615868 0. .0003137

rsl2711546 0. .0003137

rs6735895 0. .0003137

rsll635771 0. .0003143

rslll2833 0. .0003155

rsl0196761 0. .0003161

rsl0496568 0. .0003161

rsl7006463 0. .0003161

rs6725314 0. .0003161

rs7383045 0. .0003161

rs7599933 0. .0003161

rs7770235 0. .0003161

rs7038904 0. .0003163

rs9293920 0. .0003169

rs7851775 0. .0003177

rsl0490196 0. .0003193

rs4873792 0. .0003196

rsl0510271 0. .0003201

rsl0969230 0. .0003203

rs4233172 0. .000323

rsl344506 0. .0003237

rs7864733 0. .000324

rsl0203617 0. .0003251

rsl398112 0. .0003255

rslll55742 0. .0003258

rsl2611456 0. .0003268

rsl2611457 0. .0003268

rsl7039719 0. .0003268

rs6761493 0. .0003268

rsl355827 0. .000327

rs7239670 0. .000327

rsll084821 0. .0003278

rsl6962574 0. .000328

rs2025129 0. .0003285

rs4323568 0. .0003289

rs6070587 0. .000329

Page 6 Appendi xB . txt rs657426 0. .0003301

rs4685648 0. .0003311

rs7482394 0. .0003312

rs2889741 0. .0003316

rsl0214511 0. .0003325

rsl885235 0. .0003327

rsl0756650 0. .0003332

rs4804787 0. .0003338

rsll920714 0. .0003353

rsl580569 0. .0003353

rs4855516 0. .0003353

rs4855517 0. .0003353

rs4855518 0. .0003353

rs6549147 0. .0003353

rs6764924 0. .0003353

rsl0123940 0. .0003357

rs9468414 0. .000336

rsl335957 0. .0003361

rs2786266 0. .0003361

rs2312587 0. .0003365

rs6477537 0. .0003375

rsl513686 0. .0003391

rsl3146442 0. .0003393

rs7866684 0. .0003402

rsll085134 0. .0003422

rs2901113 0. .0003445

rs2901114 0. .0003445

rs4560873 0. .000345

rs610199 0. .000345

rslll29637 0. .0003451

rs7741525 0. .000346

rs9398017 0. .000346

rs6509137 0. .0003481

rsll920741 0. .0003485

rsl602200 0. .0003485

rsl6960857 0. .0003485

rsl0813132 0. .0003501

rs2067951 0. .0003504

rs7747720 0. .0003517

rsl7802700 0. .0003522

rs4490947 0. .0003524

rs2327566 0. .0003534

rs2492274 0. .0003552

rs2492276 0. .0003552

rsl6942896 0. .0003579

rsl2662569 0. .000358

rs4570560 0. .0003585

rsl895320 0. .0003596

rsl3078681 0. .0003605

rsl3065961 0. .0003615

rsl3076600 0. .0003615

rsl474276 0. .0003618

rs6482531 0. .0003633

rsl0969240 0. .0003635

rsl7698983 0. .0003653

rs2327567 0. .0003653

rs6914579 0. .0003656

rsl593872 0. .0003657

rs9482270 0. .0003671

rs902246 0. .0003679

rs902247 0. .0003679

rs7894273 0. .0003693

rs7899880 0. .0003693

rs7475020 0. .0003694

rsl3300699 0. .0003697

rsl885236 0. .0003697

rsl807403 0. .0003706

rsl0830220 0. .0003715

rs4688519 0. .0003733

rs6509134 0. .0003734

Page 7 Appendi xB . txt rsl041752 0. .0003735

rs2828109 0. .0003735

rs2828110 0. .0003735

rs7998892 0. .000375

rs319919 0. .0003754

rsl0510604 0. .0003758

rsl0481554 0. .0003772

rsl556463 0. .0003788

rs6570450 0. .0003794

rs9403262 0. .0003794

rs4884452 0. .0003805

rs9526845 0. .0003815

rs8087567 0. .0003825

rs6888545 0. .0003832

rsl2904291 0. .0003857

rs7193895 0. .0003858

rs9630225 0. .0003862

rs6502586 0. .0003884

rsl0518270 0. .0003892

rsl893692 0. .0003893

rsl0176760 0. .0003912

rsl943579 0. .0003952

rsl943580 0. .0003952

rsl943581 0. .0003952

rs2591098 0. .0003952

rs2591099 0. .0003952

rs2733134 0. .0003952

rsl943582 0. .0003962

rs2212962 0. .0003962

rs6932538 0. .0004003

rs9341408 0. .0004005

rs597462 0. .0004006

rs8129099 0. .000401

rsll970254 0. .0004022

rs2236426 0. .000403

rsl7108636 0. .0004039

rs2417752 0. .0004066

rsll946608 0. .0004076

rs4468712 0. .0004082

rsl387731 0. .0004093

rsl602199 0. .0004093

rs9812027 0. .0004093

rs991807 0. .0004093

rs991808 0. .0004093

rsl965118 0. .0004134

rs8041853 0. .0004134

rs876535 0. .0004141

rs2088403 0. .0004147

rs789900 0. .0004147

rs761937 0. .0004165

rsll886348 0. .0004167

rsll891264 0. .0004167

rs6716674 0. .0004167

rs6745850 0. .0004167

rs2328194 0. .0004175

rs9321803 0. .0004175

rs4609047 0. .0004194

rsl349710 0. .0004197

rs9321804 0. .0004208

rs7095668 0. .0004211

rsl435684 0. .0004226

rs6911913 0. .0004246

