WO2012024189A2 - Compositions et procédés de détermination de la prédisposition à développer un syndrome métabolique - Google Patents

Compositions et procédés de détermination de la prédisposition à développer un syndrome métabolique Download PDF

Info

Publication number
WO2012024189A2
WO2012024189A2 PCT/US2011/047632 US2011047632W WO2012024189A2 WO 2012024189 A2 WO2012024189 A2 WO 2012024189A2 US 2011047632 W US2011047632 W US 2011047632W WO 2012024189 A2 WO2012024189 A2 WO 2012024189A2
Authority
WO
WIPO (PCT)
Prior art keywords
sirt3
seq
metabolic syndrome
allele
snp
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2011/047632
Other languages
English (en)
Other versions
WO2012024189A3 (fr
Inventor
Eric M. Verdin
Matthew D. Hirschey
Bradley E. Aouizerat
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
J David Gladstone Institutes
University of California Berkeley
University of California San Diego UCSD
Original Assignee
J David Gladstone Institutes
University of California Berkeley
University of California San Diego UCSD
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by J David Gladstone Institutes, University of California Berkeley, University of California San Diego UCSD filed Critical J David Gladstone Institutes
Publication of WO2012024189A2 publication Critical patent/WO2012024189A2/fr
Publication of WO2012024189A3 publication Critical patent/WO2012024189A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • Metabolic Syndrome is characterized by an accumulation of risk factors for cardiovascular disease, stroke and/or diabetes mellitus type II.
  • the risk factors that characterize Metabolic Syndrome include an increased amount of adipose tissue inside the abdominal cavity (abdominal obesity), insulin resistance with increased risk of developing diabetes, hyperinsulinemia, high levels of blood fats, increased blood pressure, and elevated serum lipids.
  • the present disclosure provides a method of detecting a single nucleotide
  • SNP polymorphism
  • the present disclosure further provides nucleic acid reagents and kits for determining a subject's genotype with respect to a single nucleotide polymorphism at rsl 1246020 in a sirtuin-3 (SIRT3) gene.
  • Figures 1 A-C depict the effect of a chronic high-fat diet feeding on global
  • Figures 2A-G depict diet-induced obesity and insulin resistance in mice lacking
  • Figures 3A-E depict development of hepatic steatosis and inflammation in
  • SIRT3KO mice fed a high-fat diet fed a high-fat diet.
  • Figures 4A-C depict high expression and activity of hepatic SCD 1 in SERT3 O mice.
  • Figures 5A-D depict association of an SNP in the human SIRT3 gene with
  • Figures 6A-E depict the effect of the rs 1 1246020 SNP on SIRT3 enzymatic activity.
  • Figure 7 depicts an amino acid sequence of human SIRT3. Val-208 is in bold and underlined.
  • Figure 8 depicts genomic and cDNA nucleotide sequences in a human SIRT3 coding region. The codon encoding Val-208 is in bold and underlined.
  • Figures 9A-L depict a SIRT3 human genomic nucleotide sequence.
  • Figures 10A and 10B depict a human SIRT3 cDNA sequence.
  • metabolic syndrome is a term that is understood in the art, and refers to metabolic abnormalities, including central obesity, insulin resistance, hyperlipidemia, hyperglycemia, hypertension, and hepatic steatosis.
  • Foundation definition of metabolic syndrome is central obesity (body mass index > 30 kg/m 2 ) and two or more of: 1 ) triglycerides > 150 mg/dL; 2) high density lipoprotein (HDL) ⁇ 40 mg/kL in males, ⁇ 50 mg/dL in females, or specific treatment for low HDL; 3) elevated blood pressure, e.g., systolic BP > 130 mm Hg or diastolic BP >85 mm Hg, or treatment for elevated BP, or previous diagnosis of elevated BP; and 4) fasting blood glucose >100 mg/dL or previous diagnosis of type 2 diabetes.
  • HDL high density lipoprotein
  • a "nucleic acid,” “polynucleotide,” or “oligonucleotide” is a polymeric form of nucleotides of any length, may be DNA or RNA, and may be single- or double-stranded. Nucleic acids may include promoters or other regulatory sequences. Oligonucleotides are usually prepared by synthetic means. Nucleic acids include segments of DNA, or their complements spanning or flanking a polymorphic site in a SIRT3 gene, as described herein.
  • the segments can be between 5 and 100 contiguous bases, and often range from a lower limit of 5, 10, 12, 15, 20, or 25 nucleotides to an upper limit of 10, 15, 20, 25, 30, 50 or 100 nucleotides (where the upper limit is greater than the lower limit). Nucleic acids between 5- 10, 5-20, 10-20, 12-30, 15-30, 10-50, 20-50 or 20- 100 bases are common.
  • the polymorphic site can occur within any position of the segment.
  • a reference to the sequence of one strand of a double-stranded nucleic acid defines the complementary sequence and except where otherwise clear from context, a reference to one strand of a nucleic acid also refers to its complement.
  • nucleic acid (e.g., RNA) molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the use of phosphorothioate or 2'-0-methyl rather than phosphodiesterase linkages within the backbone of the molecule.
  • Modified nucleic acids include peptide nucleic acids (PNAs) and nucleic acids with nontraditional bases such as inosine, queosine and wybutosine and acetyl-, methyl-, thio- and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
  • PNAs peptide nucleic acids
  • nucleic acids with nontraditional bases such as inosine, queosine and wybutosine and acetyl-, methyl-, thio- and similarly modified forms of adenine, cyt
  • Hybridization probes are nucleic acids capable of binding in a base-specific manner to a complementary strand of nucleic acid. Such probes include nucleic acids and peptide nucleic acids. Hybridization may be performed under stringent conditions which are known in the art. For example, see, e.g., Berger and Kimmel ( 1987) Methods In Enzymology, Vol. 152: Guide To Molecular Cloning Techniques, San Diego:
  • probe includes primers. Probes and primers are sometimes referred to as "oligonucleotides.”
  • primer refers to a single-stranded oligonucleotide capable of acting as a point of initiation of template-directed DNA synthesis under appropriate conditions, in an appropriate buffer and at a suitable temperature.
  • the appropriate length of a primer depends on the intended use of the primer but typically ranges from 15 to 30 nucleotides.
  • a primer sequence need not be exactly complementary to a template but must be sufficiently complementary to hybridize with a template.
  • primer site refers to the area of the target DNA to which a primer hybridizes.
  • primer pair means a set of primers including a 5' upstream primer, which hybridizes to the 5' end of the DNA sequence to be amplified and a 3' downstream primer, which hybridizes to the complement of the 3' end of the sequence to be amplified.
  • Exemplary hybridization conditions for short probes and primers is about 5 to 12 degrees C below the calculated Tm.
  • allele and “allelic variant” refer to alternative forms of a gene including introns, exons, intron/exon junctions and 3' and/or 5' untranslated regions that are associated with a gene or portions thereof. Generally, alleles occupy the same locus or position on homologous chromosomes. When a subject has two identical alleles of a gene, the subject is said to be homozygous for the gene or allele. When a subject has two different alleles of a gene, the subject is said to be heterozygous for the gene.
  • isolated refers to a nucleic acid separated from macromolecules or other contaminants that may be present in the natural source of the nucleic acid, or that may be present during recombinant or chemical synthesis of the nucleic acid.
  • An isolated nucleic acid can be purified, e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or greater than 99%, pure.
  • the present disclosure provides a method of detecting a single nucleotide
  • SNP polymorphism
  • the present disclosure further provides nucleic acid reagents and kits for determining a subject's genotype with respect to a single nucleotide polymorphism at rs l 1246020 in a sirtuin-3 (SIRT3) gene.
  • a polymorphic site, rs l 1246020, has been identified in a human SIRT3 gene, in which a polymorphism is associated with a propensity to develop metabolic syndrome.
  • a codon encoding Val-208 of human SIRT3 when mutated such that the codon encodes He, is associated with a propensity to develop metabolic syndrome.
  • the codon encoding Val-208 of SIRT3 corresponds to nucleotides 3365-3367 of the genomic sequence and nucleotides 656-658 of the mRNA sequence, as depicted in Figure 8.
  • the genomic sequence depicted in Figure 8 (and in Figures 9A-L) is from GenBank
  • a G to A mutation occurs in the first nucleotide of the codon encoding Val-208 of human SIRT3.
  • the gtc codon underlined and in bold in Figure 8, which encodes Val-208 is mutated to ate, which encodes isoleucine.
  • nucleotide 3365 of the human genomic SIRT3 gene depicted in Figures 9A-L or depicted in Figure 8 is mutated from G to A in the genome of an individual, the individual has an increased risk of developing metabolic syndrome.
  • nucleotide 3365 of the human genomic SIRT3 gene depicted in Figures 9A-L or depicted in Figure 8 is mutated from G to A in the genome of an individual, the individual has a propensity to develop metabolic syndrome.
  • the genome of an individual comprises a SIRT3 allele encoding a SIRT3 polypeptide having Val-208. In some embodiments, the genome of an individual comprises a SIRT3 allele encoding a SIRT3 polypeptide having Ile-208. In some embodiments, the individual is homozygous for a SNP that is indicative of an increased propensity to develop metabolic syndrome. In some embodiments, the individual is heterozygous for a SNP that is indicative of an increased propensity to develop metabolic syndrome.
  • the presence of an SNP that is associated with a propensity in an individual to develop metabolic syndrome indicates that the individual has an at least about 25%, at least about 50%, at least about 2-fold, at least about 5-fold, or at least about 10-fold, increased risk of developing metabolic syndrome, compared to the risk that an individual who does not have the SNP will develop metabolic syndrome.
  • the present disclosure provides a method of detecting a single nucleotide
  • the method involves analyzing a polynucleotide sample from the individual for the presence of a SNP at rs l 1246020 in a sirtuin-3 (SIRT3) gene, where the SNP is associated with metabolic syndrome, and where the presence of the polymorphism is indicative of a polymorphism associated with metabolic syndrome.
  • the method involves analyzing a sample from the individual for the presence of a SIRT3 polypeptide that has a V208I substitution; the presence of the V208I substitution is indicative of a polymorphism associated with metabolic syndrome.
  • the method involves analyzing a sample from the individual for enzymatic activity of a SIRT3 polypeptide in the sample; reduced SDRT3 enzymatic activity that is associated with a V208I substitution is indicative of a polymorphism associated with metabolic syndrome.
  • a SNP in a SIRT3 gene can be detected by one or more of the following
  • the SNP is detected by hybridizing nucleic acid from an
  • a nucleic acid probe that includes the SNP is also referred to as an allele-specific probe.
  • genomic DNA is analyzed.
  • genomic DNA can be obtained from peripheral blood leukocytes collected from a patient.
  • suitable samples include saliva, cheek scrapings, organ biopsy samples, whole blood, buccal samples, tissue biopsy samples, and the like.
  • RNA or cDNA can be assayed.
  • Suitable methods include, but are not limited to, use of allele-specific probes; use of allele-specific primers; direct sequence analysis; denaturing gradient gel electrophoresis (DGGE) analysis; single-strand conformation polymorphism (SSCP) analysis; and denaturing high performance liquid chromatography (DHPLC) analysis.
  • DGGE denaturing gradient gel electrophoresis
  • SSCP single-strand conformation polymorphism
  • DPLC denaturing high performance liquid chromatography
  • Other well known methods to detect polymorphisms in DNA include use of: Molecular Beacons technology (see, e.g., Piatek et al., 1998; Nat. Biotechnol. 16:359-63; Tyagi, and Kramer, 1996, Nat. Biotechnology 14:303-308; and Tyagi, et al., 1998, Nat.
