EP1601972A2 - Association de proteines polymorphes d'ancrage de kinase a des phenotypes cardiaques et procedes correspondants - Google Patents

Association de proteines polymorphes d'ancrage de kinase a des phenotypes cardiaques et procedes correspondants

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Publication number
EP1601972A2
EP1601972A2 EP04718045A EP04718045A EP1601972A2 EP 1601972 A2 EP1601972 A2 EP 1601972A2 EP 04718045 A EP04718045 A EP 04718045A EP 04718045 A EP04718045 A EP 04718045A EP 1601972 A2 EP1601972 A2 EP 1601972A2
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European Patent Office
Prior art keywords
seq
subject
nucleotide
interval
ekg
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English (en)
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Andreas Braun
Stefan M. Kammerer
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Sequenom Inc
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Sequenom Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels

Definitions

  • Protein phosphorylation is an important mechanism for enzyme regulation and the transduction of extracellular signals across the cell membrane in eukaryotic cells.
  • a wide variety of cellular substrates including enzymes, membrane receptors, ion channels and transcription factors, can be phosphorylated in response to extracellular signals that interact with cells.
  • a key enzyme in the phosphorylation of cellular proteins in response to hormones and neurotransmitters is cyclic AMP (cAMP)-dependent protein kinase (PKA).
  • cAMP cyclic AMP
  • PKA cyclic AMP
  • PKA Upon activation by cAMP, PKA thus mediates a variety of cellular responses to such extracellular signals.
  • An array of PKA isozymes are expressed in mammalian cells.
  • the PKAs usually exist as inactive tetrameres containing a regulatory (R) subunit dimer and two catalytic (C) subunits.
  • R regulatory
  • C catalytic
  • Genes encoding three C subunits (C , C ? and Cy) and four R subunits (Rl ⁇ , R ⁇ , Rll ⁇ and Rll ?) have been identified (see Takio et a/. (1 982) Proc. Nail. Acad. Sci. U.S. A. 75:2544-2548; Lee et a/. (1 983) Proc. Nat/. Acad. Sci. U.S. A. 50:3608-361 2; Jahnsen et a/. ( 1 996) J. Biol. Chem. 261 : 1 2352-1 2361 ; Clegg et a/. (1 988) Proc. Nat/. Acad. Sci. U.S. A. 55:3703-3707; and
  • AKAPs A-kinase anchoring proteins
  • Anchoring not only places the kinase close to preferred substrates, but also positions the PKA holoenzyme at sites where it can optimally respond to fluctuations in the second messenger cAMP (Mochly-Rosen (1 995) Science 255:247-251 ; Faux and Scott (1 996) Trends Biochem. Sci. 27:31 2-31 5; Hubbard and Cohen ( 1 993) Trends Biochem. Sci. 75: 1 72-1 77) .
  • Rll subunits of PKA bind to AKAPs with nanomolar affinity (Carr et al. (1 992) J. Biol. Chem. 257: 1 3376-1 3382), and many AKAP-RII complexes have been isolated from cell extracts.
  • Rl subunits of PKA bind to AKAPs with only micromolar affinity (Burton et al. (1997) Proc. Nat/. Acad. Sci. U.S.A. 54: 1 1067-1 1072) .
  • Evidence of binding of a PKA Rl subunit to an AKAP has been reported (Miki and Eddy (1 998) J. Biol. Chem 273:34384-
  • AKAPs More than 20 AKAPs have been reported in different tissues and species.
  • Complementary DNAs (cDNAs) encoding AKAPs have been isolated from diverse species, ranging from Caenorhabditis elegans and Drosophilia to human (see, e.g. , Colledge and Scott (1 999) Trends Cell Biol. 5:21 6-221 ) .
  • Regions within AKAPs that mediate association with Rll subunits of PKA have been identified. These regions of approximately 10-1 8 amino acid residues vary substantially in primary sequence, but secondary structure predictions indicate that they are likely to form an amphipathic helix with hydrophobic residues aligned along one face of the helix and charged residues along the other (Carr et al.
  • AKAPs also have the ability to bind to multiple proteins, including other signaling enzymes.
  • AKAP79 binds to PKA, protein kinase C (PKC) and the protein phosphatase calcineurin (PP2B) (Coghlan et al. (1 995) Science 257: 108-1 1 2 and Klauck et al. ( 1 996) Science 277: 1 589-1 592) . Therefore, the targeting of AKAP79 to neuronal postsynaptic membranes brings together enzymes with opposite catalytic activities in a single complex.
  • PKA protein kinase C
  • P2B protein phosphatase calcineurin
  • AKAPs thus serve as potential regulatory mechanisms that increase the selectivity and intensity of a cAMP-mediated response. There is a need, therefore, to identify and elucidate the structural and functional properties of AKAPs in order to gain a complete understanding of the important role these proteins play in the basic functioning of cells.
  • the methods include assessment of the presence or absence of an allele of the an AKAP gene and other aspects, including determining EKG features, or methods in which the AKAP gene is predictive of treatment outcome or response.
  • Methods provided herein include steps of conducting an EKG examination; determining the EKG-PR-interval in the subject. If the EKG-PR-interval is decreased, then identity of an amino acid present in the subject at position 646 of AKAP1 0/D-AKAP2 (SEQ ID NO:2) or a nucleotide present at position corresponding to nucleotide 2073 of SEQ ID NO: 1 is assessed.
  • the presence of Val at position 646 of SEQ ID NO:2 or the presence of a -G- at nucleotide position 2073 of SEQ ID NO: 1 indicates increased susceptibility to a disease or disorder.
  • the disease or disorder can be selected from among a variety of diseases and discorders, including, but are not limited to, cardiovascular disorders, cardiac disease, proliferative disorders, neurological disorders, neurodegenerative disorders, obesity, diabetes and peripheral retinopathies.
  • the EKG-PR-interval in the subject can be compared to a predetermined age-matched standard EKG-PR-interval to determine whether it is decreased.
  • Also provided herein are methods of assessing the susceptibility of a subject to a disease or disorder associated with the cardiovascular system comprising determining the amino acid at position 646 of AKAP1 0/D-AKAP2 (SEQ ID NO:2) or the nucleotide present at position corresponding to nucleotide 2073 of SEQ ID NO: 1 , wherein the presence of Val at position 646 of SEQ ID NO:2 or the presence of a -G- at nucleotide position 2073 of SEQ ID NO: 1 , indicates increased susceptibility to a disease or disorder associated with the cardiovascular system.
  • Also provided herein are methods of diagnosing a disease or disorder associated with the cardiovascular system comprising detecting the presence of Val at 646 of D-AKAP2 (SEQ ID NO:2) or the presence of a G at a nucleotide position corresponding to nucleotide 2073 of SEQ ID NO: 1 , wherein the presence of Val at position 646 of SEQ ID NO:2 or the presence of a -G- at nucleotide position 2073 of SEQ ID NO: 1 , indicates the presence of a disease or disorder associated with the cardiovascular system.
  • the disease or disorder can be from among, but not limited to, one or more of the group consisting of: atrial fibrillation, sick sinus syndrome, sudden cardiac arrest, ventricular arrythmia, ventricular fibrillation, ventricular tachycardia, Wolf-Parkinson- White (WPW) Syndrome, Lown-Ganong-Levin (LGL) Syndrome, hypertension.
  • atrial fibrillation sick sinus syndrome
  • sudden cardiac arrest ventricular arrythmia
  • ventricular fibrillation ventricular tachycardia
  • WPW Wolf-Parkinson- White
  • LGL Lown-Ganong-Levin
  • determining responsiveness of a subject to one or more /?-blocking agents comprising detecting for the subject the presence or absence of Val at position 646 of SEQ ID NO:2 or a -G- nucleotide at a position corresponding to position 2073 of SEQ ID NO: 1 , wherein the presence of a Val at position 646 of SEQ ID NO:2 or a -G- at nucleotide 2073 of SEQ ID NO: 1 , is indicative of an increased likelihood that a subject has a modulated response to one or more ⁇ -blocking agents compared to a subject who does not have the allelic variant.
  • the modulated response is a decreased response to one or more /?-blocking agents compared to a subject who does not have the allelic variant.
  • the decreased response is a non-response to one or more ⁇ -blocking agents compared to a subject who does not have the allelic variant.
  • the modulated response is an increased response to one or more ⁇ -blocking agents compared to a subject who does not have the allelic variant.
  • the ?-blocker can be an antagonist of a ?-adrenergic receptor.
  • the ?-blocker is an agonist of a /?-adrenergic receptor.
  • Also provided herein are methods for determining responsiveness of a subject to one or more ⁇ -blocking agents comprising detecting the presence or absence of Val at position 646 of SEQ ID NO:2 or a -G- nucleotide at a position corresponding to position 2073 of SEQ ID NO: 1 , wherein the presence of a Val at position 646 of SEQ ID NO:2 or a -G- at nucleotide 2073 of SEQ ID NO: 1 , is indicative of an increased likelihood that a subject has an increased response to one or more ⁇ -blocking agents compared to a subject who does not have the allelic variant.
  • methods for determining responsiveness of a subject to one or more ⁇ -blocking agents comprising detecting for the subject the presence or absence of Val at position 646 of SEQ ID NO:2 or a -G- nucleotide at a position corresponding to position 2073 of SEQ ID NO: 1 , wherein the presence of a Val at position 646 of SEQ ID NO:2 or a -G- at nucleotide 2073 of SEQ ID NO: 1 , is indicative of an increased likelihood that a subject is non-responsive to one or more /?- blocking agents compared to a subject who does not have the allelic variant.
  • Also provided herein are methods for determining responsiveness of a subject to one or more ⁇ -blocking agents comprising detecting the presence or absence of Val at position 646 of SEQ ID NO:2 or a -G- nucleotide at a position corresponding to position 2073 of SEQ ID NO: 1 , wherein the presence of a Val at position 646 of SEQ ID NO:2 or a -G- at nucleotide 2073 of SEQ ID NO: 1 , is indicative of an increased likelihood that a subject is hyper-responsive to one or more ⁇ -blocking agents compared to a subject who does not have the allelic variant.
  • methods for indicating susceptibility of a subject to acquired long Q-T syndrome comprising detecting the presence or absence of Val at position 646 of SEQ ID NO:2 or presence or absence of a -G- nucleotide at a position corresponding to position 2073 of SEQ ID NO: 1 , wherein the presence of a Val at position 646 of SEQ ID NO:2 or a -G- at nucleotide 2073 of SEQ ID NO: 1 , is indicative of increased susceptibility to acquired long Q-T syndrome, compared to the susceptibility of a subject who does not have the allelic variant.
  • the detecting step can be effected by a method selected from the group consisting of allele specific hybridization, primer specific extension, oligonucleotide ligation assay, restriction enzyme site analysis and single-stranded conformation polymorphism analysis.
  • the detecting step can comprise mass spectrometry.
  • detection can be effected by detecting a signal moiety selected from the group consisting of radioisotopes, enzymes, antigens, antibodies, spectrophotometric reagents, chemiluminescent reagents, fluorescent reagents and other light producing reagents.