rs9390445 0. .0004246

rsll24329 0. .0004265

rsl0977739 0. .0004278

rsl2610472 0. .0004282

rs3826941 0. .0004282

rs4742595 0. .0004283

rsl368299 0. .0004288

rs8109615 0. .0004305

Page 8 AppendixB.txt rs662041 0. .0004335

rsl7403480 0. .000434

rs789887 0. .000436

rs789888 0. .000436

rs9428429 0. .0004363

rs4703645 0. .00044

rslll22898 0. .0004414

rs3860383 0. .0004414

rs7605443 0. .0004414

rs4912173 0. .0004421

rs8028380 0. .0004424

rsl547872 0. .0004433

rs7504838 0. .0004433

rs7765349 0. .0004446

rs9596651 0. .000445

rsll854288 0. .0004461

rs3759846 0. .0004461

rs6570443 0. .0004465

rsl332207 0. .0004476

rsll073855 0. .0004481

rs8036314 0. .0004481

rs8028882 0. .0004486

rs9806190 0. .0004486

rsll016936 0. .0004502

rs4503789 0. .0004521

rs9376586 0. .0004523

rsl344196 0. .0004524

rs6468799 0. .0004528

rs7015311 0. .0004528

rs3757508 0. .0004533

rs3948710 0. .0004533

rsl0204078 0. .0004539

rsl0207199 0. .0004539

rs6945109 0. .0004539

rsl0867221 0. .0004545

rslll37747 0. .0004545

rsl2339181 0. .0004545

rsl452990 0. .000456

rs6570441 0. .000456

rs9403252 0. .000456

rslll37748 0. .0004567

rs3761743 0. .0004571

rs370177 0. .0004578

rs403461 0. .0004578

rsl7112249 0. .0004586

rslll37755 0. .00046

rs769289 0. .00046

rs8109313 0. .0004607

rsl0835824 0. .0004609

rsl2986567 0. .0004619

rsl6831392 0. .0004628

rsll853075 0. .0004629

rsl6960864 0. .0004629

rsl0508710 0. .0004638

rsl2248210 0. .0004638

rsl2252825 0. .0004638

rsl2253015 0. .0004638

rsl2260321 0. .0004638

rsl2263561 0. .0004638

rsl2267071 0. .0004638

rsl2765561 0. .0004638

rsl2766929 0. .0004638

rsl2767114 0. .0004638

rsl2768879 0. .0004638

rsl2770526 0. .0004638

rsl2771414 0. .0004638

rsl2773856 0. .0004638

rsl2776584 0. .0004638

rsl2781790 0. .0004638

rsl521029 0. .0004638

Page 9 Appendi xB . txt rsl521030 0. .0004638

rsl521032 0. .0004638

rsl546702 0. .0004638

rsl7739106 0. .0004638

rs2176937 0. .0004638

rs3758441 0. .0004638

rs3758443 0. .0004638

rs3758444 0. .0004638

rs3817419 0. .0004638

rs4581343 0. .0004638

rs4631780 0. .0004638

rs5012431 0. .0004638

rs7084483 0. .0004638

rs7095982 0. .0004638

rs7096274 0. .0004638

rs7097387 0. .0004638

rs7475023 0. .0004638

rs7475188 0. .0004638

rsl352638 0. .0004646

rs4484195 0. .0004646

rsll049887 0. .0004688

rsl480630 0. .0004688

rsl452989 0. .0004689

rs7872398 0. .0004689

rs7989297 0. .0004704

rsl2243741 0. .0004719

rsl2248769 0. .0004719

rsl2249006 0. .0004719

rsl2263998 0. .0004719

rsl2780744 0. .0004719

rs7893838 0. .0004719

rs7915315 0. .0004719

rs3937845 0. .0004723

rs4771400 0. .0004723

rs4855515 0. .0004723

rs7621668 0. .0004723

rs7635448 0. .0004723

rsl489995 0. .0004733

rs6748226 0. .000474

rsll897514 0. .0004765

rs2170466 0. .0004776

rs7529541 0. .0004783

rsl2676502 0. .0004809

rsl0497107 0. .0004811

rsl6831399 0. .0004811

rs7452495 0. .0004842

rs7500154 0. .0004854

rs399355 0. .0004878

rsl3208784 0. .0004884

rsll073091 0. .0004921

rsll073092 0. .0004921

rsll635913 0. .0004921

rs6495766 0. .0004921

rs8031712 0. .0004921

rs8041062 0. .0004921

rs8041339 0. .0004921

rs4298240 0. .0004923

rs4704170 0. .0004923

rsl0765819 0. .0004936

rs6885454 0. .0004946

rs9390449 0. .0004947

rsl6831410 0. .0004961

rs6560561 0. .0004971

rs9403836 0. .0004984

rsll33174 0. .0004993

rsl0969259 0. .0004994

rs679094 0. .0004994

rsl2289316 0. .0005001

rs2070435 0. .0005002

rsl3264258 0. .0005011

Page 10 Appendi xB . txt rs7042831 0. .0005017

rsl414121 0. .000503

rsl6855165 0. .0005031

rsl873263 0. .0005049

rsl7406994 0. .0005058

rs6468794 0. .0005058

rs6468796 0. .0005058

rsl0093531 0. .0005069

rsl0107399 0. .0005069

rsll987293 0. .0005069

rsll989758 0. .0005069

rsll855819 0. .0005077

rs2356558 0. .000508

rs4544345 0. .000508

rs4140424 0. .0005085

rs6076819 0. .0005085

rs6076820 0. .0005085

rs6076823 0. .0005085