  • allele-specific probes for analyzing polymorphisms are described by e.g., Saiki et al., 1986; Dattagupta, EP 235,726, Saiki, WO 89/1 1548. Briefly, allele-specific probes are designed to hybridize to a segment of target DNA from one individual but not to the corresponding segment from another individual, if the two segments represent different polymorphic forms. Hybridization conditions are chosen that are sufficiently stringent so that a given probe essentially hybridizes to only one of two alleles. Typically, allele-specific probes are designed to hybridize to a segment of target DNA such that the polymorphic site aligns with a central position of the probe.
  • Allele-specific probes are often used in pairs, one member of a pair designed to hybridize to the reference allele of a target sequence and the other member designed to hybridize to the variant allele.
  • Several pairs of probes can be immobilized on the same support for simultaneous analysis of multiple polymorphisms within the same target gene sequence.
  • Exemplary allele-specific probes for analyzing a SI T3 SNP that is predictive of metabolic syndrome include:
  • SIRT3 gene (nucleotide sequences corresponding to SEQ ED NO:4 given):
  • Exemplary allele-specific probes comprising the minor "A” allele at nt 3365 of a human SERT3 gene (nucleotide sequences corresponding to SEQ ID NO:4 given):
  • allele-specific primers for analyzing polymorphisms are described by, e.g., WO 93/22456. Briefly, allele-specific primers are designed to hybridize to a site on target DNA overlapping a polymorphism and to prime DNA amplification according to standard PCR protocols only when the primer exhibits perfect complementarity to the particular allelic form. A single-base mismatch prevents DNA amplification and no detectable PCR product is formed. The method works best when the polymorphic site is at the extreme 3'-end of the primer, because this position is most destabilizing to elongation from the primer.
  • SNP in human SIRT3 include:
  • control primers for the "G" allele include:
  • primers are used in standard PCR protocols in conjunction with another common primer that hybridizes to the complementary strand of the SIRT3 gene at a specified location from the polymorphism.
  • the common primers are chosen such that the resulting PCR products can vary from about 100 to about 300 bases in length, or about 150 to about 250 bases in length, although smaller (about 50 to about 100 bases in length) or larger (about 300 to about 500 bases in length) PCR products are possible.
  • the length of the primers can vary from about 10 to 30 bases in length, or about 15 to 25 bases in length.
  • sequences of the common primers can be determined by inspection of the human SIRT3 genomic sequence, which is found under GenBank accession number NC_00001 1.9 and depicted in Figures 9A-L.
  • Suitable methods for detecting polymorphisms include those that involve
  • amplifying DNA or RNA from target samples e.g., amplifying the segments of the SIRT3 gene of an individual using SIRT3-specific primers
  • amplifying may result in the generation of SIRT3 allele-specific oligonucleotides, which span the single nucleotide polymorphic sites in the SIRT3 gene.
  • SIRT3-specific primer sequences and SIRT3 allele-specific oligonucleotides may be derived from the coding (exons) or non-coding (promoter, 5' untranslated, introns or 3' untranslated) regions of the SIRT3 gene.
  • Exemplary, non-limiting, primer pairs that amplify a region including the
  • rs l 1246020 polymorphic site include (where the nucleotides correspond to the numbering depicted in Figure 10A):
  • Amplification products generated using PCR can be analyzed by the use of
  • DGGE denaturing gradient gel electrophoresis
  • Alleles of target sequences can be differentiated using single-strand conformation polymorphism (SSCP) analysis. Different alleles can be identified based on sequence- and structure-dependent electrophoretic migration of single stranded PCR products. Amplified PCR products can be generated according to standard protocols, and heated or otherwise denatured to form single stranded products, which may refold or form secondary structures that are partially dependent on base sequence.
  • SSCP single-strand conformation polymorphism
  • DPLC performance liquid chromatography
  • a SIRT3 variant with a V208I substitution is detected.
  • a SIRT3 variant that has a V208I substitution is also referred to herein as "SIRT3-V208I.”
  • a SIRT3-V208I polypeptide can be detected using an antibody that can distinguish between a SIRT3-V208I polypeptide and a wild-type human SIRT3 polypeptide, e.g., a SIRT3 polypeptide that has a valine at position 208.
  • the antibody is one that can distinguish between a first and a second SIRT3 polypeptide, where the first and the second SIRT3 polypeptides differ in amino acid sequence only at amino acid 208, where the first SIRT3 polypeptide has a valine at amino acid 208; and the second SIRT3 polypeptide has an isoleucine at amino acid 208.
  • the antibody is one that can distinguish between a first and a second SIRT3 polypeptide, where the first SIRT3 polypeptide comprises the amino acid sequence as depicted in Figure 7, with a valine at amino acid 208 ("SIRT3-V208"); and the second SIRT3 polypeptide comprises the amino acid sequence as depicted in Figure 7, but has an isoleucine instead of a valine at amino acid 208 ("SIRT3-I208").
  • a suitable antibody can distinguish between SERT3- V208 and SIRT3-I208, e.g., a suitable antibody binds SIRT3-I208 with an affinity of at least about 10 "7 M, at least about 5 x 10 "7 M, at least about 10 ⁇ 8 M, at least about 5 x 10 "8 M, at least about 10 ⁇ 9 M, or greater than 10 "9 M; and binds SIRT3-V208 with an affinity of less than 10 "7 M (e.g., binds SIRT3-V208 with an affinity of 10 "6 M, 10 "5 M, 10 "4 M, or less).
  • SIRT3-V208I polypeptide In vitro methods for detection of a SIRT3-V208I polypeptide include, but are not limited to, enzyme linked immunosorbent assays (ELISAs), radioimmunoassays (RIA), Western blots, immunoprecipitation, immunofluorescence, and protein arrays/chips (e.g., arrays of antibodies or aptamers).
  • ELISAs enzyme linked immunosorbent assays
  • RIA radioimmunoassays
  • Western blots immunoprecipitation
  • immunofluorescence e.g., arrays of antibodies or aptamers
  • an "antibody specific for a SIRT3-V208I polypeptide” includes both monoclonal antibodies and polyclonal antibodies, as well as antigen-reactive proteolytic fragments of such antibodies, such as Fab, F(ab)' 2 , and Fv fragments.
  • an "antibody specific for a SIRT3-V208I polypeptide” further includes any of a variety of engineered antigen-binding molecules such as a chimeric antibody (U.S. Pat. Nos. 4,816,567 and 4,816,397; Morrison et al., Proc. Natl. Acad. Sci.
  • an isolated polypeptide e.g., a SIRT3-V208I polypeptide
  • a portion thereof e.g., a fragment of a SIRT3-V208I polypeptide that includes amino acid 208
  • a mammalian organism such as a rat, rabbit, hamster or mouse.
  • an antigenic peptide fragment e.g., a peptide fragment including amino acid 208
  • a fusion protein can be used.
  • a protein used as an immunogen may be naturally-occurring, synthetic or recombinantly produced, and may be administered in combination with an adjuvant, including but not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substance such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and the like.
  • an adjuvant including but not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substance such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and the like.
  • Monoclonal antibodies can be produced by hybridoma technology (Kohler and Creetch).
  • the immortalized cell lines can be created in vitro by fusing two different cell types, typically lymphocytes, and tumor cells.
  • the hybridoma cells may be cultivated in vitro or in vivo. Additionally, fully human antibodies can be generated by transgenic animals (He et al., J. Immunol., 169:595, 2002).
  • Fd phage and Fd phagemid technologies may be used to generate and select recombinant antibodies in vitro
  • Antibodies can be prepared against regions or discrete fragments of a SIRT3-
  • V208I polypeptide Such fragments can be selected on a physical property, such as fragments corresponding to regions that are located on the surface of the protein, e.g., hydrophilic regions, or can be selected based on sequence uniqueness, but should include amino acid 208 (e.g., amino acid 208-1).
  • An antigenic fragment will typically comprise at least about 8- 10 contiguous amino acid residues in which at least one of the amino acid residues is an amino acid affected by a SNP disclosed herein (e.g., amino acid 208).
  • the antigenic peptide can comprise, however, at least 12, 14, 16, 20, 25, 50, 100 (or any other number in-between) or more amino acid residues, provided that at le.ast one amino acid is affected by a SNP disclosed herein.
  • SIRT3-V208I polypeptide e.g., an antibody specific for a SIRT3-V208I polypeptide
  • Detectable substances include, but are not limited to, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, and luciferase
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin
  • suitable fluorescent materials include umbelliferone, fluorescein; fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin
  • an example of a luminescent material includes luminol
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin
  • suitable radioactive material include l 25 I, 13 I I, 35 S, and 3 H.
  • Detection of a SIRT3-V208I polypeptide in a biological sample obtained from an individual can be carried out by: a) contacting the biological sample with an antibody, as described above, that can distinguish between a SIRT3-V208I polypeptide and a wild- type human SIRT3 polypeptide; and b) detecting binding of the antibody with a SIRT3 polypeptide present in the biological sample. Detection of binding indicates the presence in the sample of a SIRT3-V208I polypeptide, and indicates that the individual has a propensity to develop metabolic syndrome.
  • the antibody comprises a detectable label.
  • the detection method can include a positive control, e.g., an antibody that binds a SIRT3 epitope that is shared between a SIRT3-V208 polypeptide and a SIRT3-I208 polypeptide.
  • a positive control e.g., an antibody that binds a SIRT3 epitope that is shared between a SIRT3-V208 polypeptide and a SIRT3-I208 polypeptide.
  • a subject method involves detecting SIRT3
  • a biological sample can be contacted with an acetylated SIRT3 substrate in the presence of NAD + ; and the amount of released acetyl groups quantified.
  • Suitable SIRT3 substrates include acetylated histone H4 N-terminal peptides (e.g., histone H4 amino acids 1 -23, amino acids 1-24, amino acids 1 -25, and the like).
  • the substrate can include a radiolabelled acetyl group, such that, upon action of an enzymatically active SIRT3 polypeptide, the radioactive acetyl group is released and can be measured. See, e.g., Emiliani et al. (1998) Proc. Natl. Acad. Sci. USA 95:2795.
  • deacetylase assays are performed in 100 ⁇ of deacetylase buffer (4 mM, gCl 2 , 0.2 mM dithiothreitol, 50 mM Tris-HCl, pH 8.5) containing 50 ng of recombinant SERT3, NAD + and [ 3 H] histone H4 peptide substrate.
  • the substrate can be prepared by in vitro acetylation of a histone H4 N-terminal peptide (amino acids 1-25; N-msgrgkggkglgkggakrhrkvlrd-C; SEQ ED NO:29) with
  • a positive control can include a wild-type SIRT3 polypeptide, e.g., a SIRT3 polypeptide comprising a valine at amino acid 208, e.g., a SIRT3 polypeptide comprising the amino acid sequence depicted in Figure 7.
  • a negative control can include a SIRT3 polypeptide that is catalytically inactive, e.g., SIRT3-H248Y.
  • SE T3-V208I has enzymatic activity in deacetylating an acetylated H4 histone
  • SIRT3-V208 N-terminal peptide that is substantially lower than the enzymatic activity of SIRT3- V208.
  • detection of SIRT3 enzymatic activity (catalytic efficiency) in a biological sample from an individual that is at least about 15%, at least about 20%, at least about 25%, at least about 30%, or at least about 35%, lower than the enzymatic activity of wild-type SIRT3 (SIRT3-V208) indicates that the individual could have a SIRT3 polymorphism associated with metabolic syndrome.
  • the present disclosure provides methods of determining a predisposition of an individual to develop metabolic syndrome.