  • the subject is heterozygous -GA- at a position corresponding to nucleotide 2073 of SEQ ID NO: 1 or heterozygous Val/lle at a position corresponding to position 646 of SEQ ID NO:2.
  • the subject is homozygous -GG- at a position corresponding to nucleotide 2073 of SEQ ID NO: 1 or homozygous Val/Val at a position corresponding to position 646 of SEQ ID NO:2.
  • Another non-synonymous D-AKAP2 variation retrieved from dbSNP has been verified.
  • the G-A transversion in exon 4 results in an Arg to His substitution at position 249 of SEQ ID NO:2 (R249H; corresponding to a G to A transversion at nucleotide 883 of SEQ ID NO: 1 encoding human D-AKAP2).
  • the Arg at residue 249 of SEQ ID NO:2 was found to be in complete linkage disequilibrium with the He at position 646 of SEQ ID NO:646, occurring together in every case, and therefore shows the same age effect.
  • the subject can also be assayed for the genotype at position 883 of SEQ ID NO: 1 .
  • the genotype -G- at position 883 of SEQ ID NO: 1 corresponds to genotype -A- at nucleotide 2073 of SEQ ID NO: 1 and vice versa.
  • the genotype - A- at position 883 of SEQ ID NO: 1 corresponds to genotype -G- at nucleotide 2073 of SEQ ID NO: 1 and vice versa.
  • kits for practicing the methods can include reagents for assessing genotype of an AKAP allele and also reagents and/or components for conducting an EKG and/or a ⁇ -blocking agent.
  • a kit for assessing genotype can include a primer or probe that specifically hybridizes adjacent to or at a polymorphic region spanning a position corresponding to position 2073 of SEQ ID NO 1 or 3 of an AKAP10 allele or the complement thereof and a second primer or probe that specifically hybridizes adjacent to or at a polymorphic region spanning a position corresponding to positions selected from the group consisting of position 83587 of SEQ ID NO 1 3 or 1 7, position 1 29600 of SEQ ID NO 14 or 1 7, and position 1 56,277 of SEQ ID NO 1 8 or 1 7 of an AKAP10 allele or the complement thereof.
  • Primers include, but are not limited to, nucleic acids consisting essentially of the nucleotide sequence of SEQ ID NO: 8, SEQ ID NO: 1 5, SEQ ID NO: 1 9 and SEQ ID NO 20.
  • Other genotyping components of the kit can include a first primer or probe that specifically hybridizes adjacent to or at a polymorphic region spanning a position corresponding to position 883 of SEQ ID NO 1 or 3 of an AKAP10 allele or the complement thereof and a second primer or probe that specifically hybridizes adjacent to or at a polymorphic region spanning a position corresponding to positions selected from the group consisting of position 83587 of SEQ ID NO 13 or 17, position 129600 of SEQ ID NO 14 or 17, and position 156,277 of SEQ ID NO 18 or 17 of an AKAP10 allele or the complement thereof.
  • kits optionally contain instructions for performing assays, interpreting results or for aiding in peforming the methods.
  • the kits also can include at least one didieoxynucleotide such as ddA, ddC, ddG.
  • Figure 1 shows the results of an analysis of covariance that was conducted to test the effect of the genotypes on PR mean levels.
  • Age was included as a covariate in the model, which was significantly associated with PR mean up to a third order polynomial.
  • the relationship between age and PR mean was genotype-dependent, and therefore interaction terms between genotype and age were included.
  • the predicted values from the resulting model are shown in Figure 1 for each genotype.
  • EKG examination or “ECG examination” refers to the well-known electrocardiogram examination that generates an electrical recording of the heart and is conducted on human subjects to investigate heart function and heart disease.
  • PR-interval or "EKG-PR-interval” in the context of an electrocardiogram (EKG or ECG) analysis, is the time (typically expressed herein in units of milliseconds) elapsed between the beginning of the P wave to the beginning of the next QRS complex. It corresponds to the time lag from the onset of atrial depolarization to the onset of ventricular depolarization. This time lag allows atrial systole to occur, filling the ventricles before ventricular systole. Most of the delay occurs in the AV node.
  • the PR interval is longer with high vagal tone.
  • a prolonged PR interval corresponds to impaired AV conduction.
  • the normal range of PR-intervals from about 1 20 to 200 milliseconds. It is well-known that each square on a EKG readout (graph) corresponds to 40 milliseconds.
  • the phrase "predetermined standard” refers to an average of a multiplicity of EKG-PR-intervals that can be empirically determined from a specifically chosen group of individuals.
  • the group of individuals can be selected irrespective of disease status, e.g., from a healthy patient database.
  • the standard can be obtained from a group of control age-matched subjects that do not have a particular disease, such as heart disease.
  • the predetermined standard can be obtained from a known age-matched control that is homozygous -AA- at a position corresponding to nucleotide 2073 of SEQ ID NO: 1 or homozygous lle/lle at a position corresponding to position 646 of SEQ ID NO:2.
  • age-matched standard EKG-PR-interval refers to the average PR-interval (also referred to herein as "PRmean") for a multiplicity of subjects of the same age.
  • the average PR- interval can be obtained from controls having the same genotype, such as -AA- homozygotes and/or -GA- heterozygotes at a position corresponding to nucleotide 2073 of SEQ ID NO: 1 . Accordingly, the EKG-PR-interval can be stratified by age and/or genotype.
  • the EKG-PR-interval of the subject being examined can be compared to PRmean of either: a group of control subjects of the same age having the -AA- homozygous genotype at a position corresponding to nucleotide 2073 of SEQ ID NO: 1 ; a group of control subjects of the same age having the -GA- heterozygous genotype at a position corresponding to nucleotide 2073 of SEQ ID NO: 1 ; both groups of control subjects of the same age having either the -AA- homozygous or the -GA- homozygous genotype at a position corresponding to nucleotide 2073 of SEQ ID
  • the EKG-PR-interval of the subject being examined can be compared to the PRmean of a group of control subjects of the same age having any genotype at a position corresponding to nucleotide 2073 of SEQ ID NO: 1 .
  • disease or disorders is meant to encompass all genetic or physiological irregularities that can be attributed to a particular body organ or physiological or cellular system.
  • disorder or disease associated with a decreased EKG- PR-interval or grammatical variations thereof, refers to any disease or disorder that exhibits a decreased PR-interval as one of its characteristics.
  • disorders and/or diseases contemplated herein as associated with decreased EKG-PR-intervals include, but are not limited to, those involving alterations in cellular protein phosphorylation and/or signal transduction.
  • disorders and diseases are: neurodegeneratives diseases, such as Alzheimer's Disease, cardiovascular disorders, cardiac disorders, particularly disorders associated with altered left ventricular function, cardiomyopathies, proliferative disorders, bipolar disorder and other neurological disorders, obesity, diabetes and certain peripheral retinopathies, such as retinitis pigmentosa.
  • cardiovascular disorders or cardiac disease collectively encompass all cardiovascular abnormalities, such as, but not limited to congenital heart disease, cardiac arrhythmia, brachycardia, atrial fibrillation, sick sinus syndrome, sudden cardiac arrest, ventricular arrythmia, ventricular fibrillation, ventricular tachycardia, Wolf-Parkinson- White (WPW) Syndrome, Lown-Ganong-Levin (LGL) Syndrome, hypertension, familial cardiac myxomas and Carney complex.
  • WPW Wolf-Parkinson- White
  • LGL Lown-Ganong-Levin
  • a "decreased" EKG-PR-interval or grammatical variations thereof refers to a PR-interval that is lower than the average PR-interval (e.g., PRmean) for subjects of the same age group.
  • the average PR-interval e.g., PRmean
  • the average PR-interval increases with age for subjects having either a homozygous -AA- or heterozygous -GA- genotype at a position corresponding to nucleotide 2073 of SEQ ID NO: 1 encoding the AKAP1 0/D-AKAP2 protein.
  • the average PR-interval for the -GA- heterozygotes increases with age as with the -AA- homozygotes
  • the average PR-interval e.g., PRmean
  • the EKG-PR-interval of the subject being examined is compared to the PRmean (e.g., average PR-interval) of age-matched -AA- homozygotes.
  • This embodiment serves as a preliminary screen for subjects that have either 1 or 2 copies of the I646V variant (e.g., screen for potential -GA- heterozygotes or -AA- homozygotes at a nucleotide position corresponding to nucleotide 2073 of SEQ ID NO: 1 ) .
  • the EKG-PR-interval of the subject being examined is compared to the PRmean (e.g., average PR- interval) of age-matched -GA- heterozygotes.
  • This embodiment serves as preliminary screen for subjects that have 2 copies of the I646V variant, e.g., subjects that are -GG- homozygotes.
  • the EKG-PR-interval is not decreased relative to the age-matched average PR-interval (PRmean) for - AA- homozygotes and/or -GA- heterozygotes at a position corresponding to nucleotide 2073 of SEQ ID NO: 1 , then there is no need to determine the genotype of the subject.
  • PRmean age-matched average PR-interval
  • a Q-T interval is the time from electrocardiogram Q wave to the end of the T wave corresponding to electrical systole. This interval represents the time required for depolarization and repolarization to occur. In long QT syndrome, the duration of repolarization is longer than normal. Thus, the QT-interval is prolonged. An interval above 440 milliseconds (msec) is considered prolonged. An interval at or above 480 milliseconds in females or 470 milliseconds in males typically is sufficient to diagnose a subject as having long QT syndrome.
  • sequencing refers to the process of determining a nucleotide sequence and can be performed using any method known to those of skill in the art.
  • the region of interest from the samples can be isolated, such as by PCR or restriction fragments, hybridization or other suitable method known to those of skill in the art, and sequenced.
  • sequencing analysis can be carried out using mass spectrometry (see, e.g. , U.S. Patent Nos. 5,547,835, 5,622,824, 5,851 ,765, and 5,928,906) .
  • Nucleic acids can also be sequenced by hybridization (see, e.g. , U.S. Patent Nos.
  • sequencing can be performed using other known methods, such as set forth in U.S. Patent Nos. 5,525,464;
  • polymorphism refers to the coexistence of more than one form of a gene or portion thereof.
  • a portion of a gene of which there are at least two different forms, i.e., two different nucleotide sequences, is referred to as a "polymorphic region of a gene" .
  • a polymorphic region can be a single nucleotide, the identity of which differs in different alleles.
  • a polymorphic region can also be several nucleotides in length.
  • polymorphic gene refers to a gene having at least one polymorphic region.
  • allele which is used interchangeably herein with
  • allelic variant refers to alternative forms of a gene or portions thereof. Alleles occupy the same locus or position on homologous chromosomes. When a subject has two identical alleles of a gene, the subject is the to be homozygous for the gene or allele. When a subject has two different alleles of a gene, the subject is the to be heterozygous for the gene. Alleles of a specific gene can differ from each other in a single nucleotide, or several nucleotides, and can include substitutions, deletions, and insertions of nucleotides. An allele of a gene can also be a form of a gene containing a mutation.
  • allelic allele refers to an allele that is represented in the greatest frequency for a given population. The allele or alleles that are present in lesser frequency are referred to as allelic variants.
  • allelic variants refers to allelic variants.