rs6516026 0. .0005085

rs234595 0. .0005095

rsl0152716 0. .0005105

rsl0519999 0. .0005105

rsl0520001 0. .0005105

rsll637062 0. .0005105

rsll853444 0. .0005105

rsll853446 0. .0005105

rsl455047 0. .0005105

rsl6960879 0. .0005105

rsl6960916 0. .0005105

rs8038618 0. .0005105

rs8040254 0. .0005105

rsl0488687 0. .0005112

rs6039529 0. .0005141

rs6056764 0. .0005141

rs341870 0. .0005146

rsl0515405 0. .0005147

rsl349541 0. .0005178

rs4241220 0. .0005178

rs9858963 0. .0005182

rs6936461 0. .0005183

rs9497673 0. .0005183

rs3118045 0. .0005216

rs212293 0. .0005221

rs567990 0. .000524

rsll632343 0. .000525

rsll636113 0. .000525

rsl455045 0. .000525

rs8034582 0. .000525

rs8034766 0. .000525

rs6576603 0. .0005251

rs7165604 0. .0005251

rsl0107160 0. .0005258

rsl2203394 0. .000526

rsl0129671 0. .000528

rsl0483588 0. .000528

rsl2586169 0. .000528

rsl2588980 0. .000528

rsl3075703 0. .0005282

rsl354439 0. .0005282

rs7871453 0. .0005323

rs2850287 0. .0005334

rs534815 0. .0005352

rs2277882 0. .0005383

rs7043954 0. .0005383

rsll629935 0. .0005422

rsl431603 0. .0005461

rs2163996 0. .000547

rs4373347 0. .000547

rsl452988 0. .0005474

rsl489992 0. .0005476

Page 11 Appendi xB . txt rsl7537990 0. .0005476

rsl0491673 0. .0005485

rsl408508 0. .0005485

rsl0767906 0. .0005493

rsl2221893 0. .0005493

rsl0418179 0. .0005497

rs6689022 0. .0005542

rs7539805 0. .0005542

rsl0064203 0. .0005544

rs6495767 0. .0005545

rs684371 0. .0005599

rs2828132 0. .0005603

rs4896533 0. .0005623

rsll676548 0. .0005633

rsl489993 0. .0005669

rsll947379 0. .0005687

rsl746338 0. .0005689

rs3823764 0. .0005695

rsl7154523 0. .0005714

rs6468793 0. .0005725

rsl2176199 0. .0005741

rsl3269411 0. .0005775

rsl405252 0. .0005776

rs6916676 0. .0005796

rsll923978 0. .0005808

rs6711537 0. .0005816

rs6730732 0. .0005816

rsl0969221 0. .0005818

rsl2376718 0. .0005818

rsl6851002 0. .0005824

rs9946863 0. .0005827

rsll678315 0. .0005834

rsl529013 0. .0005852

rsl3257327 0. .0005855

rs4666283 0. .0005865

rsl3377546 0. .0005889

rs6810192 0. .0005952

rsl595286 0. .0005958

rs7646856 0. .0005984

rs2138823 0. .0005987

rs2138824 0. .0005987

rs2176431 0. .0005987

rs8104694 0. .0005987

rs663651 0. .0005994

rs4131293 0. .0005996

rs4131455 0. .0006005

rsll31497 0. .0006007

rs6570784 0. .000601

rs4284720 0. .0006015

rsl70347 0. .000603

rs296323 0. .0006035

rs296328 0. .0006035

rs296335 0. .0006035

rs4740981 0. .0006042

rsl943545 0. .0006062

rsl954972 0. .0006062

rs2591104 0. .0006062

rs2733113 0. .0006062

rs2850276 0. .0006062

rsl943555 0. .0006064

rsl332204 0. .0006065

rsl332206 0. .0006065

rs3902451 0. .0006065

rs6914395 0. .0006067

rsl901515 0. .0006075

rsll237338 0. .0006098

rsl540023 0. .0006098

rsl7429236 0. .0006098

rsl7501198 0. .0006098

rsl943546 0. .0006098

Page 12 Appendi xB . txt rs2097049 0. .0006098

rs2591102 0. .0006098

rs9990383 0. .0006098

rsl0106459 0. .0006138

rs2278155 0. .0006145

rs967444 0. .0006151

rs414509 0. .0006181

rs4839651 0. .0006194

rs6440181 0. .0006194

rsl3258631 0. .0006196

rs890343 0. .000621

rs4326011 0. .0006213

rsl481110 0. .0006225

rsll607512 0. .000624

rsl567819 0. .0006251

rs4734595 0. .0006254

rs7667878 0. .0006261

rslll07212 0. .0006279

rs9490520 0. .0006285

rs8086418 0. .0006292

rsl0781390 0. .0006298

rs9749801 0. .0006324

rs4452425 0. .0006327

rs944559 0. .0006327

rsl0813114 0. .0006347

rs3918322 0. .000635

rs7319214 0. .0006355

rsll56491 0. .000637

rs2504906 0. .0006382

rsll097562 0. .0006383

rsl0813115 0. .0006391

rsl7709654 0. .0006391

rs7745871 0. .0006407

rs6468797 0. .0006434

rs6468798 0. .0006434

rs7925289 0. .0006499

rs6663538 0. .0006506

rsl575054 0. .0006533

rsl7090631 0. .0006562

rs6449263 0. .0006564

rsl2279880 0. .0006572

rsl823507 0. .0006572

rs7115801 0. .0006572

rsl7671496 0. .0006632

rs6453116 0. .0006632