  • the methods generally involving detecting in polynucleotide sample obtained from the individual the presence of a SNP in a SIRT3 gene at rs l 1246020, where the presence of the polymorphism is indicative of predisposition of the individual to develop metabolic syndrome.
  • a SNP at rs l 1246020 that is indicative of a predisposition of an individual to develop metabolic syndrome includes a "A” instead of a "G” at nucleotide 3365 of SEQ ID NO:4 (or nucleotide 125 of SEQ ID NO:3).
  • the method involves analyzing a sample from the individual for the presence of a SIRT3 polypeptide that has a V208I substitution; the presence of the V208I substitution is indicative of a predisposition of the individual to develop metabolic syndrome.
  • the method involves analyzing a sample from the individual for enzymatic activity of a SIRT3 polypeptide in the sample; reduced SIRT3 enzymatic activity that is associated with a V208I substitution is indicative of a predisposition of the individual to develop metabolic syndrome.
  • a G to A mutation occurs in the first nucleotide of the codon encoding Val-208 of human SIRT3.
  • the gtc codon underlined and in bold in Figure 8, which encodes Val-208 is mutated to ate, which encodes isoleucine.
  • nucleotide 3365 of the human genomic SIRT3 gene depicted in Figures 9A-L or depicted in Figure 8 is mutated from G to A in the genome of an individual, the individual has an increased risk of developing metabolic syndrome.
  • nucleotide 3365 of the human genomic SIRT3 gene depicted in Figures 9A-L or depicted in Figure 8 is mutated from G to A in the genome of an individual, the individual has a propensity to develop metabolic syndrome.
  • the genome of an individual comprises a SERT3 allele encoding a SIRT3 polypeptide having Val-208. In some embodiments, the genome of an individual comprises a SIRT3 allele encoding a SIRT3 polypeptide having Ile-208. In some embodiments, the individual is homozygous for a SNP that is indicative of an increased propensity to develop metabolic syndrome. In some embodiments, the individual is heterozygous for a SNP that is indicative of an increased propensity to develop metabolic syndrome.
  • the presence of an SNP that is associated with a propensity in an individual to develop metabolic syndrome indicates that the individual has an at least about 25%, at least about 50%, at least about 2-fold; at least about 5-fold, or at least about 10-fold, increased risk of developing metabolic syndrome, compared to the risk that an individual who does not have the SNP will develop metabolic syndrome.
  • predisposition to developing metabolic syndrome can be genotyped to determine which allele(s) is/are present at any given genetic region (e.g., SNP position) of interest by methods well known in the art.
  • the neighboring sequence can be used to design SNP detection reagents such as oligonucleotide probes, which may optionally be implemented in a kit format.
  • SNP detection reagents such as oligonucleotide probes, which may optionally be implemented in a kit format.
  • Exemplary suitable SNP genotyping methods are described in Chen et al., "Single nucleotide polymorphism genotyping: biochemistry, protocol, cost and throughput", Pharmacogenomics J. 2003; 3(2):77-96; Kwok et al., "Detection of single nucleotide polymorphisms", Curr Issues Mol. Biol.
  • Common SNP genotyping methods include, but are not limited to, TaqMan assays, molecular beacon assays, nucleic acid arrays, allele-specific primer extension, allele-specific PCR, arrayed primer extension, homogeneous primer extension assays, primer extension with detection by mass spectrometry, pyrosequencing, multiplex primer extension sorted on genetic arrays, ligation with rolling circle amplification, homogeneous ligation, oligonucleotide ligation assay (OLA) (U.S. Pat. No. 4,883,750; and U.S. Pat. No.
  • multiplex ligation reaction sorted on genetic arrays restriction-fragment length polymorphism, single base extension-tag assays, and the Invader assay.
  • detection mechanisms such as, for example, luminescence or chemiluminescence detection, fluorescence detection, time-resolved fluorescence detection, fluorescence resonance energy transfer, fluorescence polarization, mass spectrometry, and electrical detection.
  • Various methods for detecting polymorphisms include, but are not limited to, methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230: 1242 (1985); Cotton et al., PNAS 85:4397 (1988); and Saleeba et al., Meth. Enzymol. 217:286-295 ( 1992)), comparison of the electrophoretic mobility of variant and wild type nucleic acid molecules (Orita et al., PNAS 86:2766 ( 1989); Cotton et al., Mutat. Res. 285: 125- 144 ( 1993); and Hayashi et al, Genet. Anal. Tech.
  • SNP genotyping is performed using a TaqMan assay, which is also known as the 5' nuclease assay (U.S. Pat. Nos. 5,210,015 and 5,538,848).
  • the TaqMan assay detects the accumulation of a specific amplified product during PCR.
  • the TaqMan assay utilizes an oligonucleotide probe labeled with a fluorescent reporter dye and a quencher dye.
  • the reporter dye is excited by irradiation at an appropriate wavelength, it transfers energy to the quencher dye in the same probe via a process called fluorescence resonance energy transfer (FRET). When attached to the probe, the excited reporter dye does not emit a signal.
  • FRET fluorescence resonance energy transfer
  • the proximity of the quencher dye to the reporter dye in the intact probe maintains a reduced fluorescence for the reporter.
  • the reporter dye and quencher dye may be at the 5' most and the 3' most ends, respectively, or vice versa.
  • the reporter dye may be at the 5' or 3' most end while the quencher dye is attached to an internal nucleotide, or vice versa.
  • both the reporter and the quencher may be attached to internal nucleotides at a distance from each other such that fluorescence of the reporter is reduced.
  • DNA polymerase cleaves the probe, thereby separating the reporter dye and the quencher dye and resulting in increased fluorescence of the reporter. Accumulation of PCR product is detected directly by monitoring the increase in fluorescence of the reporter dye.
  • the DNA polymerase cleaves the probe between the reporter dye and the quencher dye only if the probe hybridizes to the target SNP-containing template which is amplified during PCR, and the probe is designed to hybridize to the target SNP site only if a particular SNP allele is present.
  • Suitable TaqMan primer and probe sequences can readily be determined using the SNP and associated nucleic acid sequence information provided herein.
  • a number of computer programs such as Primer Express (Applied Biosystems, Foster City, Calif.), can be used to rapidly obtain optimal primer/probe sets.
  • modifications of the Taqman assay well known in the art such as the use of Molecular Beacon probes (U.S. Pat. Nos. 5, 1 18,801 and 5,312,728) and other variant formats (U.S. Pat. Nos. 5,866,336 and 6, 1 17,635).
  • Another exemplary method suitable for genotyping a metabolic syndrome- associated SNP is the use of two oligonucleotide probes in an OLA.
  • one probe hybridizes to a segment of a target nucleic acid with its 3' most end aligned with the SNP site.
  • a second probe hybridizes to an adjacent segment of the target nucleic acid molecule directly 3' to the first probe.
  • the two juxtaposed probes hybridize to the target nucleic acid molecule, and are ligated in the presence of a linking agent such as a ligase if there is perfect complementarity between the 3' most nucleotide of the first probe with the SNP site. If there is a mismatch, ligation would not occur.
  • the ligated probes are separated from the target nucleic acid molecule, and detected as indicators of the presence of a SNP.
  • Mass spectrometry takes advantage of the unique mass of each of the four nucleotides of DNA. SNPs can be unambiguously genotyped by mass spectrometry by measuring the differences in the mass of nucleic acids having alternative SNP alleles.
  • MALDI-TOF Microx Assisted Laser Desorption Ionization— Time of Flight
  • mass spectrometry technology is preferred for extremely precise determinations of molecular mass, such as SNPs.
  • Numerous approaches to SNP analysis have been developed based on mass spectrometry. Suitable mass spectrometry- based methods of SNP genotyping include primer extension assays, which can also be utilized in combination with other approaches, such as traditional gel-based formats and microarrays.
  • a primer extension assay can involve designing and annealing a primer to a template PCR amplicon upstream (5') from a target SNP position.
  • a mix of dideoxynucleotide triphosphates (ddNTPs) and/or deoxynucleotide triphosphates (dNTPs) are added to a reaction mixture containing template (e.g., a SNP- containing nucleic acid molecule which has typically been amplified, such as by PCR), primer, and DNA polymerase.
  • template e.g., a SNP- containing nucleic acid molecule which has typically been amplified, such as by PCR
  • primer e.g., a SNP- containing nucleic acid molecule which has typically been amplified, such as by PCR
  • DNA polymerase e.g., a SNP- containing nucleic acid molecule which has typically been amplified, such as by PCR
  • the primer can be either immediately adjacent (i.e., the nucleotide at the 3' end of the primer hybridizes to the nucleotide next to the target SNP site) or two or more nucleotides removed from the SNP position. If the primer is several nucleotides removed from the target SNP position, the only limitation is that the template sequence between the 3' end of the primer and the SNP position cannot contain a nucleotide of the same type as the one to be detected, or this will cause premature termination of the extension primer. Alternatively, if all four ddNTPs alone, with no dNTPs, are added to the reaction mixture, the primer will always be extended by only one nucleotide, corresponding to the target SNP position.
  • primers are designed to bind one nucleotide upstream from the SNP position (i.e., the nucleotide at the 3' end of the primer hybridizes to the nucleotide that is immediately adjacent to the target SNP site on the 5' side of the target SNP site).
  • Extension by only one nucleotide minimizes the overall mass of the extended primer, thereby increasing the resolution of mass differences between alternative SNP nucleotides.
  • mass-tagged ddNTPs can be employed in the primer extension reactions in place of unmodified ddNTPs. This increases the mass difference between primers extended with these ddNTPs, thereby providing increased sensitivity and accuracy, and is particularly useful for typing heterozygous base positions.
  • Mass-tagging also alleviates the need for intensive sample- preparation procedures and decreases the necessary resolving power of the mass spectrometer.
  • the extended primers can then be purified and analyzed by MALDI-TOF mass
  • the products from the primer extension reaction are combined with light absorbing crystals that form a matrix.
  • the matrix is then hit with an energy source such as a laser to ionize and desorb the nucleic acid molecules into the gas-phase.
  • the ionized molecules are then ejected into a flight tube and accelerated down the tube towards a detector.
  • the time between the ionization event, such as a laser pulse, and collision of the molecule with the detector is the time of flight of that molecule.
  • the time of flight is precisely correlated with the mass-to-charge ratio (m/z) of the ionized molecule.
  • a metabolic syndrome-associated SNP can also be detected by direct DNA
  • SNP include single-strand conformational polymorphism (SSCP), and denaturing gradient gel electrophoresis (DGGE) (Myers et al., Nature 313:495 ( 1985)).
  • SSCP identifies base differences by alteration in electrophoretic migration of single stranded PCR products.
  • Single-stranded PCR products can be generated by heating or otherwise denaturing double stranded PCR products.
  • Single-stranded nucleic acids may refold or form secondary structures that are partially dependent on the base sequence.
  • the different electrophoretic mobilities of single-stranded amplification products are related to base-sequence differences at SNP positions.
  • DGGE differentiates SNP alleles based on the different sequence-dependent stabilities and melting properties inherent in polymorphic DNA and the corresponding differences in electrophoretic migration patterns in a denaturing gradient gel (Erlich, ed., PCR Technology, Principles and Applications for DNA Amplification, W.H. Freeman and Co, New York, 1992, Chapter 7).