  • associated refers to coincidence with the development or manifestation of a disease, condition or phenotype. Association can be due to, but is not limited to, genes responsible for housekeeping functions whose alteration can provide the foundation for a variety of diseases and conditions, those that are part of a pathway that is involved in a specific disease, condition or phenotype and those that indirectly contribute to the manifestation of a disease, condition or phenotype.
  • the term “subject” refers to mammals and in particular human beings.
  • the term “gene” or “recombinant gene” refers to a nucleic acid molecule comprising an open reading frame and including at least one exon and (optionally) an intron sequence.
  • a gene can be either RNA or DNA. Genes can include regions preceding and following the coding region (leader and trailer) .
  • intron refers to a DNA sequence present in a given gene which is spliced out during mRNA maturation.
  • nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: x refers to the nucleotide sequence of the complementary strand of a nucleic acid strand having SEQ ID NO: x.
  • complementary strand is used herein interchangeably with the term “complement” .
  • the complement of a nucleic acid strand can be the complement of a coding strand or the complement of a non-coding strand.
  • the complement of a nucleic acid having SEQ ID NO: x refers to the complementary strand of the strand having SEQ ID NO: x or to any nucleic acid having the nucleotide sequence of the complementary strand of SEQ ID NO: x.
  • the complement of this nucleic acid is a nucleic acid having a nucleotide sequence which is complementary to that of SEQ ID NO: x.
  • coding sequence refers to that portion of a gene that encodes an amino acid sequence of a protein.
  • sense strand refers to that strand of a double-stranded nucleic acid molecule that has the sequence of the mRNA that encodes the amino acid sequence encoded by the double- stranded nucleic acid molecule.
  • antisense strand refers to that strand of a double-stranded nucleic acid molecule that is the complement of the sequence of the mRNA that encodes the amino acid sequence encoded by the double-stranded nucleic acid molecule.
  • amino acids which occur in the various amino acid sequences appearing herein, are identified according to their well- known, three-letter or one-letter abbreviations.
  • amino acid residue refers to an amino acid formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages. The amino acid residues described herein are typically in the
  • L isomeric form. Residues in the “D” isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property is retained by the polypeptide.
  • NH 2 refers to the free amino group present at the amino terminus of a polypeptide.
  • COOH refers to the free carboxy group present at the carboxyl terminus of a polypeptide.
  • amino acid residue sequences represented herein by formulae have a left to right orientation in the conventional direction of amino-terminus to carboxyl-terminus.
  • amino acid residue is broadly defined to include the amino acids listed in the Table of Correspondence and modified and unusual amino acids, such as those referred to in 37 C.F.R. ⁇ ⁇ 1 .821 - 1 .822, and incorporated herein by reference.
  • a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues or to an amino-terminal group such as NH 2 or to a carboxyl-terminal group such as COOH.
  • a DNA or nucleic acid homolog refers to a nucleic acid that includes a preselected conserved nucleotide sequence, such as a sequence encoding a therapeutic polypeptide.
  • substantially homologous is meant having at least 80%, typically at least 90%, or at least 95% homology therewith or a less percentage of homology or identity and conserved biological activity or function.
  • the terms "homology” and “identity” are often used interchangeably. In this regard, percent homology or identity can be determined, for example, by comparing sequence information using a GAP computer program.
  • the GAP program uses the alignment method of Needleman and Wunsch (J. Mol. Biol. 48:443 (1 970), as revised by Smith and Waterman (Adv. Appl. Math. 2:482 ( 1 981 ) .
  • the GAP program defines similarity as the number of aligned symbols (i.e., nucleotides or amino acids) which are similar, divided by the total number of symbols in the shorter of the two sequences.
  • the default parameters for the GAP program can include: (1 ) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745 (1 986), as described by Schwartz and Dayhoff, eds., A TLAS OF PROTEIN SEQUENCE AND STRUCTURE, National Biomedical Research Foundation, pp. 353-358 (1 979); (2) a penalty of 3.0 for each gap and an additional 0.1 0 penalty for each symbol in each gap; and (3) no penalty for end gaps.
  • nucleic acid molecules Whether any two nucleic acid molecules have nucleotide sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% "identical” can be determined using known computer algorithms such as the "FAST A” program, using for example, the default parameters as in Pearson and Lipman, Proc. Nat/. Acad. Sci. USA 55:2444 (1 988). Alternatively the BLAST function of the National Center for Biotechnology Information database can be used to determine identity
  • sequences are aligned so that the highest order match is obtained. "Identity" per se has an art-recognized meaning and can be calculated using published techniques.
  • identity is well known to skilled artisans (Carillo, H. & Lipton, D., SIAM J Applied Math 45: 1073 (1 988)). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1 994, and Carillo, H. & Lipton, D., SIAM J Applied Math 45: 1073 (1 988).
  • Typical computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al., Nucleic Acids Research 12(/):387 (1 984)), BLASTP, BLASTN, FASTA (Atschul, S.F., et a/., J Mo/ec Biol 215:403 (1 990)) .
  • identity represents a comparison between a test and a reference polypeptide or polynucleotide.
  • a test polypeptide can be defined as any polypeptide that is 90% or more identical to a reference polypeptide.
  • the term at least "90% identical to” refers to percent identities from 90 to 99.99 relative to the reference polypeptides. Identity at a level of 90% or more is indicative of the fact that, assuming for exemplification purposes a test and reference polypeptide length of 1 00 amino acids are compared. No more than 1 0% (i.e., 10 out of 100) amino acids in the test polypeptide differs from that of the reference polypeptides. Similar comparisons can be made between a test and reference polynucleotides. Such differences can be represented as point mutations randomly distributed over the entire length of an amino acid sequence or they can be clustered in one or more locations of varying length up to the maximum allowable, e.g.
  • stringency conditions refer to the washing conditions for removing the non-specific probes and conditions that are equivalent to either high, medium, or low stringency as described below: 1 ) high stringency: 0.1 x SSPE, 0.1 % SDS, 65 °C 2) medium stringency: 0.2 x SSPE, 0.1 % SDS, 50°C
  • heterologous DNA is DNA that encodes RNA and proteins that are not normally produced in vivo by the cell in which it is expressed or that mediates or encodes mediators that alter expression of endogenous DNA by affecting transcription, translation, or other regulatable biochemical processes or is not present in the exact orientation or position as the counterpart DNA in a wildtype cell.
  • Heterologous DNA can also be referred to as foreign DNA. Any DNA that one of skill in the art would recognize or consider as heterologous or foreign to the cell in which is expressed is herein encompassed by heterologous DNA.
  • heterologous DNA examples include, but are not limited to, DNA that encodes traceable marker proteins, such as a protein that confers drug resistance, DNA that encodes therapeutically effective substances, such as anti-cancer agents, enzymes and hormones, and DNA that encodes other types of proteins, such as antibodies.
  • Antibodies that are encoded by heterologous DNA can be secreted or expressed on the surface of the cell in which the heterologous DNA has been introduced.
  • isolated with reference to a nucleic acid molecule or polypeptide or other biomolecule means that the nucleic acid or polypeptide has separated from the genetic environment from which the polypeptide or nucleic acid were obtained. It can also mean altered from the natural state. For example, a polynucleotide or a polypeptide naturally present in a living animal is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term is employed herein. Thus, a polypeptide or polynucleotide produced and/or contained within a recombinant host cell is considered isolated.
  • isolated polypeptide or an “isolated polynucleotide” are polypeptides or polynucleotides that have been purified, partially or substantially, from a recombinant host cell or from a native source.
  • a recombinantly produced version of a compound can be substantially purified by the one-step method described in Smith and Johnson, Gene 57/31 -40 (1 988).
  • isolated and purified are sometimes used interchangeably.
  • isolated is meant that the nucleic acid is free of the coding sequences of those genes that, in the naturally-occurring genome of the organism (if any) immediately flank the gene encoding the nucleic acid of interest.
  • Isolated DNA can be single-stranded or double-stranded, and can be genomic DNA, cDNA, recombinant hybrid DNA, or synthetic DNA. It can be identical to a native DNA sequence, or can differ from such sequence by the deletion, addition, or substitution of one or more nucleotides.
  • Isolated or purified as it refers to preparations made from biological cells or hosts means any cell extract containing the indicated DNA or protein including a crude extract of the DNA or protein of interest.
  • a purified preparation can be obtained following an individual technique or a series of preparative or biochemical techniques and the DNA or protein of interest can be present at various degrees of purity in these preparations.
  • the procedures can include for example, but are not limited to, ammonium sulfate fractionation, gel filtration, ion exchange change chromatography, affinity chromatography, density gradient centrifugation and electrophoresis.
  • a preparation of DNA or protein that is "substantially pure” or “isolated” should be understood to mean a preparation free from naturally occurring materials with which such DNA or protein is normally associated in nature. "Essentially pure” should be understood to mean a “highly” purified preparation that contains at least 95% of the DNA or protein of interest.
  • a cell extract that contains the DNA or protein of interest should be understood to mean a homogenate preparation or cell-free preparation obtained from cells that express the protein or contain the DNA of interest.
  • the term “cell extract” is intended to include culture media, especially spent culture media from which the cells have been removed.
  • receptor refers to a biologically active molecule that specifically binds to (or with) other molecules.
  • the term “receptor protein” can be used to more specifically indicate the proteinaceous nature of a specific receptor.
  • recombinant refers to any progeny formed as the result of genetic engineering.
  • a promoter region refers to the portion of DNA of a gene that controls transcription of the DNA to which it is operatively linked.
  • the promoter region includes specific sequences of DNA that are sufficient for RNA polymerase recognition, binding and transcription initiation. This portion of the promoter region is referred to as the promoter.
  • the promoter region includes sequences that modulate this recognition, binding and transcription initiation activity of the RNA polymerase. These sequences can be cis acting or can be responsive to trans acting factors. Promoters, depending upon the nature of the regulation, can be constitutive or regulated.
  • operatively linked generally means the sequences or segments have been covalently joined into one piece of DNA, whether in single or double stranded form, whereby control or regulatory sequences on one segment control or permit expression or replication or other such control of other segments.
  • the two segments are not necessarily contiguous.
  • a DNA sequence and a regulatory sequence(s) are connected in such a way to control or permit gene expression when the appropriate molecular, e.g., transcriptional activator proteins, are bound to the regulatory sequence(s) .
  • production by recombinant means by using recombinant DNA methods means the use of the well known methods of molecular biology for expressing proteins encoded by cloned DNA, including cloning expression of genes and methods, such as gene shuffling and phage display with screening for desired specificities.
  • conjugated refers stable attachment, such ionic or covalent attachment.
  • composition refers to any mixture of two or more products or compounds. It can be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.
  • a combination refers to any association between two or more items.
  • substantially identical to a product means sufficiently similar so that the property of interest is sufficiently unchanged so that the substantially identical product can be used in place of the product.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • One typical vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication.
  • Typical vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked.
  • Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors" .
  • expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer generally to circular double stranded DNA loops which, in their vector form are not bound to the chromosome.
  • Plasmid and “vector” are used interchangeably as the plasmid is the most commonly used form of vector.
  • Other such other forms of expression vectors that serve equivalent functions and that become known in the art subsequently hereto.