rs6890143 0. .0006632

rs6890320 0. .0006632

rs6893216 0. .0006632

rs717587 0. .0006632

rs6668923 0. .0006636

rsl408507 0. .0006639

rs319924 0. .000664

rs319925 0. .000664

rs6100193 0. .000664

rs2121777 0. .0006647

rs7681041 0. .0006666

rsl0876268 0. .0006675

rsl532763 0. .0006693

rs2209168 0. .0006702

rs2588844 0. .0006712

rs8122333 0. .0006718

rsl0497108 0. .0006725

rsl0497111 0. .0006725

rsl6823807 0. .0006725

rsl0961885 0. .000673

rsl407978 0. .000673

rs427608 0. .0006739

rs451388 0. .0006739

rs3754205 0. .0006745

rs9965748 0. .0006781

Page 13 Appendi xB . txt rs875429 0. .0006792

rs7871676 0. .0006819

rsl7752458 0. .0006822

rs7745473 0. .0006822

rsl0464926 0. .0006833

rsl0464928 0. .0006833

rsll787096 0. .0006833

rsl2679840 0. .0006833

rsl2680350 0. .0006833

rsl543010 0. .0006833

rs9293488 0. .000684

rsl2711699 0. .0006843

rs2421357 0. .0006843

rsl974868 0. .0006848

rs2305317 0. .0006858

rsl2066808 0. .0006862

rsl6840486 0. .0006874

rs292209 0. .0006876

rs9376605 0. .0006901

rs756274 0. .0006926

rs7336398 0. .0006934

rs7039335 0. .0006974

rs627387 0. .0006978

rsll261299 0. .0006982

rs7534106 0. .0006986

rs493412 0. .0006989

rsl7054953 0. .0006996

rs403668 0. .0006996

rs4368235 0. .0007

rs7106157 0. .0007007

rsl862245 0. .0007025

rs986861 0. .0007037

rs7452823 0. .0007051

rsl2441919 0. .0007053

rs6688496 0. .0007057

rslll06529 0. .0007058

rs6934306 0. .000709

rs978960 0. .000709

rs598741 0. .0007093

rs622020 0. .0007093

rs7046871 0. .0007102

rs9536632 0. .0007103

rsl3243392 0. .0007121

rsl2319366 0. .0007123

rs601519 0. .0007132

rsl0790449 0. .0007143

rs4729093 0. .0007143

rs6468795 0. .0007144

rsl6831413 0. .0007156

rsl2368136 0. .0007159

rsl480639 0. .0007159

rsl0876257 0. .000718

rsl2149168 0. .0007181

rs2854516 0. .0007225

rs4236869 0. .0007253

rs7345130 0. .0007269

rs3935754 0. .0007284

rsll892222 0. .0007302

rs4803668 0. .0007322

rs4242641 0. .0007326

rs4895591 0. .0007326

rs6570456 0. .0007326

rsl0786282 0. .000733

rsl0736107 0. .0007335

rs6721161 0. .0007336

rsl2308231 0. .0007337

rsl013507 0. .0007338

rsl225740 0. .0007341

rsl0206342 0. .0007383

rs4517379 0. .0007383

Page 14 Appendi xB . txt rs632496 0. .0007383

rs7760482 0. .0007387

rsl342404 0. .0007392

rs7420862 0. .0007428

rs275737 0. .0007437

rs2588849 0. .0007438

rs7029062 0. .0007456

rsl0501696 0. .0007463

rs6686154 0. .0007466

rs7537518 0. .0007466

rs873982 0. .0007486

rsl0455430 0. .00075

rsl333201 0. .00075

rs7996728 0. .0007502

rsl949914 0. .0007516

rs2063034 0. .000753

rs2016496 0. .0007532

rs7431080 0. .0007557

rsl573515 0. .0007565

rsl945295 0. .0007565

rs226478 0. .0007591

rs438666 0. .0007592

rsl3092390 0. .0007605

rs9937891 0. .000761

rs4603646 0. .0007621

rsl925558 0. .0007634

rs7914124 0. .0007634

rsl7061059 0. .0007639

rs6560559 0. .0007642

rs440980 0. .0007662

rsl406538 0. .0007681

rs9526913 0. .0007699

rsl513681 0. .0007704

rs9952567 0. .0007704

rs7835485 0. .0007712

rsl7704557 0. .0007715

rs8036702 0. .0007715

rs8037124 0. .0007715

rsl2681932 0. .000774

rsll682773 0. .0007748

rs9399593 0. .000775

rsl393914 0. .0007753

rsll698603 0. .0007779

rsl2625034 0. .0007779

rsl2625431 0. .0007779

rsl2625628 0. .0007779

rsl2625665 0. .0007779

rsl438106 0. .0007779

rsl550266 0. .0007779

rs4254564 0. .0007779

rs4353709 0. .0007779

rs6034368 0. .0007779

rs6034370 0. .0007779

rs6043726 0. .0007779

rs7509079 0. .0007779

rs585721 0. .0007782

rs7222072 0. .0007793

rs4880981 0. .0007826

rsl2063812 0. .000783

rs4839649 0. .0007863

rsll618716 0. .0007868

rsl448822 0. .000787

rs7087620 0. .000788

rs4749914 0. .0007883

rsll245624 0. .0007902

rs3096735 0. .0007905

rs2023256 0. .0007907

rs4710440 0. .0007935

rs6936487 0. .0007936

rs7656857 0. .000794

Page 15 AppendixB.txt rs6914649 0. .0007951

rsl0876266 0. .0007952

rsl0876267 0. .0007952