  • Sequence-specific ribozymes can also be used to detect a metabolic syndrome-associated SNP based on the development or loss of a ribozyme cleavage site. Perfectly matched sequences can be distinguished from mismatched sequences by nuclease cleavage digestion assays or by differences in melting temperature. If the SNP affects a restriction enzyme cleavage site, the SNP can be identified by alterations in restriction enzyme digestion patterns, and the corresponding changes in nucleic acid fragment lengths determined by gel electrophoresis.
  • SNP genotyping can include the steps of, for example, collecting a biological sample from a human subject (e.g., sample of tissues, cells, fluids, secretions, etc.), isolating nucleic acids (e.g., genomic DNA, mRNA or both) from the cells of the sample, contacting the nucleic acids with one or more primers which specifically hybridize to a region of the isolated nucleic acid containing a target SNP under conditions such that hybridization and amplification of the target nucleic acid region occurs, and determining the nucleotide present at the SNP position of interest, or, in some assays, detecting the presence or absence of an amplification product (assays can be designed so that hybridization and/or amplification will only occur if a particular SNP allele is present or absent).
  • the size of the amplification product is detected and compared to the length of a control sample; for example, deletions and insertions can be detected by a change in size of the amp
  • a SIRT3 variant with a V208I substitution is detected.
  • a SIRT3 variant with a V208I substitution is detected.
  • a SIRT3 variant that has a V208I substitution is also referred to herein as "SIRT3-V208I.”
  • a SIRT3-V208I polypeptide can be detected using an antibody that can distinguish between a SIRT3- V208I polypeptide and a wild-type human SIRT3 polypeptide, e.g., a SIRT3 polypeptide that has a valine at position 208.
  • the antibody is one that can distinguish between a first and a second SIRT3 polypeptide, where the first and the second SIRT3 polypeptides differ in amino acid sequence only at amino acid 208, where the first SIRT3 polypeptide has a valine at amino acid 208; and the second SIRT3 polypeptide has an isoleucine at amino acid 208.
  • the antibody is one that can distinguish between a first and a second SIRT3 polypeptide, where the first SIRT3 polypeptide comprises the amino acid sequence as depicted in Figure 7, with a valine at amino acid 208 ("SIRT3-V208"); and the second SIRT3 polypeptide comprises the amino acid sequence as depicted in Figure 7, but has an isoleucine instead of a valine at amino acid 208 ("SIRT3-I208").
  • a suitable antibody can distinguish between SIRT3-V208 and SIRT3-I208, e.g., a suitable antibody binds SIRT3-I208 with an affinity of at least about 10 "7 M, at least about 5 x 10 "7 M, at least about 10 "8 M, at least about 5 x 10 "8 M, at least about 10 "9 M, or greater than 10 "9 M; and binds SIRT3-V208 with an affinity of less than 10 "7 M.
  • Detection of a SIRT3-V208I polypeptide in a biological sample obtained from an individual can be carried out by: a) contacting the biological sample with an antibody, as described above, that can distinguish between a SIRT3-V208I polypeptide and a wild- type human SIRT3 polypeptide; and b) detecting binding of the antibody with a SIRT3 polypeptide present in the biological sample. Detection of binding indicates the presence in the sample of a SIRT3-V208I polypeptide, and indicates that the individual has a propensity to develop metabolic syndrome.
  • the antibody comprises a detectable label.
  • the detection method can include a positive control, e.g., an antibody that binds a SIRT3 epitope that is shared between a SIRT3-V208 polypeptide and a SIRT3-I208 polypeptide.
  • a positive control e.g., an antibody that binds a SIRT3 epitope that is shared between a SIRT3-V208 polypeptide and a SIRT3-I208 polypeptide.
  • a subject method involves detecting SIRT3
  • enzymatic activity in a biological sample obtained from an individual. Detection of enzymatic activity can be carried out independently of, or in addition to, detection of a SERT3-V208I polypeptide.
  • a biological sample can be contacted with an acetylated SIRT3 substrate in the presence of NAD + ; and the amount of released acetyl groups quantified.
  • Suitable SIRT3 substrates include acetylated histone H4 N-terminal peptides (e.g., histone H4 amino acids 1 -23, amino acids 1 -24, amino acids 1 -25, and the like).
  • the substrate can include a radiolabeled acetyl group, such that, upon action of an enzymatically active SIRT3 polypeptide, the radioactive acetyl group is released and can be measured. See, e.g., Emiliani et al. ( 1998) Proc. Natl. Acad. Sci. USA 95:2795.
  • deacetylase assays are performed in 100 ⁇ of deacetylase buffer (4 mM, MgCl 2 , 0.2 mM dithiothreitol, 50 mM Tris-HCl, pH 8.5) containing 50 ng of recombinant SIRT3, NAD + and [ 3 H] histone H4 peptide substrate.
  • the substrate can be prepared by in vitro acetylation of a histone H4 N-terminal peptide (amino acids 1-25; N-msgrgkggkglgkggakrhrkvlrd-C; SEQ ED NO:29) with radiolabeled acetyl-CoA and recombinant P300/CBP-associated factor (PCAF; see, e.g., GenBank Accession Nos. NP_003875 and XP_001003318) (Heltweg et al. (2005) Methods 36:332).
  • PCAF P300/CBP-associated factor
  • Deacetylation reactions can be conducted at 37 °C under gentle agitation, and stopped by adding 25 ⁇ of stop solution (0.2 M HC1, 0.32 M acetic acid). Radioactivity is extracted into 500 ⁇ ethyl acetate by vortexing for 15 seconds. After centrifugation at 14,000g for 5 min, 450 ⁇ of the ethyl acetate fraction is mixed with 5 ml of scintillation fluid, and the radioactivity was measured with a liquid scintillation counter.
  • stop solution 0.2 M HC1, 0.32 M acetic acid
  • a positive control can include a wild-type SIRT3 polypeptide, e.g., a SIRT3 polypeptide comprising a valine at amino acid 208, e.g., a SIRT3 polypeptide comprising the amino acid sequence depicted in Figure 7.
  • a negative control can include a SIRT3 polypeptide that is catalytically inactive, e.g., SIRT3-H248Y.
  • SIRT3-V208I has enzymatic activity in deacetylating an acetylated H4 histone N-terminal peptide that is substantially lower than the enzymatic activity of SIRT3- V208.
  • detection of SIRT3 enzymatic activity (catalytic efficiency) in a biological sample from an individual that is at least about 15%, at least about 20%, at least about 25%, at least about 30%, or at least about 35%, lower than the enzymatic activity of wild-type SIRT3 (SIRT3-V208) indicates that the individual has a propensity to develop metabolic syndrome.
  • the results of a test can be provided in the form of a "report".
  • a tangible report can optionally be generated as part of a testing process (which may be interchangeably referred to herein as “reporting”, or as “providing” a report, "producing” a report, or “generating” a report). Examples of tangible reports may include, but are not limited to, reports on paper (such as computer-generated printouts of test results) or equivalent formats and reports stored on computer readable medium (such as a compact disk (CD), computer hard drive, or computer network server, etc.).
  • Reports e.g., those stored on computer readable medium, can be part of a database (such as a database of patient records, which may be a "secure database” that has security features that limit access to the report, such as to allow only the patient and the patient's medical practitioner(s) to view the report, for example).
  • a database such as a database of patient records, which may be a "secure database” that has security features that limit access to the report, such as to allow only the patient and the patient's medical practitioner(s) to view the report, for example.
  • reports can also be displayed on a computer screen (or the display of another electronic device or instrument).
  • a report can further be "transmitted” or "communicated” (these terms may be used herein interchangeably), such as to the individual who was tested, a medical practitioner (e.g., a doctor, nurse, clinical laboratory practitioner, genetic counselor, etc.), a healthcare organization, a clinical laboratory, and/or any other party intended to view or possess the report.
  • a medical practitioner e.g., a doctor, nurse, clinical laboratory practitioner, genetic counselor, etc.
  • the act of "transmitting” or “communicating” a report can be by any means known in the art, based on the form of the report.
  • reporting can include delivering a report ("pushing") and/or retrieving ("pulling") a report.
  • reports can be
  • transmitted/communicated by such means as being physically transferred between parties (such as for reports in paper format), such as by being physically delivered from one party to another, or by being transmitted electronically or in signal form (e.g., via e- mail or over the internet, by facsimile, and/or by any wired or wireless communication methods known in the art), such as by being retrieved from a database stored on a computer network server, etc.
  • an individual may be identified as predisposed to developing metabolic syndrome.
  • the individual may be counseled by a medical personnel to do one or more of the following: 1 ) modify the diet of the individual, e.g., to reduce caloric intake, to alter the quality and quantity of the diet, etc.; 2) prescribe an exercise regimen; and 3) undergo frequent monitoring to monitor one or more of: a) fasting blood glucose levels; b) triglyceride levels; c) HDL levels; d) blood pressure; and e) BMI.
  • the individual may be advised to undergo treatment to ameliorate one or more manifestations of metabolic syndrome, e.g., reducing fasting blood glucose levels, reducing triglyceride levels, increasing HDL levels, reducing blood pressure, and reducing BMI.
  • the present disclosure provides reagents, devices, and kits for detecting SIRT3 SNPs associated with increased risk of developing metabolic syndrome. Although particularly suited for screening for risk of developing metabolic syndrome in a subject, it will be understood that in certain embodiments these reagents, devices and kits can be used for analysis of SIRT3 polymorphisms for any purpose, including research applications.
  • the present disclosure provides SNP detection reagents for detecting a metabolic syndrome-associated SNP in a human SERT3 gene.
  • a subject SNP detection reagent includes an allele-specific probe, and allele-specific primer, and a primer pair that specifically amplifies a region in a human SERT3 gene that contains a metabolic syndrome-associated SNP.
  • a subject SNP detection reagent can have a length of from about 15 nucleotides (nt) to about 250 nt, e.g., from about 15 nt to about 20 nt, from about 20 nt to about 25 nt, from about 25 nt to about 30 nt, from about 30 nt to about 40 nt, from about 40 nt to about 50 nt, from abut 50 nt to about 75 nt, from about 75 nt to about 100 nt, from about 100 nt to about 150 nt, from about 150 nt to about 200 nt, or from about 200 nt to about 250 nt.
  • nt nucleotides
  • a subject SNP detection reagent is an allele-specific
  • SIRT3 gene (nucleotide sequences corresponding to SEQ ID NO:4 given):
  • a subject SNP detection reagent can include a pair of allele-specific probes, e.g., where the first member (the "reference” member) of the pair of allele-specific probe includes a "G" at a position corresponding to nt 125 of SEQ ID NO:3, or nt 3365 of SEQ ID NO:4; and where the second member of the pair includes an "A" at a position corresponding to nt 125 of SEQ ID NO:3, or nt 3365 of SEQ ID NO:4.
  • Exemplary, non-limiting allele-specific probe pairs include:
  • a subject SNP detection reagent is an allele-specific
  • exemplary, non-limiting allele-specific primers for analyzing a metabolic syndrome-associated SNP in human SIRT3 include:
  • a subject SNP detection reagent comprises pairs of allele- specific primers, where the first member (the "reference” member) of the pair of allele- specific probe includes a "G” at a position corresponding to nt 125 of SEQ ID NO:3, or nt 3365 of SEQ ID NO:4; and where the second member of the pair includes an "A" at a position corresponding to nt 125 of SEQ ID NO:3, or nt 3365 of SEQ ID NO:4.