  • indicating means that the presence or absence of an allelic variant can be one of many factors that are considered when a subject's predisposition to a disease or disorder is evaluated. Thus a predisposition to a disease or disorder is not necessarily conclusively determined by only ascertaining the presence or absence of one or more allelic variants, but the presence of one of more of such variants is among a number of factors considered.
  • predisposition to develop a disease or disorder means that a subject having a particular genotype and/or haplotype has a higher likelihood than one not having such a genotype and/or haplotype for developing a particular disease or disorder.
  • morbidity refers to conditions, such as diseases or disorders, that compromise the health and well-being of an organism, such as an animal.
  • Morbidity susceptibility or morbidity-associated genes are genes that, when altered, for example, by a variation in nucleotide sequence, facilitate the expression of a specific disease clinical phenotype.
  • morbidity susceptibility genes have the potential, upon alteration, of increasing the likelihood or general risk that an organism will develop a specific disease.
  • telomere length refers to the statistical likelihood that an organism, particularly an animal, will not survive a full predicted lifespan.
  • a trait or a marker, such as a polymorphism associated with increased mortality is observed at a lower frequency in older than younger segments of a population.
  • transgenic animal refers to any animal, typically a non-human animal, e.g. a mammal, bird or an amphibian, in which one or more of the cells of the animal contain heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.
  • the nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
  • the term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule.
  • transgenic animal also includes those recombinant animals in which gene disruption of one or more genes is caused by human intervention, including recombination and antisense techniques.
  • target nucleic acid refers to a nucleic acid molecule which contains all or a portion of a polymorphic region of a gene of interest.
  • signal moiety refers to any moiety that allows for the detection of a nucleic acid molecule. Included are moieties covalently attached to nucleic acids and those that are not.
  • molecule that modulates or effects the biological activity of an AKAP10 protein refers to any drug, small molecule, nucleic acid (sense and antisense), ribozyme, protein, peptide, lipid, carbohydrate etc. or combination thereof, that directly or indirectly changes, alters, abolishes, increases or decreases a biological activity attributed to AKAP10 protein.
  • biological activity of an AKAP10 protein refers to, but is not limited to, binding of AKAP10 to protein kinase A or its subunits, localization of AKAP10 protein to a subcellular site, e.g., the mitochondria, localization of protein kinase A to the mitochondria and binding of AKAP1 0 protein to other proteins including other signaling enzymes.
  • combining refers to contacting the biologically active agent with a cell or animal such that the agent is introduced into the cell or animal.
  • a cell any method that results in an agent traversing the plasma membrane is useful.
  • an animal any of the standard routes of administration of an agent, e.g. oral, rectal, transmucosal, intestinal, intravenous, intraperitoneal, intraventricular, subcutaneous, intramuscular, etc. , can be used.
  • composition refers to any mixture. It can be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.
  • a combination refers to any association between two or among more items.
  • kit refers to a package that contains a combination, such as one or more primers or probes used to amplify or detect polymorphic regions of AKAP1 0 genes, optionally including instructions and/or reagents for their use.
  • solid support refers to a support substrate or matrix, such as silica, polymeric materials or glass. At least one surface of the support can be partially planar. Regions of the support can be physically separated, for example with trenches, grooves, well or the like. Some examples of solid supports include slides and beads. Supports are of such composition so as to allow for the immobilization or attachment of nucleic acids and other molecules such that these molecules retain their binding ability.
  • array refers to a collection of elements, such as nucleic acids, containing three or more members. An addressable array is one in which the members of the array are identifiable, typically by position on a solid support. Hence, in general the members of the array will be immobilized to discrete identifiable loci on the surface of a solid phase.
  • hybridizes refers to hybridization of a probe or primer only to a target sequence preferentially to a non-target sequence.
  • Those of skill in the art are familiar with parameters that affect hybridization; such as temperature, probe or primer length and composition, buffer composition and salt concentration and can readily adjust these parameters to achieve specific hybridization of a nucleic acid to a target sequence.
  • nucleic acid refers to polynucleotides such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) .
  • the term should also be understood to include, as equivalents, derivatives, variants and analogs of either RNA or DNA made from nucleotide analogs, single (sense or antisense) and double-stranded polynucleotides.
  • Deoxyribonucleotides include deoxyadenosine, deoxycytidine, deoxyguanosine and deoxythymidine.
  • the uracil base is uridine.
  • mass spectrometry encompasses any suitable mass spectrometric format known to those of skill in the art.
  • Such formats include, but are not limited to, Matrix-Assisted Laser Desorption/lonization, Time-of-Flight (MALDI-TOF), Electrospray (ES), IR- MALDI (see, e.g. , published International PCT Application No. WO 99/5731 8 and U.S. Patent No. 5, 1 1 8,937) Ion Cyclotron Resonance (ICR), Fourier Transform and combinations thereof.
  • MALDI particular UV and IR, are among the typical formats.
  • "at a position corresponding to” refers to a position of interest (i.e.
  • base number or residue number in a nucleic acid molecule or protein relative to the position in another reference nucleic acid molecule or protein.
  • Corresponding positions can be determined by comparing and aligning sequences to maximize the number of matching nucleotides or residues, for example, such that identity between the sequences is greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%.
  • the position of interest is then given the number assigned in the reference nucleic acid molecule. For example, it is shown herein that a particular polymorphism in AKAP1 0 occurs at nucleotide 2073 of SEQ ID No. 1 .
  • the sequences are aligned and then the position that lines up with 2073 is identified. Since various alleles can be of different length, the position designate 2073 can not be nucleotide 2073, but instead is at a position that "corresponds" to the position in the reference sequence.
  • probe refers to a nucleic acid molecule including DNA, RNA and analogs thereof, including protein nucleic acids (PNA), and mixtures thereof. Such molecules are typically of a length such that they are statistically unique (i.e. , occur only once) in the genome of interest. Generally, for a probe or primer to be unique in the human genome, it contains at least 14, 1 6 or contiguous nucleotides of a sequence complementary to or identical to a gene of interest. Probes and primers can be 1 0, 20, 30, 50, 100 or more nucleic acids long.
  • antisense nucleic acid molecule refers to a molecule encoding a sequence complementary to at least a portion of an RNA molecule.
  • the sequence is sufficiently complementary to be able to hybridize with the RNA, typically under moderate or high stringency conditions to form a stable duplex.
  • the ability to hybridize depends on the degree of complementarity and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA it can contain and still form a stable duplex.
  • One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
  • a "variant protein” refers to a protein encoded by an allelic variant of a AKAP10 gene which results in a change of an amino acid residue at a particular position relative to that position in the protein encoded by the predominant allele.
  • signal transduction refers to the propagation of a signal.
  • an extracellular signal is transmitted through the cell membrane to become an intracellular signal. This signal can then stimulate a cellular response.
  • the term also encompasses signals that are propagated entirely within a cell.
  • the polypeptide molecules involved in signal transduction processes are typically receptor and non-receptor protein kinases, receptor and non-receptor protein phosphatases, nucleotide exchange factors and transcription factors.
  • One of the key biochemical mechanisms involved in signal transduction is protein phosphorylation.
  • AKAP10 proteins are involved in signal transduction as they bind to protein kinase A (PKA) and are though to anchor the kinase at a location, e.g.
  • adjacent refers to a position 5' to the site of a single nucleotide polymorphism (SNP) such that there could be unpaired nucleotides between that position and the site of the SNP.
  • SNP single nucleotide polymorphism
  • immediate adjacent refers to a position 5' to the site of a single nucleotide polymorphism (SNP) such that there are no unpaired nucleotides between that position and the site of the SNP.
  • SNP single nucleotide polymorphism
  • binding to PKA refers to the interaction of the PKA binding domain of an AKAP10 protein and the regulatory subunits Rl and/or Rll of the protein kinase A holoenzyme.
  • Methods herein include a step a identifying the prescence of a particular allele of an A-kinase anchoring protein (AKAP) genes.
  • AKAP A-kinase anchoring protein
  • polymorphic sequences encoding an A-kinase anchoring protein (AKAP) genes and polymorphic AKAP proteins encoded by polymorphic AKAP gene sequences are used in methods provided herein. These polymorphic sequences are based on differences in AKAP genes within and among different organisms, including humans.
  • AKAPs provide a mechanism for regulating ubiquitous cAMP-dependent kinase (PKA) activity by tethering PKA to specific subcellular locations thereby segregating it with particular components in a given signaling pathway and contributing to specificity in cellular responses to extracellular signals.
  • PKA ubiquitous cAMP-dependent kinase
  • polymorphisms in AKAP gene sequences can affect the proper functioning of cells and systems within organisms and could be directly linked with certain disorders or could predispose an organism to a variety of diseases and disorders, especially those involving alterations in cellular protein phosphorylation and/or signal transduction.
  • disorders and diseases include, but are not limited to, neurodegeneratives diseases, such as Alzheimer's Disease, cardiovascular disorders, cardiac disorders, particularly disorders associated with altered left ventricular function, cardiomyopathies, proliferative disorders, bipolar disorder and other neurological disorders, obesity, diabetes and certain peripheral retinopathies, such as retinitis pigmentosa.
  • AKAP gene polymorphisms such as those described herein, provides for the identification and development of diagnostic and prognostic methods, also provided herein, and the development of drug therapies and treatment regimens. Furthermore, polymorphisms of AKAP genes aid in the study of AKAP protein structure and function, which also contributes to the development of diagnostic methods and therapies. 1 . AKAP10
  • the AKAP10 protein is primarily located in mitochondria.
  • the sequence of a human AKAP10 cDNA (also referred to as D-AKAP2) is available in the GenBank database, at accession numbers AF037439 and NM 007202, and is provided in SEQ. ID. NO: 1 .
  • the AKAP10 gene is located on chromosome 1 7.
  • mouse D-AKAP2 cDNA The sequence of a mouse D-AKAP2 cDNA is also available in the GenBank database (see accession number AF021 833).
  • the mouse D- AKAP2 protein contains an RGS domain near the amino terminus that is characteristic of proteins that interact with G ⁇ subunits and possess GTPase activating protein-like activity (Huang et al. (1 997) Proc. Natl. Acad. Sci. U.S.A. 54: 1 1 1 84-1 1 1 89).
  • the human AKAP1 0 protein also has sequences homologous to RGS domains.
  • the carboxy-terminal 40 residues of the mouse D-AKAP2 protein are responsible for the interaction with the regulatory subunits of PKA. This sequence is fairly well conserved between the mouse D-AKAP2 and human AKAP10 proteins.
  • Polymorphisms of AKAP genes that alter gene expression, regulation, protein structure and/or protein function are more likely to have a significant effect on the regulation of enzyme (particularly PKA) activity, cellular transduction of signals and responses thereto and on the basic functioning of cells than polymorphisms that do not alter gene and/or protein function.
  • Included in the polymorphic AKAPs provided herein are human AKAP10 proteins containing differing amino acid residues at position number 646 of SEQ. ID. No. 2.
  • Amino acid 646 of the human AKAP1 0 protein (SEQ. ID. NO: 2) is located in the carboxy-terminal region of the protein within a segment that participates in the binding of R-subunits of PKAs. This segment includes the carboxy-terminal 40 amino acids.