rsl3121574 0. .0007987

rs7067673 0. .0007992

rs3735530 0. .0008004

rsll845831 0. .0008031

rsl7278718 0. .000804

rsl2005037 0. .0008041

rsl2454081 0. .0008046

rsl2149202 0. .000806

rsl943530 0. .0008061

rslll67538 0. .0008066

rsl2519170 0. .0008066

rs4814547 0. .0008091

rsl513679 0. .0008093

rsl3277059 0. .0008098

rsl0748673 0. .00081

rs2015439 0. .00081

rsl0027730 0. .0008101

rs268899 0. .0008104

rs2225595 0. .0008127

rs7136664 0. .0008146

rs985652 0. .0008156

rsl2510520 0. .0008166

rsl2510610 0. .0008166

rsl3338089 0. .000817

rsl7483533 0. .0008171

rs402617 0. .0008206

rs569420 0. .0008214

rs4319886 0. .0008219

rsl0818876 0. .0008236

rs2895621 0. .0008248

rs7600875 0. .0008261

rsl3123933 0. .0008268

rs7601126 0. .0008273

rsll25335 0. .0008274

rsl2297679 0. .0008288

rsl2303248 0. .0008288

rsl2309427 0. .0008288

rsl388688 0. .0008288

rsl2352641 0. .0008295

rsl889054 0. .0008314

rs2303021 0. .0008315

rs2303022 0. .0008315

rslll27237 0. .0008317

rsl073373 0. .000832

rs2892324 0. .000832

rslll48252 0. .0008322

rsll620062 0. .0008322

rs4886018 0. .0008322

rs7166935 0. .0008325

rsl3105679 0. .000833

rsl6836521 0. .0008337

rs4371301 0. .0008384

rs7616888 0. .0008394

rs2186392 0. .0008397

rs9946213 0. .0008399

rsl924036 0. .0008422

rsl2625207 0. .0008429

rs6043688 0. .0008429

rs4485297 0. .000843

rsl009359 0. .0008444

rsll782123 0. .0008448

rsllll886 0. .0008465

rsl497482 0. .0008473

rsl0257555 0. .0008487

rs980608 0. .0008491

rs949391 0. .0008498

rsl6940979 0. .00085

Page 16 Appendi xB . txt rs6730351 0. .0008508

rslll07218 0. .0008509

rsl7280309 0. .0008524

rs4921281 0. .0008532

rs7664389 0. .0008544

rs2123451 0. .0008552

rs4896534 0. .0008552

rs6905756 0. .0008552

rs9376601 0. .0008552

rs9403280 0. .0008552

rs7338409 0. .0008561

rsl7478082 0. .0008573

rsl2619716 0. .0008574

rsl335307 0. .0008576

rsl722545 0. .0008578

rsl722546 0. .0008578

rs6848514 0. .0008578

rsl2466324 0. .0008581

rsll07436 0. .0008615

rsl2677525 0. .0008626

rs2609115 0. .0008649

rs2301721 0. .0008656

rsll040444 0. .0008659

rs605323 0. .0008672

rs607160 0. .0008672

rsl6950049 0. .0008694

rs6557545 0. .0008695

rs9327842 0. .0008696

rs2248674 0. .0008704

rs4671750 0. .0008714

rs912507 0. .0008723

rslll88735 0. .0008737

rs317221 0. .0008738

rsl2292560 0. .0008766

rs2159106 0. .0008769

rsl557336 0. .0008787

rsl893305 0. .0008788

rsl3360698 0. .0008789

rsl037293 0. .0008799

rsl2149210 0. .00088

rsl907153 0. .00088

rsl701916 0. .0008816

rsl2472272 0. .0008822

rs7418932 0. .0008822

rsll639204 0. .0008825

rs7179938 0. .0008825

rs7179940 0. .0008825

rs6689433 0. .0008838

rs6123426 0. .0008852

rs912512 0. .0008852

rs2387616 0. .0008854

rsl2205984 0. .000886

rs450855 0. .000886

rs2081725 0. .0008887

rsl350072 0. .0008922

rs4949165 0. .0008926

rs6934166 0. .0008931

rs6911292 0. .0008934

rs9657649 0. .000894

rsl2292039 0. .0008947

rs7179747 0. .0008966

rsll668398 0. .0008968

rs4848983 0. .0008984

rsl2797590 0. .0008991

rsl0786281 0. .0009004

rs319920 0. .0009027

rs3792775 0. .000903

rs7999587 0. .0009054

rsl370500 0. .0009058

rs7164397 0. .0009065

Page 17 Appendi xB . txt rsl2643660 0. .0009071

rs6468792 0. .0009071

rs6987380 0. .0009071

rs7769835 0. .0009082

rslll90728 0. .0009084

rs906190 0. .0009101

rsl345170 0. .0009108

rs4839650 0. .0009108

rsl7764682 0. .0009125

rs2636723 0. .000916

rs3742297 0. .0009183

rsl2049174 0. .0009201

rs7829940 0. .0009232

rsl0209582 0. .0009239

rsl2052554 0. .0009239

rsl2053338 0. .0009239

rsl3419898 0. .0009239

rsl3429785 0. .0009239

rs2045041 0. .0009239

rs6746493 0. .0009239

rsl64734 0. .0009241

rsl64735 0. .0009241

rs351689 0. .0009241

rs351693 0. .0009241

rs6838150 0. .0009247

rs914804 0. .0009255

rsl971167 0. .0009265

rs2013356 0. .0009265

rs9965899 0. .0009265

rs2414594 0. .000927