  • a subject SNP detection reagent comprises a pair of
  • the first member of the pair is an allele-specific primer, as described above, and the second member of the pair hybridizes to a human SIRT3 gene at a location upstream or downstream of the location to which the first member hybridizes, such that, under standard polymerase chain reaction conditions, the first and the second members of the primer pair amplify a segment of the human SIRT3 gene comprising nucleotide 3365 of SEQ ID NO:4, e.g., the first and the second members of the primer pair amplify a segment of the human SIRT3 gene that includes from 1 nt to 300 nt (e.g., from 1 nt to 5 nt, from 5 nt to 10 nt, from 10 nt to 15 nt, from 15 nt to 20 nt from 20 nt to 25 nt, from 25 nt to 50 nt, from 50 nt to 100 nt, from 100 nt to 200 nt, from 100 nt to
  • Exemplary primer pairs, where the forward primer is an allele-specific primer include: 3' (SEQ ID NO:24)
  • a subject SNP detection reagent comprises a pair of
  • the first member of the primer pair hybridizes to a region that is 5' of nucleotide 3365 of the human SIRT3 gene as set forth in SEQ ID NO:4, where the second member of the primer pair hybridizes to a region that is 3' of of nucleotide 3365 of the human SIRT3 gene as set forth in SEQ ID NO:4, where the such that, under standard polymerase chain reaction conditions, the first and the second members of the primer pair amplify a segment of the human SIRT3 gene comprising nucleotide 3365 of SEQ ID NO:4, e.g., the first and the second members of the primer pair amplify a segment of the human SIRT3 gene that includes from 5 nt to 300 nt 5' (e.g., from 5 nt to 10 nt, from 10 nt to 15 nt, from 15 nt to 20 nt from 20 nt to 25 nt, from 25 nt to 50 nt, from 50
  • primer pairs that amplify a region including the
  • rs l 1246020 polymorphic site include (where the nucleotides correspond to the numbering depicted in Figure 10A):
  • a subject SNP detection reagent can comprise a detectable label, e.g., a radiolabel, a fluorogenic dye, etc.
  • a subject SNP detection reagent is labeled with a fluorogenic reporter dye that emits a detectable signal.
  • Suitable reporter dyes include, but are not limited to, Acridine, AMCA, BODIPY, Cascade Blue, Cy2, Cy3, Cy5, Cy7, Dabcyl, Edans, Eosin, Erythrosin, Fluorescein, 6- Fam, Tet, Joe, Hex, Oregon Green, Rhodamine, Rhodol Green, Tamra, Rox, and Texas Red.
  • Suitable fluorogenic dyes include, e.g., 5-carboxyfluorescein, 6- carboxyfluorescein, 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein, ⁇ , ⁇ , ⁇ ', ⁇ '- tetramethyl-6-carboxy rhodamine, 6-carboxyrhodamine X, 4,7,2',4',5',7'-hexachloro-6- carboxyfluorescein, 4,7,2',4',5',7'-hexachloro-5-carboxyfluorescein, 2',4',5',7'- tetrachloro-5-carboxyfluorescein, 4,7,2',7'-tetrachloro-6-carboxyfluorescein, 1 ',2',7',8'- dibenzo-4,7-dichloro-5-carboxyfluorescein, and 1 ',
  • a subject SNP detection reagent can be further labeled with a quencher dye such as Tamra, e.g., when the reagent is used as a self-quenching probe such as a TaqMan (U.S. Pat. Nos. 5,210,015 and 5,538,848) or Molecular Beacon probe (U.S. Pat. Nos. 5, 1 18,801 and 5,312,728), or other stemless or linear beacon probe (Livak et al., 1995, PCR Method Appl. 4:357-362; Tyagi et al., 1996, Nature Biotechnology 14: 303-308; Nazarenko et al., 1997, Nucl. Acids Res. 25:2516-2521 ; U.S. Pat. Nos. 5,866,336 and 6, 1 17,635).
  • a quencher dye such as Tamra
  • a subject SNP detection reagent is in some embodiments immobilized on a
  • Suitable substrates include, e.g., glass; plastic; paper, nylon, nitrocellulose, or other type of membrane (e.g., which membrane may be in the form of a test strip); a filter; a chip; or any other suitable solid support.
  • Subject reagents include, e.g., allele-specific primers, primer pairs for
  • kits contains a first container containing a probe, primer, or primer pair for a metabolic syndrome- associated SIRT3 gene SNP, as described above, and a second container containing a reference probe, primer, or primer pair, e.g., for detecting the reference allele
  • kits comprising an allele-specific oligonucleotide that hybridizes to a human SIRT3 gene comprising a metabolic syndrome-associated SIRT3 gene SNP.
  • the kits may contain one or more pairs of SIRT3 allele-specific oligonucleotides hybridizing to different forms of a polymorphism.
  • the SIRT3 allele-specific oligonucleotides may include sequences derived from the coding (exons) or non-coding (promoter, 5' untranslated, introns or 3' untranslated) region of the SIRT3 gene.
  • the SIRT3 allele-specific oligonucleotides may be provided immobilized on a substrate.
  • a subject kit can include at least one SIRT3-specific primer that hybridizes to a region spanning or adjacent to a metabolic syndrome-related polymorphism in the human SIRT3 gene.
  • the SIRT3-specific primers may include sequences derived from the coding (exons) or non-coding (promoter, 5' untranslated, introns or 3' untranslated) region of the SIRT3 gene.
  • a subject kit can contain one or more pairs of SIRT3-specific primers that hybridize to opposite strands of nucleic acid adjacent to a metabolic syndrome-associated polymorphism in the SBRT3 gene. In the presence of appropriate buffers and enzymes, the SIRT3-specific primer pairs are useful in amplifying specific polymorphisms in the SIRT3 gene.
  • a subject kit can include, in addition to a SNP detection reagent, one or more biochemical reagents, containers, packages such as packaging intended for commercial sale, substrates to which SNP detection reagents are attached, electronic hardware components, etc.
  • a subject SNP detection kit contains one or more
  • detection reagents and other components e.g., a buffer, enzymes such as DNA polymerases or ligases, chain extension nucleotides such as deoxynucleotide
  • a subject kit can further contain means for determining the amount of a target nucleic acid, and means for comparing the amount with a standard, and can comprise instructions for using the kit to detect the SNP- containing nucleic acid.
  • a subject SNP detection kit can include components that are used to prepare nucleic acids from a test sample for the subsequent amplification and/or detection of a SNP-containing nucleic acid molecule.
  • sample preparation components can be used to produce nucleic acid extracts (including DNA and/or RNA) from any bodily fluids (such as blood, serum, plasma, urine, saliva, phlegm, gastric juices, semen, tears, sweat, etc.), skin, hair, cells (especially nucleated cells; e.g., peripheral blood
  • test samples used in a subject method will vary based on such factors as the assay format, nature of the detection method, and the specific tissues, cells or extracts used as the test sample to be assayed.
  • Methods of preparing nucleic acids and cell extracts are well known in the art and can be readily adapted to obtain a sample that is compatible with the system utilized. Automated sample preparation systems for extracting nucleic acids from a test sample are commercially available.
  • a subject SNP detection kit is a compartmentalized kit.
  • a compartmentalized kit includes any kit in which reagents are contained in separate containers. Such containers include, for example, small glass containers, plastic containers, strips of plastic, glass or paper, or arraying material such as silica. Such containers allow one to efficiently transfer reagents from one compartment to another compartment such that the test samples and reagents are not cross-contaminated, or from one container to another vessel not included in the kit, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another or to another vessel.
  • Such containers may include, for example, one or more containers which will accept the test sample, one or more containers which contain at least one probe or other SNP detection reagent for detecting a subject SNP, one or more containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and one or more containers which contain the reagents used to reveal the presence of the bound probe or other SNP detection reagents.
  • the kit can optionally further comprise compartments and/or reagents for, for example, nucleic acid amplification or other enzymatic reactions such as primer extension reactions, hybridization, ligation, electrophoresis (preferably capillary electrophoresis), mass spectrometry, and/or laser- induced fluorescent detection.
  • kits may also include instructions for using the kit.
  • Exemplary compartmentalized kits include microfluidic devices known in the art (see, e.g., Weigl et al., "Lab-on-a-chip for drug development", Adv Drug Deliv Rev. 2003 Feb. 24; 55(3):349-77).
  • the containers may be referred to as, for example, microfluidic "compartments", “chambers”, or "channels.”
  • Microfluidic devices which can also be referred to as "lab-on-a-chip” systems, biomedical micro-electro-mechanical systems (bioMEMs), or multicomponent integrated systems, are suitable for inclusion in a subject SNP detection kit.
  • Such systems miniaturize and compartmentalize processes such as probe/target hybridization, nucleic acid amplification, and capillary electrophoresis reactions in a single functional device.
  • Such microfluidic devices typically utilize detection reagents in at least one aspect of the system, and such detection reagents may be used to detect a subject SNP.
  • detection reagents may be used to detect a subject SNP.
  • microfluidic systems comprise a pattern of microchannels designed onto a glass, silicon, quartz, or plastic wafer included on a microchip.
  • the movements of the samples may be controlled by electric, electroosmotic or hydrostatic forces applied across different areas of the microchip to create functional microscopic valves and pumps with no moving parts. Varying the voltage can be used as a means to control the liquid flow at intersections between the micro-machined channels and to change the liquid flow rate for pumping across different sections of the microchip. See, for example, U.S. Pat. Nos. 6, 153,073, and 6,156, 181.
  • an exemplary microfluidic system may integrate, for
  • nucleic acid amplification for example, nucleic acid amplification, primer extension, capillary electrophoresis, and a detection method such as laser induced fluorescence detection.
  • a detection method such as laser induced fluorescence detection.
  • nucleic acid samples are amplified, e.g., by PCR.
  • the amplification products are subjected to automated primer extension reactions using ddNTPs (specific fluorescence for each ddNTP) and the appropriate oligonucleotide primers to carry out primer extension reactions which hybridize just upstream of the targeted SNP.
  • ddNTPs specific fluorescence for each ddNTP
  • the primers are separated from the unincorporated fluorescent ddNTPs by capillary electrophoresis.
  • the separation medium used in capillary electrophoresis can be, for example, polyacrylamide, poly(ethylene glycol) or dextran.
  • the incorporated ddNTPs in the single nucleotide primer extension products are identified by laser- induced fluorescence detection.
  • Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pi, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c, subcutaneous(ly); rt, room temperature; and the like.
  • Antibodies used were specific for ATPase subunit a and ⁇ (Invitrogen Molecular Probes, Carlsbad, CA), monoclonal and polyclonal acetyllysine (Cell Signaling
  • mice were used for SCD1 mouse studies. wt (C57B1/6), SERT3KO (C57Bl/6-5iriJ “A ;, SCD 1 KO (fi6A 29-Scdl' m ' Nlam n, The Jackson Laboratory, Bar Harbor, ME), or dKO (C57Bl/6-Sci/7 " " Si>/J "A ), mice were used. Mice were sacrificed at 7:00 h for fed mouse studies, or transferred to a new cage without food for 24 h from 7:00 h to 7:00 h, and then sacrificed for fasted mouse studies.
  • Murine SDR 3 cDNA was cloned into pShuttle-IRES-GFP- 1 vector
  • Adenoviruses were recombined and produced using pAdeasy Adenoviral System (Stratagene/Agilent, Santa Clara, CA). After amplification with Ad-293 as packaging cell line, virus was purified using cesium chloride gradient ultra-centrifugation and dialyzed into PBS plus 10% glycerol as described ⁇ 40). For injection, 3-month old male mice were injected via tail vein with adenovirus over-expression either green fluorescent protein (GFP) (control) or SBRT3 at dose of 5 x 10 9 plaque forming units (PFU)/g body weight, as described ⁇ 41). The mice were monitored for signs of distress, and recovered under observation. On the sixth day after virus injection, the mice were sacrificed and livers were removed and measured for total lipids.