  • AKAP10 protein is an isoleucine.
  • Polymorphic human AKAP10 proteins provided herein have the amino acid sequence set forth in SEQ. ID. NO: 2 but contain residues other than isoleucine at amino acid position 646 of the protein.
  • polymorphic human AKAP10 proteins provided herein have the amino acid sequence set forth in SEQ. ID. NO: 2 but contain residues other than isoleucine at amino acid position 646 of the protein.
  • AKAP10 proteins provided herein, the amino acid at position 646 of SEQ.
  • ID. NO: 2 is a valine (as set forth in SEQ. ID. NO: 4), leucine or phenylalanine residue. a. An A to G transition at nucleotide 2073 of the human
  • an allelic variant of the human AKAP10 gene is at the polymorphic site at position 2073 of the coding sequence (see
  • SEQ. ID. NO: 3 encodes a valine at position 646 of the AKAP1 0 protein.
  • This allelic variant has been found to vary in frequency in DNA samples from younger and older segments of a healthy population.
  • This allele has the A at position 2073 of the AKAP1 0 gene coding sequence of SEQ. ID. NO: 1 changed to a G, giving rise to the sequence set forth in SEQ. ID. NO: 3. Consequently, the codon for amino acid 646 changes from ATT, coding for isoleucine, to GTT, coding for valine.
  • AKAP1 0-1 additional variants are represented by the presence of A or G at nucleotide position 1 56277 of SEQ ID NO: 1 7.
  • AKAP10-7 additional variants are represented by the presence of C or T at nucleotide position 1 29600 of SEQ ID NO: 1 7.
  • C Association of AKAP10-5 I646V variant with Cardiac Traits The SNPs found to be associated with age were analyzed for association with disease-related quantitative traits in a twin collection. To identify traits correlated with the observed age association of the I646V SNP, a cohort of 41 7 fasting Caucasian twin pairs with extensive coverage for a variety of disease-related traits was utilized. The analysis was conducted using a quantitative transmission-disequilibrium test
  • methods of comparing a subjects PR-interval to the PRmean of an age-matched control group of either one or both of an -AA- homozygous or -GA- heterozygous genotype at a nucleotide corresponding to position 2073 of SEQ ID NO: 1 can identify subjects that have a higher likelihood of possessing a -GG- homozygous genotype at that position.
  • that subject's particular genotype can be determined using the methods provided herein at a position corresponding to nucleotide 2073 of SEQ ID NO: 1 .
  • those subjects having the -GG- genotype have an increase susceptibility to a disease or disorder, such as a cardiovascular disease or disorder.
  • methods of comparing a subjects PR-interval to the PRmean of an age- matched control group of an -AA- homozygous genotype at a nucleotide corresponding to position 2073 of SEQ ID NO: 1 can identify subjects that have a higher likelihood of possessing at least one -G- allele at that position.
  • SNPs in the D-AKAP2 gene have been identified herein that are associated with morbidity using a genome-wide association study from an age-stratified healthy population and 6,500 gene-based SNPs.
  • the combined genetic and biochemical evidence points to the lle/Val variant as the functional polymorphism.
  • the Val variant is contemplated to be the deleterious allele in a Caucasian-American cohort, and this finding is replicated in Hispanic-Americans.
  • the variant maps to the conserved AKB domain of the D-AKAP2 gene. It has been found that the I646V variation impacts the binding to PKA in an isoform-specific manner both in vitro and in cells.
  • the lie variant have been found to bind three-fold weaker to the Rla isoform than to the Val variant. At the cellular level, this affinity difference results in a dramatic decrease in compartmentalization of Rla for the He variant.
  • the lle/Val variant has been found to be critical for binding to only the Rla isoform of P
  • the Rl and Rll isoforms of PKA have distinct functions.
  • the Rl isoform has been implicated in a variety of biological functions such as cell proliferation, tumor suppression, immune regulation, and embryonic development.
  • the Rla isoform plays a significant role in maintaining cAMP-regulation of PKA as evidenced by the embryonic lethality of mice deficient in the gene. Interestingly, these mice have defects in cardiac morphogenesis.
  • the identified correlation of the I646V variant e.g., -G- at position
  • the I646V polymorphism is a predisposing factor for a disease or disorder, such as a cardiovascular disease or disorder manifesting a cardiac phenotype.
  • a disease or disorder such as a cardiovascular disease or disorder manifesting a cardiac phenotype.
  • individuals homozygous for the Val variant exhibit shorter depolarization intervals of the atrium (PR) as compared to individuals homozygous for He.
  • PR atrium
  • AKAP-mediated PKA signaling in normal cardiac function suggest a lead into the pathogenesis coded for by this functional variant.
  • AKAP-mediated targeting of PKA in cardiac myocytes has been implicated in regulating cell contractility (Fink et al. , Cir.
  • Stimulation of the ⁇ -adrenergic signaling pathway in cardiac myocytes results in activation of PKA and phosphorylation of a variety of PKA substrates, including the sarcolemmal L-type Ca2 + channel, the ryanodine receptor (RyR), phospholamban (PLB) of the sarcoplasmic reticulum (SR), the myofibrillar proteins troponin I (Tnl) and myosin binding protein C (MBP-C) (Holroyde et al., Biochim. Biophys.
  • PKA has broad substrate specificity, it can be highly selective by targeting of PKA to distinct subcellular locations via interaction with AKAPs (Colledge et al., Trends Cell Biol., 19:216-221 , 1999).
  • Three AKAPs have been shown to interact with PKA in cardiac myocytes, muscle-selective AKAP (mAKAP), AKAP18 and Yotiao.
  • mAKAP targets PKA to the perinuclear region of differentiated myocytes, coordinating both PKA and phosphodiesterase activity in a single complex (Kapiloff et al., J. Cell Sci., 114:3167-3176, 2001).
  • AKAP18 couples PKA to L-type Ca2+ channels, which enhances Ca2+ influx through the channel following ?-adrenergic stimulation (Gray et al., J. Biol. Chem..272:6297-6302, 1997).
  • Yotiao previously associated with NMDA receptors, has been shown to interact with the KCNQ1-KCNE1 K+ channel subunits in human hearts (Marx et al., Science, 295:496- 499, 2002). This channel is responsible for the slow delayed rectifier current that repolarizes the myocyte membrane and controls action potential duration.
  • D-AKAP2 (AKAP10-5) contains a PDZ binding motif (TKL) at the C-terminus (Fig. 3a), which is contemplated herein to serve as a targeting domain to membrane-bound receptors or ion-channels (Harris et al. , J. Cell Sci. , 1 14:321 9-3231 , 2001 ), and two RGS domains, which are contemplated herein to coordinate upstream G alpha signaling with downstream PKA signaling. It is contemplated herein that D-AKAP2 is part of a signaling complex associated with a cardiac ion-channel.
  • TKL PDZ binding motif
  • the D-AKAP2 variants is contemplated herein to impact the phosphorylation state of the ion-channel by recruiting different amounts of PKA-Rla and thereby modulate heart contraction. This model is in agreement with the observed association with an EKG phenotype.
  • the shorter depolarization intervals for Val/Val homozygous individuals is contemplated herein to be due to increased activation of ion-channels in cardiac myocytes.
  • methods for indicating increased susceptibility of a subject to a disease or disorder comprising: conducting an EKG examination; determining the EKG-PR-interval in the subject, wherein, if the EKG-PR-interval is decreased, then determining the amino acid present in the subject at position 646 of AKAP1 0/D-AKAP2 (SEQ ID NO:2) or the nucleotide present at position corresponding to nucleotide 2073 of SEQ ID NO: 1 , wherein the presence of Val at position 646 of SEQ ID NO:2 or the presence of a -G- at nucleotide position 2073 of SEQ ID NO: 1 , indicates increased susceptibility to a disease or disorder.
  • the disease or disorder can be selected from among cardiovascular disorders, cardiac disease, proliferative disorders, neurological disorders, neurodegenerative disorders, obesity, diabetes and peripheral retinopathies.
  • the EKG-PR-interval in the subject is compared to a predetermined age-matched standard EKG-PR-interval.
  • the predetermined standard EKG-PR-interval can be obtained from a known age-matched control group that is homozygous -AA- at a position corresponding to nucleotide 2073 of SEQ ID NO: 1 or homozygous He/He at a position corresponding to position 646 of SEQ ID NO:2.
  • the predetermined standard EKG-PR-interval can be obtained from a known age-matched control group that is heterozygous -GA- at a position corresponding to nucleotide 2073 of SEQ ID NO: 1 or heterozygous
  • the predetermined standard EKG-PR-interval can be obtained from a known age-matched control group that is selected from either homozygous -AA- at a position corresponding to nucleotide 2073 of SEQ ID NO:1 or homozygous He/He at a position corresponding to position 646 of SEQ ID NO:2; or heterozygous -GA- at a position corresponding to nucleotide 2073 of SEQ ID NO: 1 or heterozygous Val/lle at a position corresponding to position 646 of SEQ ID NO:2.
  • the predetermined standard EKG-PR-interval is obtained from a control age- matched subject without heart disease.
  • the subject is heterozygous -GA- at a position corresponding to nucleotide 2073 of SEQ ID NO: 1 or heterozygous Val/lle at a position corresponding to position 646 of SEQ ID NO:2.
  • the subject is homozygous -GG- at a position corresponding to nucleotide 2073 of SEQ ID NO: 1 or homozygous Val/Val at a position corresponding to position 646 of SEQ ID NO:2.
  • Also provided are methods for indicating increased susceptibility of a subject to a disease or disorder associated with the cardiovascular system comprising: conducting an EKG exam; determining the EKG-PR-interval in the subject, wherein, if the EKG-PR-interval is decreased, then determining the amino acid present at position 646 of AKAP10/D- AKAP2 (SEQ ID NO:2) or the nucleotide present at position corresponding to nucleotide 2073 of SEQ ID NO: 1 , wherein the presence of Val at position 646 of SEQ ID NO:2 or the presence of a -G- at nucleotide position 2073 of SEQ ID NO: 1 , indicates increased susceptibility to a disease or disorder associated with the cardiovascular system.
  • the EKG-PR-interval in the subject can be compared to a predetermined age-matched standard EKG-PR-interval to determine whether it is decreased.
  • the predetermined standard EKG-PR-interval can be obtained from a known age-matched control that is homozygous - AA- at a position corresponding to nucleotide 2073 of SEQ ID NO: 1 or homozygous He/He at a position corresponding to position 646 of SEQ ID NO:2.
  • the predetermined standard EKG-PR-interval can be obtained from a known age-matched control group that is heterozygous -GA- at a position corresponding to nucleotide 2073 of SEQ ID NO: 1 or heterozygous Val/lle at a position corresponding to position 646 of SEQ ID NO:2.
  • the predetermined standard EKG-PR-interval can be obtained from a known age-matched control group that is selected from either homozygous -AA- at a position corresponding to nucleotide 2073 of SEQ ID NO: 1 or homozygous He/He at a position corresponding to position 646 of SEQ ID NO:2; or heterozygous -GA- at a position corresponding to nucleotide 2073 of SEQ ID NO: 1 or heterozygous Val/lle at a position corresponding to position 646 of SEQ ID NO:2.