rs2414597 0. .000927

rs2414598 0. .000927

rs2414599 0. .000927

rsl539948 0. .0009274

rsll46931 0. .000928

rsl0076638 0. .0009285

rs7742698 0. .0009289

rsl6962582 0. .0009298

rs7131359 0. .0009319

rs2764609 0. .0009358

rs4483986 0. .0009381

rs4591380 0. .0009382

rs6750501 0. .0009382

rs2256121 0. .0009393

rsl0091905 0. .0009414

rs9482272 0. .0009415

rs7764184 0. .0009423

rsl533172 0. .0009442

rs7096228 0. .0009443

rs4659424 0. .0009459

rs2120054 0. .0009466

rs771620 0. .0009467

rs7608301 0. .0009486

rs608539 0. .0009512

rs611671 0. .0009512

rs9388315 0. .0009517

rs3767999 0. .0009525

rsl0079948 0. .0009534

rsl2995362 0. .0009537

rs2835027 0. .0009553

rsl0876271 0. .0009555

rsl0876272 0. .0009555

rs6545476 0. .0009566

rs4769474 0. .0009571

rs4769475 0. .0009571

rs5022638 0. .0009583

rslll37756 0. .0009589

rslll37757 0. .0009589

rsl049868 0. .0009592

rsl654559 0. .0009616

Page 18 Appendi xB . txt rsl701921 0. .0009616

rs996609 0. .0009628

rs9371462 0. .0009667

rs6921058 0. .0009668

rsl0795234 0. .000967

rsl561231 0. .0009679

rs2118283 0. .0009679

rs2164715 0. .0009679

rs6723870 0. .0009679

rsl0049352 0. .0009692

rsl2476575 0. .0009699

rsl335304 0. .000972

rs6678949 0. .0009722

rsl7593128 0. .0009731

rsl0876270 0. .0009755

rsl7627505 0. .0009756

rs319923 0. .0009756

rs9313918 0. .0009761

rs761228 0. .0009773

rs9977734 0. .0009774

rs7993748 0. .000978

rs9946774 0. .0009786

rsl0882777 0. .0009788

rs4282632 0. .0009797

rs4744313 0. .0009797

rs7239336 0. .0009806

rs4395124 0. .0009808

rsll694900 0. .0009822

rs4799513 0. .0009828

rs6854195 0. .0009836

rsl015669 0. .0009842

rs7714352 0. .0009842

rs7714503 0. .0009842

rs2154652 0. .0009843

rs3864151 0. .0009844

rs7585690 0. .0009844

rs7586111 0. .0009844

rs4767012 0. .0009847

rsl749689 0. .0009848

rsl0765183 0. .0009851

rs7929619 0. .0009851

rs7932640 0. .0009851

rsl613720 0. .0009858

rslll01266 0. .0009871

rsl0737329 0. .0009919

rs4142492 0. .0009919

rs867541 0. .0009919

rs477424 0. .0009937

rs2788872 0. .0009947

rs667213 0. .0009953

rsll747342 0. .0009956

rsl0183953 0. .0009965

rs6545479 0. .0009965

rs6715694 0. .0009965

rs7588591 0. .0009965

rsl2189092 0. .0009975

rs7709412 0. .0009975

rsl0505012 0. .0009983

rs4777023 0. .0009993

rsl981815 0. .0009999

rs834194

rs473467

rs4085813

rs24356

rs6502586

rs4924773

rs6532675

rs4482769

rs6888545

rs420828

Page 19 Appendi xB . txt rs7116127

rs2658787

rs7383045

rs4895590

rs7475020

rs4378287

rs7475023

rs4313469

rs7475188

rs4581344

rs7481282

rs4509797

rs7482394

rs4517513

rs7504838

rs2850277

rs7509079

rs4254566

rs7926062

rs2605589

rsl0936257

rs9810082

rsll084821

rs7249031

rsll635913

rs9788669

rsl2176199

rs9371463

rsl2493604

rs9857513

Page 20

Claims

A method of treating a subject having a metabolic disorder, the method comprising:

(a) obtaining a biological sample from the subject;

(b) evaluating the sample for the presence or absence of at least one genetic indicator, wherein the at least one genetic indicator is selected from a single nucleotide polymorphism and a level of gene expression within a reference range; and

(c) performing a first metabolic procedure on the subject, if the at least one genetic indicator is absent, or (d) if the at least one genetic indicator is present, performing a second metabolic procedure on the subject, wherein the second metabolic procedure is different from the first metabolic procedure.

A method of treating a weight-related disorder in a subject comprising:

(a) obtaining a sample comprising nucleic acids from the subject;

(b) evaluating the nucleic acids for an absence or presence of one or more genetic indicators; and

(c) based on if the genetic indicator(s) is absent in (b), performing a first metabolic procedure on the subject, or if the genetic indicator(s) is present in (b), performing a second metabolic procedure on the subject, wherein the second metabolic procedure is different from the first metabolic procedure.