  • GFP green fluorescent protein
  • PFU plaque forming units
  • Glucose and insulin tolerance tests were performed according to The Jackson Laboratory protocol. For glucose tolerance tests, a 200 mg/ml glucose solution was prepared. Mice were fasted 6hs from 7:00 h to 13:00 h, and then glucose injected into the intraperitoneal space (2g /kg body weight). For insulin tolerance tests, mice were fasted 6hs from 7:00 h to 13:00 h, and pre-warmed insulin was injected into the interaperitoneal space at a concentration of 1U insulin/kg body weight. Glucose was monitored at regular intervals up until 2h with a handheld personal glucose monitoring device (Freestyle, Abbott Park, IL). Histology and Microscopy
  • mice were perfused or tissues were dissected and drop- fixed or in fresh 3% paraformaldehyde overnight. The following day, tissues were cryoprotected using a sucrose gradient ( 10% for 1 h, 20% for 2 h and 30% overnight). After cryoprotection, tissue was placed in OCT (Tissue Tek 4583) in a peel-away mold (Ted Pella, Redding, CA) and frozen using dry-ice-cooled isopentane (M32631 , Sigma- Aldrich, St. Louis, MO), and stored at -80°C.
  • OCT tissue Tek 4583
  • a peel-away mold Ted Pella, Redding, CA
  • M32631 dry-ice-cooled isopentane
  • Specimens were placed at -20°C for l h to equilibrate and 8- ⁇ sections were cut onto charged slides (Snowcoat X-tra, Surgipath, Richmond, IL). Slides were dried at RT for 5 min then stored at -20°C. For ORO staining, slides were brought to room temperature, and washed in running water for 10 min, to remove OCT. They were placed in 50% isopropanol for 3 minutes, followed by a further 3 min in 100% isopropanol and into 0.5% ORO (O-0625, Sigma-Aldrich, St. Louis, MO) in 100% isopropanol (398039-2L, Sigma-Aldrich, St. Louis, MO) for 2 h.
  • ORO O-0625, Sigma-Aldrich, St. Louis, MO
  • Specimens were then differentiated in three 3-min 85% isopropanol washes and running water for 10 min. Specimens were then counterstained with Mayer's hematoxylin (American Mastertech HXMMHGAL, Lodi, CA) for 10 sec, followed by bluing in running water for a further 10 min. Slides were mounted using crystal mount (American Mastertech MMC00168E, Lodi, CA) and left to dry overnight.
  • Inflammation was graded based on the average number of inflammatory aggregates in 10X field: none, 0; minimal ( ⁇ 1 inflammatory aggregate), 1 ; mild (1 inflammatory aggregate), 2; moderate (2-3 inflammatory aggregates), 3; severe (>3 inflammatory aggregates), 4.
  • Steatosis was graded based on the percent of overall fat in normal areas: none, 0; minimal ( ⁇ 5%), 1 ; mild (5-33%), 2; moderate (33-66%), 3; severe (66- 100%), 4.
  • Hepatocyte ballooning was graded based on the prevalence of cells: rare, 1 ; mild, 2; moderate, 3; severe, 4.
  • Fibrosis was graded based on the location and severity of collagen: centrizonal, 1 ;
  • centrizonal and periportal 2; bridging, 3; cirrhosis, 4.
  • electrochemiluminescent immunoassay technology from Meso Scale Discovery (MSD, Gaithersburg, MD). Measurements were performed with an MSD SI-2400 imager for the determination of multiplex panels of analytes in 96-well plates using reagent kits from MSD. Assays included a seven-plex mouse proinflammatory panel for IFN- ⁇ , IL- 10, IL- 12p70, IL- ⁇ ⁇ , IL-6, TNF-a, and KC chemokine; a biplex mouse metabolic panel for insulin and leptin; and single panels for mouse adiponectin and resistin. Additional measurements were performed by conventional spectrophotometric methodology on a Beckman-Coulter DxC600 autoanalyzer.
  • acylcarnitines esterified with hot, acidic methanol (acylcarnitines) or n-butanol (amino acids), and then analyzed by tandem mass spectrometry (Quattro Micro, Waters Corporation, Milford, MA). Acylcarnitines were assayed by adapting described methods for analysis of amino acids in dried blood spots (42, 43).
  • Lipid ester bands were scraped from the TLC plates and methylated using BF3/methanol as described by Morrison and Smith (47).
  • acyl-CoA measurements hepatic acyl CoA esters were extracted, analyzed and purified based on previously published methods (48-50).
  • the acyl CoAs were analyzed by flow injection analysis using positive electrospray ionization on Quattro micro, triple quadrupole mass spectrometer (Waters, Milford, MA) employing methanol/water (80:20, v:v) containing 30 mM ammonium hydroxide as the mobile phase.
  • Spectra were acquired in the multichannel acquisition mode monitoring the neutral loss of 507 amu (phosphoadenosine diphosphate) and scanning from m/z 750- 1060.
  • Heptadecanoyl CoA and 13C3 malonyl CoA were employed as internal standards for the long and short chain CoA esters, respectively.
  • the endogenous CoAs were quantified using calibrators prepared by spiking liver homogenates with authentic CoAs (Sigma-Aldrich, St.
  • the samples were prepared using Affymetrix WT cDNA Synthesis and Amplification Kits and WT Target Labeling and Control Reagents according to the manufacturer's instructions. Labeled cDNA samples were hybridized, stained, and scanned to Affymetrix Mouse Gene 1.0 ST arrays according to manufacturer's instructions.
  • Raw intensities from the CEL files were analyzed using Affymetrix Power Tools (APT, version 1.10.1 ) to generate an RMA [robust multi-array average (57)] intensity on a log2 scale for each probe set and various quality metrics.
  • the perfect match (PM) intensities per probe set were defined as (i) background corrected; (ii) quantile-normalized (to make the distribution of intensities the same for all arrays); and (iii) summarized for each probe set using a robust fit of linear models.
  • RNA (2 ⁇ g) was reverse transcribed with Superscript III reverse transcriptase and oligo(dT) primers (Invitrogen, Carlsbad, CA) to generate cDNA.
  • PCR Real-time quantitative polymerase chain reaction
  • PCR reaction mix contained 0.6 ⁇ of cDNA, 5 ⁇ of 2x SYBR green master mix (Qiagen, Germantown, MD), and 10 pmol of each primer. Relative mRNA abundance was normalized to the internal standard cyclophilin. Oligonucleotide primers were designed using qPrimerDepot, NCI, NIH. Primer sequences for various genes are listed in Table 1. Table 1
  • R S- GACCACCACTCCAGAAGCAT -3' (SEQ ID NO:31)
  • Agpat7 F 5'- CTACAGAAGGCTGGGCTGTC -3'
  • Cyclophilin F 5'- TGGAAGAGCACCAAGACAGACA -3'
  • Gpat2 R 5'- GTCTCCGAAAGACAGCCAAG -3'
  • Lipinl R 5'- TTCACCGTCACAAACACCTG -3'
  • Lipin2 R 5'- GCCCACATAATTCATGGTTTG -3'
  • Lipin3 R 5'- CTACTGTGGGACCCTTGGAC -3'
  • Rl 5'- CAGCCGAGCCTTGTAAGTTC -3' (SEQ ID NO:55)
  • R2 5'- CAGCCGTGCCTTGTAAGTTC -3' (SEQ ID NO:57)
  • R2 5'- ACGTCATCTGGGACATAGGC -3' (SEQ ID NO:61 )
  • HSL R 5'- TCTACCACTTTCAGCGTCACCG -3'
  • Results are given as the mean + standard error.
  • Statistical analyses represent a one-tailed Students t-test or a Wilcoxon rank-sum test and null hypotheses were rejected at 0.05.
  • linear models were fit for each gene to estimate cell- type effects and associated significance using the limma package (R/Bioconductor, Smyth 2008). Moderated t-statistics arid the associated p-values were calculated, as well as B-statis ' tics (logOdds), the log posterior odds ratio.
  • P-values were adjusted for multiple testing by controlling for false-discovery rate (FDR) using the Benjamini- Hochberg method (52) and controlling for family-wise error rate using the Bonferroni correction (adjP).
  • the design was a cross-sectional cohort study of nonalcoholic fatty liver disease.
  • NAFLD CRN Nonalcoholic Steatohepatitis Clinical Research Network
  • NIDDK National Institute of Diabetes & Digestive and Kidney Diseases
  • Demographic and clinical characteristics are presented in Table 5.
  • Demographic information collected during screening interviews as part of the registration process included age, sex, waist circumference, weight and height.
  • Body mass index (BMI) was calculated as the weight (kg) divided by the height (meters) squared.
  • Comorbid conditions i.e., hypertension, type 2 diabetes
  • Smoking status was determined by self-report.
  • Baseline lipid measurements were obtained by peripheral venipuncture after a minimum of 10 hours of fasting.
  • Serum was used to determine alanine aminotransferase (ALT, U/L), aspartate aminotransferase (AST, U/L), gamma glutamyl transferase (GGT, U L), alkaline phosphatase (ALP, U/L), glucose (mg/dL), insulin (mg/dL), total plasma triglyceride (TG, mg/dL), total plasma cholesterol (TC, mg/dL), low density lipoprotein cholesterol (LDLC, mg dL) and high density lipoprotein cholesterol (HDLC, mg/dL) using the clinical laboratories at each study site. Low-density lipoprotein cholesterol (LDL) was calculated using the Friedewald Formula.
  • the IDF criteria were refined by excluding participants with type 2 diabetes.
  • Histology data are presented in Table 5.
  • the NASH CRN Pathology Committee developed and validated a feature-based histological scoring system that encompasses the spectrum of lesions of NAFLD (55). Liver biopsy slides from subjects were read centrally by the Pathology Committee during which biopsies were rigorously evaluated according to the published scoring system (55). Steatosis grade was scored according to amount (%) of biopsy occupied using a four-point scale. A diagnosis of NAFLD required the presence of > 5% steatosis.
  • Blood collection and genotyping Blood samples were obtained by venipuncture and genomic DNA was extracted from peripheral blood lymphocytes (Invitrogen, Carlsbad, CA). Genotyping was performed blinded to clinical status; positive and negative controls were included. DNA samples were quantitated with a Nanodrop Spectrophotometer (ND- 1000) and normalized to a concentration of 50 ng/ L (diluted in 10 mM Tris/1 mM EDTA). Samples were genotyped by TaqMan allelic discrimination assay (Applied Biosystems, Foster City, CA) and processed according to the standard protocol.
  • ND- 1000 Nanodrop Spectrophotometer
  • Phenotyping http://genetics(dot)bwh(dot)harvard(dot)edu/pph/), a tool which predicts possible impact of an amino acid substitution on the structure and function of a human protein using empirical rules applied to the sequence, in addition to phylogenetic and structural information characterizing the substitution.
  • Rigorous quality control (QC) procedures were adopted to ensure high quality data for downstream analyses.
  • the first QC step was to exclude SNPs with poor quality data.
  • SNP call rate was set at 95%, resulting in the exclusion of one SNP (rs l 1246007).
  • the second QC step was to excluded SNPs with a minor allele frequency (MAF) less than 5% from subsequent analyses; 2 SNPs were excluded (rs3020901 and rs551570).
  • the third QC step involved the inclusion of replicate samples.
  • rsID reference sequence identifier
  • Position nucleotide
  • S P single nucleotide polymorphism
  • Intercooled Stata 9.2 for Windows was used for statistical analysis. Descriptive statistics and frequency distributions were generated on the sample demographic and clinical characteristics, and biochemical measurements.
  • Allele and genotype frequencies were determined by gene counting. Measures of linkage disequilibrium, D' and r2, were computed from the genotypes with Haploview 4.2 (http://www(dot)broad(dot)mit(dot)edu/mpg/haploview ). LD-based haplotype block definition was based on D' confidence interval (55). Gene structure for SIRT3 was rendered with FancyGene 1.4 (genomic sequence accession NM_012239). [00201] For association tests, four genetic models were assessed for each SNP: dominant, recessive, log additive, and codominant.
  • Permutation tests were used to adjust the type-I error rate against inflation due to testing of multiple SNPs. To account for multiple comparisons, outcome status was permuted 10,000 times to determine the likelihood that our findings were due to chance. Permutation analyses were done using Intercooled Stata 9.2 for Windows.
  • haplotype analyses were conducted in order to localize the association signal within the gene and to determine if haplotypes improved the strength of the association with the outcome. Haplotypes were constructed using the program PHASE version 2.1 (59)
  • haplotype construction procedure was repeated 5 times using different seed numbers with each cycle; only haplotypes that were inferred with probability estimates greater than or equal to 0.9 were retained for downstream analyses. Haplotypes with frequency estimates of 1 % or less were grouped into a single category.