  • the predetermined standard EKG-PR-interval can be obtained from a control age-matched subject without heart disease.
  • a decreased EKG-PR-interval is less than 1 50 for a subject 40 or more year old. In another embodiment, a decreased EKG-PR-interval is less than 1 55 for a subject 50 or more year old. In another embodiment, a decreased EKG-PR-interval is less than 1 50 for a subject 50 or more year old. In another embodiment, a decreased EKG-PR-interval is less than 1 60 for a subject 60 or more year old. In another embodiment, a decreased EKG-PR-interval is less than 1 55 for a subject 60 or more year old. In another embodiment, a decreased EKG-PR-interval is less than 1 50 for a subject 60 or more year old.
  • a decreased EKG-PR-interval for the subject is less than 146. In another embodiment, a decreased EKG-PR- interval for the subject is less than 1 30. In another embodiment, a decreased EKG-PR-interval for the subject is less than 1 20.
  • the disease or disorder can be selected from one or more of the group consisting of: atrial fibrillation, sick sinus syndrome, sudden cardiac arrest, ventricular arrythmia, ventricular fibrillation, ventricular tachycardia, Wolf-Parkinson-White (WPW) Syndrome, Lown-Ganong- Levin (LGL) Syndrome, hypertension. In one embodiment, the methods can further comprise monitoring the subject for cardiovascular disease.
  • the subject is heterozygous -GA- at a position corresponding to nucleotide 2073 of SEQ ID NO: 1 or heterozygous Val/lle at a position corresponding to position 646 of SEQ ID NO:2.
  • the subject is homozygous -GG- at a position corresponding to nucleotide 2073 of SEQ ID NO: 1 or homozygous Val/Val at a position corresponding to position 646 of SEQ ID NO:2.
  • the methods can further comprise administering to the subject prophylactic steps.
  • the methods provided herein permit the determination of the probability and risk assessment for the development of disease, in particular heart disease in an individual.
  • the methods provided herein permit the determination of the probability and risk assessment for the development of disease, in particular heart disease in an individual.
  • the genetic screening methods herein and/or family health histories it is possible to predict the probability a particular individual has for developing any one of several types of disease, such as heart disease.
  • Those individuals identified as being predisposed to developing a particular form of disease by using the methods provided herein can take prophylactic steps towards reducing the risk of the particular disease, such as a heart disease.
  • high-risk individuals identified herein can take one or more of the well- known prophylactic steps against the form of disease that they have a predisposition to develop. a. Methods of assessing the susceptibility of a subject to a disease or disorder associated with the cardiovascular system
  • Also provided herein are methods of assessing the susceptibility of a subject to a disease or disorder associated with the cardiovascular system comprising determining the amino acid at position 646 of AKAP10/D-AKAP2 (SEQ ID NO:2) or the nucleotide present at position corresponding to nucleotide 2073 of SEQ ID NO: 1 , wherein the presence of Val at position 646 of SEQ ID NO:2 or the presence of a -G- at nucleotide position 2073 of SEQ ID NO: 1 , indicates increased susceptibility to a disease or disorder associated with the cardiovascular system.
  • Also provided herein are methods of diagnosing a disease or disorder associated with the cardiovascular system comprising detecting the presence of Val at 646 of D-AKAP2 (SEQ ID NO:2) or the presence of a G at a nucleotide position corresponding to nucleotide 2073 of SEQ ID NO: 1 , wherein the presence of Val at position 646 of SEQ ID NO:2 or the presence of a -G- at nucleotide position 2073 of SEQ ID NO: 1 , indicates the presence of a disease or disorder associated with the cardiovascular system.
  • the disease or disorder can be from among one or more of the group consisting of: atrial fibrillation, sick sinus syndrome, sudden cardiac arrest, ventricular arrythmia, ventricular fibrillation, ventricular tachycardia, Wolf-Parkinson-White (WPW) Syndrome, Lown-Ganong-Levin (LGL) Syndrome, hypertension.
  • the subject is heterozygous -GA- at a position corresponding to nucleotide 2073 of SEQ ID NO: 1 or heterozygous Val/lle at a position corresponding to position 646 of SEQ ID NO:2.
  • the subject is homozygous -GG- at a position corresponding to nucleotide 2073 of SEQ ID NO: 1 or homozygous Val/Val at a position corresponding to position 646 of SEQ ID NO:2.
  • the presence of the I646V variant in a subject is contemplated herein to affect the D-AKAP2-mediated /?-adrenergic signaling pathway.
  • the presence of 1 or 2 copies of the I646V variant e.g., a -g- at nucleotide 2073 of SEQ ID NO: 1
  • Beta-blockers -adrenergic blocking drugs
  • Beta-blockers also block the impulses that can cause an arrhythmia.
  • the heterozygous presence of the I646V variant e.g., a -GA- heterozygous genotpye at a nucleotide corresponding to position 2073 of SEQ ID NO: 1
  • the heterozygous presence of the I646V variant is contemplated herein to produce a ?-blocker resistance phenotype.
  • the homozygous presence of the I646V variant (e.g., a -GG- heterozygous genotpye at a nucleotide corresponding to position 2073 of SEQ ID NO: 1 ) is contemplated herein to produce the /?-blocker resistance phenotype.
  • methods for determining responsiveness of a subject to one or more ⁇ -blocking agents comprising: detecting for the subject the presence or absence of Val at position 646 of SEQ ID NO:2 or a -G- nucleotide at a position corresponding to position 2073 of SEQ ID NO: 1 , wherein the presence of a Val at position 646 of SEQ ID NO:2 or a -G- at nucleotide 2073 of SEQ ID NO: 1 , is indicative of an increased likelihood that a subject has a modulated response to one or more ⁇ -blocking agents compared to a subject who does not have the allelic variant.
  • the modulated response is a decreased response to one or more /?-bIocking agents compared to a subject who does not have the allelic variant.
  • the decreased response is a non-response to one or more ⁇ -blocking agents compared to a subject who does not have the allelic variant.
  • the modulated response is an increased response to one or more ⁇ -blocking agents compared to a subject who does not have the allelic variant.
  • the /?- blocker is an agonist of a ?-adrenergic receptor.
  • the ?-blocker is an antagonist of a /?- adrenergic receptor. In another embodiment, the /?-blocker is an agonist of a ⁇ -adrenergic receptor.
  • ⁇ -blocking agents comprising: detecting for the subject the presence or absence of Val at position 646 of SEQ ID NO:2 or a -G- nucleotide at a position corresponding to position 2073 of SEQ ID NO: 1 , wherein the presence of a Val at position 646 of SEQ ID NO:2 or a -G- at nucleotide 2073 of SEQ ID NO: 1 , is indicative of an increased likelihood that a subject is non-responsive to one or more ?-blocking agents compared to a subject who does not have the allelic variant.
  • the ?-blocker is an antagonist of a -adrenergic receptor.
  • the /?-blocker is an agonist of a /?-adrenergic receptor.
  • Also provided herein are methods for determining responsiveness of a subject to one or more -blocking agents comprising: detecting the presence or absence of Val at position 646 of SEQ ID NO:2 or a -G- nucleotide at a position corresponding to position 2073 of SEQ ID NO: 1 , wherein the presence of a Val at position 646 of SEQ ID NO:2 or a -G- at nucleotide 2073 of SEQ ID NO: 1 , is indicative of an increased likelihood that a subject is hyper-responsive to one or more ⁇ - blocking agents compared to a subject who does not have the allelic variant.
  • the /?-block ⁇ r can be an antagonist of a /?-adr ⁇ nergic receptor.
  • the /?-blocker is an agonist of a ?-adrenergic receptor.
  • Exemplary ?-blockers well known in the art include, but are not limited to, Acebutolol, atenolol, Betaxolol, Bisoprolol, Carteolol, Carbedilol, Esmolol, Labetolol, Metoprolol, Nadolol, Penbutolol, Pindolol, Propranolol and Timolol.
  • s-blocking agents are used to treat: high blood pressure, angina, abnormal heart rythms, hypertrophic cardiomyopathy, heart failure, vasovagal fainting, migraines, essential tremor, bleeding from esophageal varices, stage fright, glaucoma and to prolong survival of heart attack patients.
  • the methods provided herein are useful to identify subjects that require or would benefit from a different treatment regimen than the use of /?-blockers for their respective disease or disorder.
  • the subject is heterozygous -GA- at a position corresponding to nucleotide 2073 of SEQ ID NO: 1 or heterozygous Val/lle at a position corresponding to position 646 of SEQ ID NO:2.
  • the subject is homozygous -GG- at a position corresponding to nucleotide 2073 of SEQ ID NO: 1 or homozygous Val/Val at a position corresponding to position 646 of SEQ ID NO:2.
  • torsade de pointes which leads to sudden loss of consciousness (syncope) and can cause sudden cardiac death.
  • the syndrome can be inherited (the genetic form) or acquired.
  • the inherited long QT Syndrome was first clearly described in 1 957. There are two variants, the autosomal dominant Romano-Ward type and the autosomal recessive Jervell and Lange Nielsen type. Even though LQTS was described almost 40 years ago, too many physicians are unaware of it. Whereas acquired long QT syndrome is most often due to the administration of medication. These medications are contraindicated in patients with the long QT syndrome.
  • LQTS long QT syndrome
  • syncope Sudden loss of consciousness
  • sudden death typically occurring during physical activity or emotional upset.
  • the syncopal episodes are often misdiagnosed as the common faint (vasovagal event) or a seizure.
  • Actual seizures are uncommon in long QT syndrome, but epilepsy is one of the common errors in diagnosis. Sudden loss of consciousness during physical exertion or during emotional excitement should strongly raise the possibility of the long QT syndrome.
  • a family history of unexplained syncope or sudden death in young people should also raise suspicion.
  • the 1646V variant in D-AKAP2 is indicative of a predisposition to the acquired form of Long Q-T syndrome. Accordingly, provided herein are methods that identify subjects who are predisposed or susceptible to acquired long Q-T syndrome. These methods are useful in identifying the class of subjects who should avoid taking particular medications, such as the well-documented group of medications that those diagnosed with long Q-T syndrome should avoid.
  • methods for indicating susceptibility of a subject to acquired long Q-T syndrome comprising: detecting the presence or absence of Val at position 646 of SEQ ID NO:2 or presence or absence of a -G- nucleotide at a position corresponding to position 2073 of SEQ ID NO: 1 , wherein the presence of a Val at position 646 of SEQ ID NO:2 or a -G- at nucleotide 2073 of SEQ ID NO: 1 , is indicative of increased susceptibility to acquired long Q- T syndrome, compared to the susceptibility of a subject who does not have the allelic variant.
  • the detecting step can be effected by a method selected from the group consisting of allele specific hybridization, primer specific extension, oligonucleotide ligation assay, restriction enzyme site analysis and single-stranded conformation polymorphism analysis.
  • the detecting step can comprise mass spectrometry.
  • the detection can be effected by detecting a signal moiety selected from the group consisting of radioisotopes, enzymes, antigens, antibodies, spectrophotometric reagents, chemiluminescent reagents, fluorescent reagents and other light producing reagents.