The method of claim 2, wherein the second metabolic procedure excludes bariatric surgery.

The method of claim 2, wherein the nucleic acids are deoxyribonucleic acids (DNA).

The method of claim 2, wherein the nucleic acids are positive for the indicator(s).

The method of claim 2, wherein the nucleic acids are negative for the indicator(s).

The method of claim 2, wherein the genetic indicator(s) is at least one single nucleotide polymorphism (SNP) selected from the SNPs shown in Appendix A (SEQ ID NOs 129- 837), Appendix B, or Appendix C.

8. The method of claim 7, wherein the at least one single nucleotide polymorphism (SNP) is selected from the SNPs shown in Appendix A. (SEQ ID NOs 129-837).

9. The method of claim 7, wherein the absence of the SNP correlates with therapeutically effective weight loss of at least 20% weight change after the metabolic procedure in the subject.

10. The method of claim 2, further comprising obtaining a clinical measurement in the

subject prior to step (c).

11. The method of claim 10, wherein the clinical measurement is at least one of a preoperative body mass index (BMI), a glucose tolerance, bile acid profile, and body composition/fat distribution of the subject.

12. The method of claim 11, wherein the clinical measurement is the BMI of the subject.

13. The method of claim 12, wherein the nucleic acids are negative for the indicator(s) and the pre-operative BMI of the subject is greater than 23 kg/m .

14. A method of treating a subject having a metabolic disorder, the method comprising:

(a) obtaining a biological sample from the subject;

(b) evaluating expression of at least one gene in the sample, wherein the gene is differentially expressed after bariatric surgery;

(c) comparing the expression level of the gene(s) evaluated in (b) to a reference range,

if expression of the gene(s) is outside the reference range, performing a first metabolic procedure on the subject, or if expression of the gene is inside the reference range, performing a second metabolic procedure on the subject.

15. The method of claim 14, wherein the second metabolic procedure excludes bariatric surgery.

16. The method of claim 14, wherein the gene(s) are selected from SEQ ID NOs 1-128.

17. The method of claim 14, wherein the reference range of gene expression is +20% of an average determined from multiple patients having undergone bariatric surgery.

18. The method of claim 14, wherein the first metabolic procedure is a surgical procedure.

19. The method of claim 18, wherein the surgical procedure is selected from the group

consisting of gastric bypass, Roux-en-Y gastric bypass (RYGB), biliopancreatic diversion, partial gastrectomy procedures such as vertical sleeve gastrectomy, adjustable gastric banding, duodenal switch, duodenojejunal bypass, vertical banded gastroplasty, intragastric balloon therapy, greater curvature plication, gastric plication, Magenstrasse and Mill, ileal transposition or interposition, small bowel transposition, biliary diversion, procedures involving anastomotic connections of the gastrointestinal tract, gastric balloon implantation and other gastric or intestinal device implantation, gastric, duodenal or intestinal endoluminal barrier implantation, gastric electrical stimulation, small bowel electrical stimulation, vagal electrical stimulation, and vagal electrical inhibition.

20. The method of claim 15, wherein the second metabolic procedure is selected from the group consisting of (a) administering hormone, neuropeptide, receptor agonists or antagonists, (b) other pharmacological or nutritional therapies, (c) activation of brown adipose tissue, and (d) providing an alternative medical device based therapy, such as, but not limited to providing duodenal endoluminal barrier.