  • mitochondria were lysed by sonication and resuspended in a low-stringency immunoprecipitation (IP) buffer (0.05% non-ionic detergent NP-40, 50 mM NaCl, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris-HCl, pH 7.4, 10 mM nicotinamide, 1 ⁇ trichostatin A, protease inhibitor cocktail (Roche)).
  • IP immunoprecipitation
  • Deacetylase assays were performed in 100 ⁇ of deacetylase buffer (4 mM, MgCl 2 , 0.2 mM dithiothreitol, 50 mM Tris-HCl, pH 8.5) containing 50 ng of recombinant SIRT3, NAD + and [ 3 H] histone H4 peptide substrate.
  • the substrate was prepared by in vitro acetylation of a histone H4 N-terminal peptide (amino acids 1-25) with radiolabeled acetyl-CoA and recombinant PCAF (Heltweg et al. (2005) Methods 36:332).
  • Deacetylation reactions were conducted at 37 °C under gentle agitation, and stopped by adding 25 ⁇ of stop solution (0.2 M HC1, 0.32 M acetic acid). Radioactivity was extracted into 500 ⁇ ethyl acetate by vortexing for 15 s. After centrifugation at 14,000g for 5 min, 450 ⁇ of the ethyl acetate fraction was mixed with 5 ml of scintillation fluid (Perkin Elmer), and the radioactivity was measured with a liquid scintillation counter (Beckman LS6000).
  • Mitochondrial protein acetylation is regulated by high-fat diet feeding (Fig. 1A).
  • Western blot analysis of hepatic mitochondrial extracts with an anti- acetllysine antibody revealed that chronic high fat feeding (13 weeks) but not acute high fat feeding (1 week) induced global mitochondrial protein acetylation.
  • High-fat diet feeding accelerates the development of metabolic abnormalities, including central obesity, insulin resistance, hyperlipidemia, hyperglycemia, hypertension, and hepatic steatosis, defined as the metabolic syndrome ⁇ 10). Because high-fat diet feeding leads to mitochondrial protein hyperacetylation, the possibility that the major nicotinamide adenine dinucleotide (NAD + )-dependent mitochondrial protein deacetylase, SIRT3
  • SIRT3 is reduced with chronic high-fat diet feeding.
  • SIRT1 -SIRT7 the primary regulator of mitochondrial protein acetylation and mice lacking SIRT3 have hyperacetylated mitochondrial proteins (3).
  • SAIRT1 -SIRT7 the primary regulator of mitochondrial protein acetylation and mice lacking SIRT3 have hyperacetylated mitochondrial proteins (3).
  • SART3 expression was initially increased in response to a one-week high-fat diet feeding in wt mice (Fig. 1 B) and mitochondria protein acetylation was unchanged (Fig. I B).
  • hepatic SIRT3 was suppressed with chronic high-fat diet feeding ( 13 weeks) compared to a standard diet (Fig. I B). Additionally, the acetylation level of a specific SIRT3 target, LCAD, was significantly increased in wt mice fed chronically on a high-fat diet. To test whether this
  • SIRT3KO mice develop diet-induced obesity and insulin resistance.
  • SIRT3KO mice which show constitutive mitochondrial protein hyperacetylation (3, 4), were placed on a high-fat diet. No early differences in weight were noted between wt and SIRT3KO mice (Fig. 2A).
  • SIRT3KO mice developed diet-induced obesity at an accelerated rate when maintained on a high-fat diet, (Fig. 2A).
  • Dual energy X-ray absorptiometry (DEXA) analyses showed that the increased weight in SIRT3KO mice was due to increased adiposity.
  • Insulin resistance is a hallmark of obesity and the metabolic syndrome (10, 13, 14).
  • Glucose tolerance and insulin sensitivity were measured in wt and SIRT3KO mice.
  • Obese 12-month-old SIRT3KO mice fed a high-fat diet exhibited hyperglycemia during glucose-tolerance testing and were insulin resistant by insulin tolerance testing (Fig. 2D, 2E).
  • glucose and insulin tolerance were measured in non-obese 12-month old standard diet fed wt and SERT3KO mice.
  • SIRT3KO mice but not organic acids or amino acids. These results are consistent with the previously demonstrated reduced fatty acid oxidation in SIRT3KO mouse livers (4).
  • SIRT3 deficiency reduces fatty acid oxidation and results in
  • hepatic lipids an effort was made to determine if SIRT3 overexpression is protective against hepatic lipid accumulation.
  • Recombinant adenoviruses containing the cDNA encoding Sirt3, or green fluorescent protein (GFP) as a control were injected into the tail veins of wt mice, and hepatic tissue homogenates were assessed for total lipid levels.
  • Hepatic triglyceride levels were 50% lower in wt mice injected with SIRT3- expressing adenovirus than in mice injected with the GFP-expressing virus (Fig. 3C).
  • Fig. 3D lymphoplasmacytic inflammation
  • SIRT3KO mice develop hyperlipidemias with high-fat diet feeding. Obesity, insulin resistance, and the metabolic syndrome often coincide with lipid abnormalities, including hypertriglyceridemia, hypercholesterolemia, and other dyslipidemias ⁇ 10).
  • Serum lipid measurements from 12-month-old SIRT3KO mice fed a high-fat diet revealed higher levels of triglyceride (97% increase) and cholesterol ( 141 % increase) than in wt mice (Table 4). Higher levels of low-density lipoproteins (LDL, 60%
  • VLDL very-low-density lipoproteins
  • Insulin (ng/ml) 0.7 ⁇ 0.2 2.7 ⁇ 0.9 ⁇ 0.05
  • Adiponectin (ng/ml) 15.6 ⁇ 0.4 14.9 ⁇ 0.9 n.s.
  • Resistin ( g/ml) 8.6 ⁇ 1.6 8.8 + 2.1 n.s.
  • HDL high-density lipoprotein
  • LDL low-density lipoprotein
  • VLDL very-low- density lipoprotein
  • SCD1 stearoyl-CoA desaturase 1
  • SCD 1 is a fatty acid synthesis enzyme that catalyzes the biosynthesis of monounsaturated long-chain acyl CoAs from saturated long-chain acyl CoAs (77).
  • SCD l has been implicated in the pathogenesis of the metabolic syndrome in mice (78) and humans (79).
  • Increased SCD l mRNA abundance was independently validated using quantitative RT-PCR and a fivefold increase in mRNA was detected in SIRT3KO mice in comparison to wt mice (Fig. 4A).
  • SCD l was the only mRNA for lipogenic genes whose expression was increased (Fig. 4A).
  • the plasma desaturation index was measured. This index represents the ratio of serum palmitoleate:palmitate ( 16: 1/16:0) or oleate:stearate ( 18: 1/18:0) and is a well- documented marker for SCD l activity (18).
  • the free fatty acid desaturation indexes for C I 6: 1 /C I 6:0 and C 18: l/C 18:0 were increased (213% and 62%, respectively) (Fig. 4B).
  • Triglyceride desaturation indices were also increased (66% for triglyceride C 16: l/C 16:0) (Fig. 4B).
  • mice lacking SCDl are protected from hepatic steatosis (20), mice lacking both SIRT3 and SCDl (dKO) were generated.
  • mice lacking both SIRT3 and SCDl were generated.
  • Fig. 4C no differences in hepatic lipids were observed between wt, SIRT3KO, SCD1 KO or dKO mice.
  • high-fat diet-induced hepatic steatosis was observed in wt mice and was exacerbated in SIRT3KO mice as described above.
  • Hepatic steatosis was absent in SCD 1 KO mice fed a high-fat diet, as previously reported (27).
  • mice lacking both SIRT3 and SCDl showed markedly reduced hepatic triglycerides with high-fat diet feeding (Fig. 4C), demonstrating loss of SCDl ameliorates hepatic steatosis induced by SIRT3 deficiency and mitochondrial protein hyperacetylation.
  • SNP single nucleotide polymorphism
  • Genotype frequencies for 13 SIRT3 SNPs were measured, seven of which passed all quality control criteria (Table 3).
  • the strength of these associations was virtually unchanged following adjustment for age, sex, and BMI.
  • GGT Gamma glutamyl transferase
  • NAFLD Nonalcoholic fatty liver disease
  • haplotype analysis of the haplotype block was performed to refine the association signal and to determine if either SNP was a surrogate of the other.
  • the four-SNP haplotypes did not improve the association signal (Fig. 5B).
  • rs7934919 appears to be a surrogate of rs l 1246020 given the associations detected were strongest for rs l 1246020, and adjusting for rs7934919 in multivariable analyses completely attenuated the association signal.
  • Fig. 1 Chronic high-fat diet feeding results in global mitochondrial
  • Mitochondria were isolated from livers of wt mice fed a standard or high-fat diet for 1 week, 5 weeks or 13 weeks (Jackson Laboratory) and analyzed for SIRT3 expression by western blot analysis with an antiserum specific for anti-SIRT3; electron transfer flavoprotein was used as a reference.
  • Fig. 2 Mice lacking SIRT3 develop diet-induced obesity and insulin resistance.
  • D, E 12-month old SERT3 O and wt mice fed a high-fat diet were tested for glucose (D) and insulin tolerance (E) and measured for blood glucose levels; inset data represent area under the curve (AUC).
  • FIG. 4 SIRT3KO mice have high expression and activity of hepatic SCD 1.
  • TG triglyceride
  • PL phospholipids
  • FFA free fatty acids
  • A Heat map detailing the pairwise LD among the 7 SNPs spanning the SIRT3 coding region; the pairwise correlations (D') are rendered within each diamond with greater LD reflected by darker shades of gray; SIRT3 gene structure is depicted above the LD heat map: eons are depicted in gray boxes, introns as connecting black lines, and untranslated regions as smaller boxes shades in pink;
  • flanking DNA sequence approximately 10 kbp of flanking DNA sequence was included in the tagSNP selection procedure; tagSNPs the promoter region is included in the diagram to the right of the transcription start site indicated by the arrow found directly above the SIRT3 gene structure diagram; SNPs rendered in bold are included in the haploblock outlined in the black triangle; the two SNPs that encode for nonsynonymous polymorphisms
  • rs l 1246020 and rs28365927 are rendered in green font color.
  • B Haplotype frequency estimates, inferred from rs l 023430, rs7934919, rs l 1246020, and rs28365927, reading from left to right in the "haplotype” column.
  • C Differences in clinical phenotypes by SIRT3 rsl 1246020 genotype.
  • P p- value for the test statistic
  • OR (95% CI), the odds ratio and 95% confidence interval for the test statistic
  • UNADJ unadjusted
  • ADJ adjusted for age, sex, and BMI unless otherwise indicated
  • NAS NAFLD Activity Score
  • * p-va ⁇ ue for the overall model i.e. steatosis, steatohepatitis, cirrhosis
  • Metabolic syndrome criteria based on the IDF guidelines, excluding the presence of type 2 diabetes (D) Working model.
  • SIRT3 functions to deacetylate mitochondrial proteins, and increase fatty acid oxidation and energy production.
  • Functional SNP in SIRT3 causes a point mutation and reduces enzymatic activity.
  • the non-synonymous point mutation encoded by rs l 1246020 results in a change of valine to isoleucine at residue 208 of the SIRT3 polypeptide.
  • the V208I polymorphism lies within the conserved catalytic deacetylase domain of SIRT3 ( Figure 6A and Frye et al. (2000) Biochem. Biophys. Res. Commun. 273:793) and could therefore affect its enzymatic activity.
  • recombinant wt SIRT3, SD T3-V208I and catalytically-inactive SIRT3-H248Y were expressed in E. coli; and their deacetylase activity in vitro was tested.
  • FIG. 6 The rs l 1246020 SNP in the human SIRT3 gene encodes a point- mutation and reduces SIRT3 enzymatic activity.
  • A Schematic of SIRT3 protein; mitochondrial targeting sequence (MTS), mitochondrial processing peptidase (MPP) site.
  • B-D Steady-state kinetic analyses of SIRT3 activity; rates of activity were measured as a function of [NAD + ] (B) or [ 3 H-histone H4 peptide] (C), as measured by organic-soluble radioactive signal;
  • E Protein levels of wt, V208I, and H248Y SIRT3 were assessed for three independent preparations (Prep 1 -3) to measure enzyme kinetic parameters.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Pathology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé de détection d'un polymorphisme mononucléotidique (SNP) associé au syndrome métabolique chez un individu ; et un procédé de détection d'une prédisposition d'un individu à développer un syndrome métabolique. La présente invention concerne en outre des réactifs acides nucléiques et des kits destinés à déterminer le génotype d'un sujet par rapport à un polymorphisme mononucléotidique au niveau du rs11246020 à l'intérieur d'un gène sirtuin-3 (SIRT3).
PCT/US2011/047632 2010-08-20 2011-08-12 Compositions et procédés de détermination de la prédisposition à développer un syndrome métabolique Ceased WO2012024189A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US37558510P 2010-08-20 2010-08-20
US61/375,585 2010-08-20