  • the subject is heterozygous -GA- at a position corresponding to nucleotide 2073 of SEQ ID NO: 1 or heterozygous Val/lle at a position corresponding to position 646 of SEQ ID NO:2.
  • the subject is homozygous -GG- at a position corresponding to nucleotide 2073 of SEQ ID NO: 1 or homozygous Val/Val at a position corresponding to position 646 of SEQ ID NO:2. 4.
  • Methods for indicating susceptibility to morbidity, increased or early mortality, or morbidity and increased or early mortality of a subject comprising: conducting an EKG exam; determining the EKG-PR-interval in the subject, wherein if the EKG- PR-interval is decreased; then determining the amino acid at position 646 of AKAP10/D-AKAP2 (SEQ ID NO:2) or the nucleotide present at position corresponding to nucleotide 2073 of SEQ ID NO: 1 , wherein the presence of Val at position 646 of SEQ ID NO:2 or the presence of a -G- at nucleotide position 2073 of SEQ ID NO: 1 , indicates increased susceptibility to morbidity, increased or early mortality, or morbidity and increased or early mortality of a subject.
  • the EKG-PR-interval in the subject can be compared to a predetermined standard EKG-PR-interval.
  • the predetermined standard EKG-PR-interval can be obtained from a known age-matched control that is homozygous -AA- at a position corresponding to nucleotide 2073 of SEQ ID NO: 1 or homozygous lle/lle at a position corresponding to position 646 of SEQ ID NO:2.
  • the detecting step can be effected by a method selected from the group consisting of allele specific hybridization, primer specific extension, oligonucleotide ligation assay, restriction enzyme site analysis and single-stranded conformation polymorphism analysis.
  • the detecting step can comprise mass spectrometry.
  • the detection step can be effected by detecting a signal moiety selected from the group consisting of radioisotopes, enzymes, antigens, antibodies, spectrophotometric reagents, chemiluminescent reagents, fluorescent reagents and other light producing reagents.
  • a signal moiety selected from the group consisting of radioisotopes, enzymes, antigens, antibodies, spectrophotometric reagents, chemiluminescent reagents, fluorescent reagents and other light producing reagents.
  • the subject is heterozygous -GA- at a position corresponding to nucleotide 2073 of SEQ ID NO: 1 or heterozygous Val/lle at a position corresponding to position 646 of SEQ ID NO:2.
  • the subject is homozygous -GG- at a position corresponding to nucleotide 2073 of SEQ ID NO: 1 or homozygous Val/Val at a position corresponding to position 646 of SEQ ID NO:2.
  • Methods of determining the presence or absence of allelic variants of a human AKAP10 gene are also provided.
  • the detection or identification of a G, C, or T nucleotide at position 2073 of the sense strand of the human AKAP10 gene coding sequence indicates the presence of an allelic variant.
  • the detection or identification of an A nucleotide at position 2073 of the sense strand of the human AKAP1 0 gene coding sequence, or the detection or identification of a T nucleotide at the same position in the antisense strand of the human AKAP1 0 gene coding sequence indicates the absence of polymorphism.
  • nucleotide other than a C at position 83587 of the SEQ ID NO: 1 7 or a nucleotide other than a G on the complementary strand detect or identify a nucleotide other than a G at position 1 29600 of the SEQ ID NO: 1 7 or a nucleotide other than a C on the complementary strand or a nucleotide other than T at position 1 56,277 of SEQ ID NO: 1 7 or a nucleotide other than A on the complementary strand
  • Nucleic acid detection methods are based in sequence-specific polynucleotides, oligonucleotides, probes and primers. Any method known to those of skill in the art for detecting a specific nucleotide within a nucleic acid sequence or for determining the identity of a specific nucleotide in a nucleic acid sequence is applicable to the methods of determining the presence or absence of an allelic variant of the AKAP10 gene.
  • Such methods include, but are not limited to, techniques utilizing nucleic acid hybridization of sequence-specific probes, nucleic acid sequencing, selective amplification, analysis of restriction enzyme digests of the nucleic acid, cleavage of mismatched heteroduplexes of nucleic acid and probe, alterations of electrophoretic mobility, primer specific extension, oligonucleotide ligation assay and single-stranded conformation polymorphism analysis.
  • primer extension reactions that specifically terminate by incorporating a dideoxynucleotide are useful for detection.
  • Several such general nucleic acid detection assays are known (see, e.g., U.S. Patent No. 6,030,778). a. Primer extension-based methods
  • a primer is prepared that specifically hybridizes adjacent to a polymorphic site in a particular nucleic acid molecule.
  • the primer is then extended in the presence of one or more dideoxynucleotides, typically with at least one of the dideoxynucleotides being the complement of the nucleotide that is polymorphic at the site.
  • the primer and/or the dideoxynucleotides can be labeled to facilitate a determination of primer extension and identity of the extended nucleotide.
  • primer extension and/or the identity of the extended nucleotide(s) are determined by mass spectrometry (see, e.g. , PCT Application Nos. PCT/US96/03651 (WO96/29431 ), PCT Application No. PCT/US97/20444 (WO 98/20166), PCT Application No. PCT/US97/201 94 (WO 98/2001 9), PCT Application No.
  • PCT/US91 /00046 (WO91 /1 3075), and U.S. Patent Nos. 5,605,798, 5,622,824, 5,856,092.
  • a typical detection method is allele specific hybridization using probes overlapping the polymorphic site and having about 5, 10, 1 5, 20, 25, or 30 nucleotides around the polymorphic region.
  • the probes can contain naturally occurring or modified nucleotides (see U.S. Patent No. 6, 1 56,501 ) .
  • oligonucleotide probes can be prepared in which the known polymorphic nucleotide is placed centrally (allele- specific probes) and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1 986) Nature 324: 163; Saiki et al. (1989) Proc. Natl Acad. Sci USA 86:6230; and Wallace et al. ( 1 979) Nucl. Acids Res. 6:3543) .
  • Allele specific oligonucleotide hybridization techniques can be used for the simultaneous detection of several nucleotide changes in different polymorphic regions.
  • oligonucleotides having nucleotide sequences of specific allelic variants are attached to a hybridizing membrane and this membrane is then hybridized with labeled sample nucleic acid. Analysis of the hybridization signal will then reveal the identity of the nucleotides of the sample nucleic acid.
  • several probes capable of hybridizing specifically to allelic variants are attached to a solid phase support, e.g., a "chip".
  • Oligonucleotides can be bound to a solid support by a variety of processes, including lithography. For example a chip can hold up to 250,000 oligonucleotides (GeneChip, Affymetrix, Santa Clara, CA) .
  • a chip includes all the allelic variants of at least one polymorphic region of a gene.
  • the solid phase support is then contacted with a test nucleic acid and hybridization to the specific probes is detected. Accordingly, the identity of numerous allelic variants of one or more genes can be identified in a simple hybridization experiment. c.
  • Nucleic acid amplification-based methods In other detection methods, it is necessary to first amplify at least a portion of an AKAP gene prior to identifying the allelic variant. Amplification can be performed, e.g. , by PCR and/or LCR, according to methods known in the art. In one embodiment, genomic DNA of a cell is exposed to two PCR primers and amplification is performed for a number of cycles sufficient to produce the required amount of amplified DNA. In typical embodiments, the primers are located between 1 50 and 350 base pairs apart.
  • Alternative amplification methods include: self sustained sequence replication (Guatelli, J. C. et al. , 1 990, Proc. Natl. Acad. Sci. U.S.A.
  • Oligonucleotides used as primers for specific amplification can carry the allelic variant of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. ( 1 989) Nucleic Acids Res. 1 7:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1 993) Tibtech 1 1 :238; Newton et al. (1 989) Nucl. Acids Res. 1 7:2503) .
  • any of a variety of sequencing reactions known in the art can be used to directly sequence at least a portion of an AKAP gene and to detect allelic variants, e.g. , mutations, by comparing the sequence of the sample sequence with the corresponding wild-type (control) sequence.
  • Exemplary sequencing reactions include those based on techniques developed by Maxam and Gilbert (Proc. Natl. Acad. Sci. USA (1 977) 74:560) or Sanger (Sanger et al.
  • Restriction enzyme digest analysis In some cases, the presence of a specific allele in nucleic acid, particularly DNA, from a subject can be shown by restriction enzyme analysis.
  • a specific nucleotide polymorphism can result in a nucleotide sequence containing a restriction site which is absent from the nucleotide sequence of another allelic variant. f. Mismatch Cleavage
  • cleavage agents such as, but not limited to, a nuclease, hydroxylamine or osmium tetroxide and with piperidine
  • cleavage agents can be used to detect mismatched bases in RNA/RNA DNA/DNA, or RNA/DNA heteroduplexes (Myers, et al. (1 985) Science 230: 1 242) .
  • the technique of "mismatch cleavage" starts by providing heteroduplexes formed by hybridizing a control nucleic acid, which is optionally labeled, e.g. , RNA or DNA, comprising a nucleotide sequence of an allelic variant with a sample nucleic acid, e.g, RNA or DNA, obtained from a tissue sample.
  • RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with S1 nuclease to enzymatically digest the mismatched regions.
  • either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine whether the control and sample nucleic acids have an identical nucleotide sequence or in which nucleotides they differ (see, for example, Cotton et al. (1 988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al. (1 992) Methods Enzymod. 21 7:286-295) . The control or sample nucleic acid is labeled for detection. g. Electrophoretic mobility alterations
  • alteration in electrophoretic mobility is used to identify the type of allelic variant in an AKAP gene.
  • SSCP single-strand conformation polymorphism
  • SSCP single-strand conformation polymorphism
  • Single-stranded DNA fragments of sample and control nucleic acids are denatured and allowed to renature.
  • the secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change.
  • the DNA fragments can be labeled or detected with labeled probes.
  • the sensitivity of the assay can be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence.
  • the subject method uses heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1 991 ) Trends Genet 7:5). h.
  • the identity of an allelic variant of a polymorphic region of an AKAP gene is obtained by analyzing the movement of a nucleic acid comprising the polymorphic region in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1 985) Nature 31 3:495) .
  • DGGE denaturing gradient gel electrophoresis
  • DNA will be modified to ensure that it does not completely denature, for example by adding a GC clamp of approxi mately 40 bp of high-melting GC-rich DNA by PCR.
  • a temperature gradient is used in place of a denaturing agent gradi ent to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1 987) Biophys Chem 265: 1275).
  • OLA Oligonucleotide ligation assay
  • identification of the allelic variant is carried out using an oligonucleotide ligation assay (OLA), as described, e.g. , in U.S. Patent No. 4,998,617 and in Landegren, U. et al. , Science 241 : 1077-1080 (1 988) .
  • OLA oligonucleotide ligation assay
  • the OLA protocol uses two oligonucleotides which are designed to be capable of hybridizing to abutting sequences of a single strand of a target.
  • One of the oligonucleotides is linked to a separation marker, e.g, . biotinylated, and the other is detectably labeled.
  • oligonucleotides will hybridize such that their termini abut, and create a ligation substrate. Ligation then permits the labeled oligonucleotide to be recovered using avidin, or another biotin ligand.