21. The method of claim 14, wherein the method further comprises obtaining a clinical

measurement in the subject prior to step (c).

22. The method of claim 21, wherein the clinical measurement is at least one of a preoperative body mass index (BMI), a glucose tolerance, bile acid profile, and body composition/fat distribution of the subject.

23. The method of claim 22, wherein the clinical measurement is the BMI of the subject.

24. A kit for assessing the presence of a single nucleotide polymorphism (SNP) shown in Appendix A (SEQ ID NOs 129-837), Appendix B, or Appendix C in a sample

comprising: a pair of primers that specifically hybridize to regions proximal to the SNP selected from Appendix A (SEQ ID NOs 129-837), Appendix B, or Appendix C; and reagents for polymerase chain reaction (PCR).

25. The method of using the kit of claim 24 comprising:

(a) obtaining a sample comprising nucleic acids from the subject;

(c) if the genetic indicator(s) is absent in (b), performing a first metabolic procedure, or if the genetic indicator(s) is present in (b), performing an alternative second metabolic procedure.

26. The method of claim 25, wherein the second metabolic procedure excludes bariatric surgery.

27. A method of treating a weight-related disorder in a subject comprising:

(a) obtaining a sample comprising nucleic acids from the subject;

(b) evaluating the nucleic acids for an absence or presence of one or more genetic indicators;

(c) predicting an outcome of performing a first metabolic procedure based on the absence or presence of the genetic indicator(s); and

(d) performing the first metabolic procedure or performing an alternative second metabolic procedure based on the predicted outcome.

28. The method of claim 27, wherein the second metabolic procedure excludes bariatric surgery.

29. The method of claim 27, wherein the genetic indicator(s) is at least one single nucleotide polymorphism (SNP) selected from Appendix A (SEQ ID NOs 129-837), Appendix B, or Appendix C.

30. The method of claim 29, wherein the outcome is a therapeutically effective weight loss and the genetic indicator(s) is absent.

31. The method of claim 29, wherein the outcome is a therapeutically effective weight loss and the metabolic procedure is performed in the absence of the genetic indicator(s).

32. The method of claim 27, wherein the outcome is therapeutically effective weight loss of at least 20% weight change.

33. The method of claim 27, wherein the alternative second metabolic procedure is

performed in the presence of the genetic indicator(s).

34. The method of claim 27, wherein the outcome is at least one of (1) amelioration of or reduction of at least one weight-related co-morbid condition and (2) an adverse metabolic event.

35. The method of claim 34, wherein the co-morbid condition is at least one of hypertension, dyslipidemia, diabetes, acid reflux, fatty liver disease, steatohepatitis, heart disease, depression, sleep apnea, asthma, osteoarthritis, compression fractures, gallstones, lymphoedema, urinary incontinence, stroke, cancer and/or other metabolic syndromes.

36. The method of claim 27, further comprising obtaining a clinical measurement in the subject prior to step (c).

37. The method of claim 36, wherein the clinical measurement is at least one of a preoperative body mass index (BMI), a glucose tolerance, bile acid profile, and body composition/fat distribution of the subject.

38. The method of claim 37, wherein the clinical measurement is the BMI of the subject.

39. The method of claim 27, wherein the step of predicting the outcome comprises inputting the subject's patient data into a metabolic procedure outcome prediction system.

40. The method of claim 39, wherein the patient data comprises the evaluation of the absence or presence of the genetic indicator(s).

41. The method of claim 40, wherein the patient data further comprises a clinical measurement of at least one of a pre-operative body mass index (BMI), a glucose tolerance, bile acid profile, and body composition/fat distribution of the subject.

42. A method of treating a metabolic disorder in a subject, the method comprising:

(a) measuring expression of the gene(s) in a sample from the subject;

(b) comparing the expression level of the gene(s) to a reference range of expression of the gene, wherein the reference range is determined from multiple patients having undergone a bariatric surgery; and

(c) administering to the subject a composition that modulates expression of the gene(s) to the mimic expression after bariatric surgery, thereby treating the metabolic disorder.

43. The method of claim 42, wherein the gene(s) are at least one of SEQ ID NOs 1-128.

44. The method of claim 42, wherein the method results in a therapeutically significant

weight loss.

45. The method of claim 44, wherein the therapeutically significant weight loss is at least a 20% body weight change or an amelioration of or reduction of at least one weight-related co-morbid condition.

46. The method of claim 45, wherein the co-morbid condition is at least one of hypertension, dyslipidemia, diabetes, acid reflux, fatty liver disease, steatohepatitis, heart disease, depression, sleep apnea, asthma, osteoarthritis, compression fractures, gallstones, lymphodema, urinary incontinence, stroke, cancer and/or other metabolic syndromes.

47. A kit for assessing genetic variation in at least one gene associated with response to a metabolic procedure in a sample comprising:

a pair of primers that specifically hybridize to an expression product of the gene(s) selected from SEQ ID NOs 1-128; and

reagents for quantitative polymerase chain reaction (qPCR). Use of a single nucleotide polymorphism to evaluate a course of treatment for a metabolic disorder.

Use of a gene expression level to evaluate a course of treatment for a metabolic disorder.