Publications (2)

Publication Number Publication Date
WO2012024189A2 true WO2012024189A2 (fr) 2012-02-23
WO2012024189A3 WO2012024189A3 (fr) 2012-05-31

Family

ID=45605616

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2011/047632 Ceased WO2012024189A2 (fr) 2010-08-20 2011-08-12 Compositions et procédés de détermination de la prédisposition à développer un syndrome métabolique

Country Status (1)

Country Link
WO (1) WO2012024189A2 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014006231A1 (fr) * 2012-07-06 2014-01-09 Randox Laboratories Ltd Association de variant génétique du facteur de croissance de l'endothélium vasculaire avec syndrome métabolique
US10172915B2 (en) 2013-10-20 2019-01-08 Duke University Methods and compositions for activation of sirtuins with Annexin A1 peptides
CN112689762A (zh) * 2018-06-01 2021-04-20 美国控股实验室公司 用于lc-ms/ms蛋白质组学基因分型的方法和系统

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2021502A4 (fr) * 2006-05-09 2010-08-25 Mas Metabolic Analytical Servi Gènes et marqueurs atypiques dans le diabète de type 2 et l'obésité

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014006231A1 (fr) * 2012-07-06 2014-01-09 Randox Laboratories Ltd Association de variant génétique du facteur de croissance de l'endothélium vasculaire avec syndrome métabolique
US10172915B2 (en) 2013-10-20 2019-01-08 Duke University Methods and compositions for activation of sirtuins with Annexin A1 peptides
CN112689762A (zh) * 2018-06-01 2021-04-20 美国控股实验室公司 用于lc-ms/ms蛋白质组学基因分型的方法和系统
US12163963B2 (en) 2018-06-01 2024-12-10 Laboratory Corporation Of America Holdings Methods and systems for LC-MS/MS proteomic genotyping

Also Published As

Publication number Publication date
WO2012024189A3 (fr) 2012-05-31

Similar Documents

Publication Publication Date Title
US11408034B2 (en) Genetic polymorphisms associated with cardiovascular diseases, methods of detection and uses thereof
JP6448149B2 (ja) 肝線維症に関連する遺伝的多型、その検出方法および使用
JP5825788B2 (ja) 脳卒中に関連した遺伝子遺伝子多型およびその検出方法および使用
Lees et al. Analysis of germline GLI1 variation implicates hedgehog signalling in the regulation of intestinal inflammatory pathways
EP2013365B1 (fr) Utilisation d'ornithine transcarbamylase (otc), en tant que marqueur de diagnostic de traumatismes cérébraux
JP2016054738A (ja) 心臓血管障害および薬物応答に関連した遺伝子多型、それらの検出方法および用途
WO2008003826A1 (fr) Nouveaux gènes et marqueurs dans l'hypertension arterielle essentielle
Chen et al. UGT1A1 sequence variants associated with risk of adult hyperbilirubinemia: a quantitative analysis
Fu et al. Loss-of-function mutation in the X-linked TBX22 promoter disrupts an ETS-1 binding site and leads to cleft palate
BRPI0809143B1 (pt) método in vitro para identificar um indivíduo que apresente um risco alterado para desenvolver doença cardíaca coronária (chd) ou aneurisma/dissecção
WO2012024189A2 (fr) Compositions et procédés de détermination de la prédisposition à développer un syndrome métabolique
Chen et al. AluYb8 insertion in the MUTYH gene is related to increased 8-OHdG in genomic DNA and could be a risk factor for type 2 diabetes in a Chinese population
Banno et al. Association of genetic polymorphisms of endothelin-converting enzyme-1 gene with hypertension in a Japanese population and rare missense mutation in preproendothelin-1 in Japanese hypertensives
Ali et al. Association study of Klotho gene polymorphism with calcium oxalate stones in the Uyghur population of Xinjiang, China
US20150315649A1 (en) Method for diagnosing and assessing risk of pancreatitis using genetic variants
US20080275728A1 (en) Method for Personalized Diet Design for Females of Asian Origin
Xaubet et al. A haplotype of cyclooxygenase-2 gene is associated with idiopathic pulmonary fibrosis
JP5904501B2 (ja) ２型糖尿病の検出方法
US20140206002A1 (en) Methods of Diagnosing Breast Cancer
Kushioka et al. Evaluation of ApoE genotyping using saliva-derived DNA
US20110177963A1 (en) Variation in the CHI3L1 Gene Influences Serum YKL-40 Levels, Asthma Risk and Lung Function
Sumaya et al. Study on the association of Glutathione S-transferase Mu 1 (GSTM1) gene polymorphism with acute decompensated heart failure with reduced ejection fraction
Wendy et al. Association between the CYP24A1 rs2762939 polymorphism and vascular calcification in Indonesian patients with chronic kidney disease on hemodialysis
EP2823054B1 (fr) Facteurs génétiques pour la pression sanguine
van der Vleuten Familial Combined Hyperlipidemia. A complex multifactoral lipid disorder.

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11818601

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11818601

Country of ref document: EP

Kind code of ref document: A2