  • Nickerson, D. A. et al. have described a nucleic acid detection assay that combines attributes of PCR and OLA (Nickerson, D. A. et al. , Proc. Natl. Acad. Sci. (U.S.A.) 87:8923-8927 (1 990) . In this method, PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA.
  • each OLA reaction can be detected by using hapten specific antibodies that are labeled with different enzyme reporters, alkaline phosphatase or horseradish peroxidase. This system permits the detection of the two alleles using a high throughput format that leads to the production of two different colors.
  • SNP detection methods Also provided are methods for detecting single nucleotide polymorphisms. Because single nucleotide polymorphisms constitute sites of variation flanked by regions of invariant sequence, their analysis requires no more than the determination of the identity of the single nucleotide present at the site of variation and it is unnecessary to determine a complete gene sequence for each patient. Several methods have been developed to facilitate the analysis of such single nucleotide polymorphisms.
  • the single base polymorphism can be detected by using a specialized exonuclease-resistant nucleotide, as disclosed, e.g. , in Mundy, C. R. (U.S. Patent No. 4,656, 1 27) .
  • a primer complementary to the allelic sequence immediately 3' to the polymorphic site is permitted to hybridize to a target molecule obtained from a particular animal or human. If the polymorphic site on the target molecule contains a nucleotide that is complementary to the particular exonuclease-resistant nucleotide derivative present, then that derivative will be incorporated onto the end of the hybridized primer.
  • a solution-based method for determining the identity of the nucleotide of a polymorphic site is employed (Cohen, D. et al. (French Patent 2,650,840; PCT Application No. WO91 /02087)).
  • a primer is employed that is complementary to allelic sequences immediately 3' to a polymorphic site. The method determines the identity of the nucleotide of that site using labeled dideoxynucleotide derivatives, which, if complementary to the nucleotide of the polymorphic site will become incorporated onto the terminus of the primer.
  • k Genetic Bit Analysis
  • GBATM Genetic Bit Analysis
  • the method of Goelet, et al. uses mixtures of labeled terminators and a primer that is complementary to the sequence 3' to a polymorphic site.
  • the labeled terminator that is incorporated is thus determined by, and complementary to, the nucleotide present in the polymorphic site of the target molecule being evaluated.
  • the method of Goelet, et al. is typically a heterogeneous phase assay, in which the primer or the target molecule is immobilized to a solid phase.
  • allelic variants of a polymorphic region located in the coding region of a gene yet other methods than those described above can be used. For example, identification of an allelic variant which encodes a mutated protein can be performed by using an antibody specifically recognizing the mutant protein in, e.g. , immunohistochemistry or immunoprecipitation. Binding assays are known in the art and involve, e.g. , obtaining cells from a subject, and performing binding experiments with a labeled lipid, to determine whether binding to the mutated form of the protein differs from binding to the wild-type protein. m.
  • the identity of the allelic variant can be determined by determining the molecular structure of the mRNA, pre-mRNA, or cDNA.
  • the molecular structure can be determined using any of the above described methods for determining the molecular structure of the genomic DNA, e.g. , sequencing and SSCP.
  • Mass spectrometric methods Nucleic acids can also be analyzed by detection methods and protocols, particularly those that rely on mass spectrometry (see, e.g. , U.S. Patent Nos. 5,605,798, 6,043,031 , 6, 1 97,498, and International Patent Application No.
  • a typical format for performing the analyses is a chip based format in which the biopolymer is linked to a solid support, such as a silicon or silicon-coated substrate, for example, in the form of an array. More typically, when analyses are performed using mass spectrometry, particularly MALDI, nanoliter volumes of sample are loaded on, such that the resulting spot is about, or smaller than, the size of the laser spot. It has been found that when this is achieved, the results from the mass spectrometric analysis are quantitative. The area under the peaks in the resulting mass spectra are proportional to concentration (when normalized and corrected for background). Methods for preparing and using such chips are described in U.S. Patent No. 6,024,925, co-pending U.S. Application Serial Nos.
  • detector e.g. , oligonucleotides or oligonucleotide mimetics.
  • detector e.g. , oligonucleotides or oligonucleotide mimetics.
  • the molecular weight differences between the detector oligonucleotides must be large enough so that simultaneous detection (multiplexing) is possible. This can be achieved either by the sequence itself (composition or length) or by the introduction of mass-modifying functionalities into the detector oligonucleotides (see below) .
  • Mass modifying moieties can be attached, for instance, to either the 5'-end of the oligonucleotide, to the nucleobase (or bases), to the phosphate backbone, and to the 2'-position of the nucleoside (nucleosides) and/or to the terminal 3'-position.
  • Examples of mass modifying moieties include, for example, a halogen, an azido, or of the type, XR, wherein X is a linking group and R is a mass-modifying functionality.
  • the mass-modifying functionality can thus be used to introduce defined mass increments into the oligonucleotide molecule.
  • the mass-modifying functionality can be located at different positions within the nucleotide moiety (see, e.g. , U.S. Patent No. 5,547,835 and International PCT Application No. WO 94/21822) .
  • the mass-modifying moiety, M can be attached either to the nucleobase, (in case of the c 7 -deazanucleosides also to C-7), to the triphosphate group at the alpha phosphate or to the 2'-position of the sugar ring of the nucleoside triphosphate.
  • Modifications introduced at the phosphodiester bond have the advantage that these modifications do not interfere with accurate Watson-Crick base-pairing and additionally allow for the one-step post-synthetic site-specific modification of the complete nucleic acid molecule e.g. , via alkylation reactions (see, e.g. , Nakamaye et al. (1 988) Nucl. Acids Res. 1 6:9947-59) .
  • Typical mass-modifying functionalities are boron-modified nucleic acids since they are better incorporated into nucleic acids by polymerases (see, e.g. , Porter et al.
  • the mass-modification can be introduced for X in XR as well as using oligo-/polyethylene glycol derivatives for R.
  • the nucleic acid molecule e.g. , detector oligonucleotide (D) or the nucleoside triphosphates, respectively.
  • the oligo/polyethylene glycols can also be monoalkylated by a lower alkyl such as, but are not limited to, methyl, ethyl, propyl, isopropyl and t-butyl.
  • a lower alkyl such as, but are not limited to, methyl, ethyl, propyl, isopropyl and t-butyl.
  • Other chemistries can be used in the mass-modified compounds (see, e.g. , those described in Oligonucleotides and Analogues, A Practical Approach, F. Eckstein, editor, IRL Press, Oxford, 1 991 ).
  • various mass-modifying functionalities, R can be selected and attached via appropriate linking chemistries, X.
  • a simple mass-modification can be achieved by substituting H for halogens, such as F, Cl, Br and/or I, or pseudohalogens such as CN, SCN, NCS, or by using different alkyl, aryl or aralkyl moieties such as methyl, ethyl, propyl, isopropyl, t-butyl, hexyl, phenyl, substituted phenyl, benzyl, or functional groups such as CH 2 F, CHF 2 , CF 3 , Si(CH 3 ) 3 , Si(CH 3 ) 2 (C 2 H 5 ), Si(CH 3 )(C 2 H 5 ) 2 , Si(C 2 H 5 ) 3 .
  • Yet another mass-modification can be obtained by attaching homo- or heteropeptides through the nucleic acid molecule (e.g. , detector (D)) or nucleoside triphosphates).
  • nucleic acid molecule e.g. , detector (D)
  • nucleoside triphosphates e.g. , nucleoside triphosphates.
  • Simple oligoamides also can be used, e.g.
  • Different mass-modified detector oligonucleotides can be used to simultaneously detect all possible variants/mutants simultaneously.
  • all four base permutations at the site of a mutation can be detected by designing and positioning a detector oligonucleotide, so that it serves as a primer for a DNA/RNA polymerase with varying combinations of elongating and terminating nucleoside triphosphates.
  • mass modifications also can be incorporated during the amplification process.
  • a different multiplex detection format is one in which differentiation is accomplished by employing different specific capture sequences which are position-specifically immobilized on a flat surface (e.g. , a 'chip array').
  • Additional methods of analyzing nucleic acids include amplification- based methods including polymerase chain reaction (PCR), ligase chain reaction (LCR), mini-PCR, rolling circle amplification, autocatalytic methods, such as those using QJ replicase, TAS, 3SR, and any other suitable method known to those of skill in the art.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • mini-PCR mini-PCR
  • rolling circle amplification such as those using QJ replicase, TAS, 3SR, and any other suitable method known to those of skill in the art.
  • polymorphisms include but are not limited to, allele specific probes, Southern analyses, and other such analyses.
  • Primers refer to nucleic acids which are capable of specifically hybridizing to a nucleic acid sequence which is adjacent to a polymorphic region of interest or to a polymorphic region and are extended.
  • a primer can be used alone in a detection method, or a primer can be used together with at least one other primer or probe in a detection method.
  • Primers can also be used to amplify at least a portion of a nucleic acid.
  • a forward primer i.e. , 5' primer
  • a reverse primer i.e. , 3' primer
  • Probes refer to nucleic acids which hybridize to the region of interest and which are not further extended.
  • a probe is a nucleic acid which hybridizes adjacent to or at a polymorphic region of an AKAP gene and which by hybridization or absence of hybridization to the DNA of a subject will be indicative of the identity of the allelic variant of the polymorphic region of the gene.
  • Typical probes have a number of nucleotides sufficient to allow specific hybridization to the target nucleotide sequence.
  • the size of a probe can have to be longer to provide sufficiently specific hybridization, as compared to a probe which is used to detect a target sequence which is present in a shorter fragment of DNA.
  • a portion of an AKAP gene can first be amplified and thus isolated from the rest of the chromosomal DNA and then hybridized to a probe. In such a situation, a shorter probe will likely provide sufficient specificity of hybridization.
  • a probe having a nucleotide sequence of about 10 nucleotides can be sufficient.
  • RNA, DNA (single-stranded or double- stranded), PNA and their analogs can be labeled with any detectable reporter or signal moiety including, but not limited to radioisotopes, enzymes, antigens, antibodies, spectrophotometric reagents, chemiluminescent reagents, fluorescent and any other light producing chemicals. Additionally, these probes can be modified without changing the substance of their purpose by terminal addition of nucleotides designed to incorporate restriction sites or other useful sequences, proteins, signal generating ligands such as acridinium esters, and/or paramagnetic particles.
  • probes can also be modified by the addition of a capture moiety (including, but not limited to para-magnetic particles, biotin, fluorescein, dioxigenin, antigens, antibodies) or attached to the walls of microtiter trays to assist in the solid phase capture and purification of these probes and any DNA or RNA hybridized to these probes.
  • a capture moiety including, but not limited to para-magnetic particles, biotin, fluorescein, dioxigenin, antigens, antibodies
  • Fluorescein can be used as a signal moiety as well as a capture moiety, the latter by interacting with an anti-fluorescein antibody.
  • Any probe, primer or antisense molecule can be prepared according to methods well known in the art and described, e.g. , in Sambrook, J. Fritsch, E.F., and Maniatis, T.
  • probes and primers can be prepared using the Polymerase Chain Reaction (PCR) using primers having an appropriate sequence.
  • Oligonucleotides can be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch (Novato, CA); Applied Biosystems (Foster City, CA) and other methods) .
  • an automated DNA synthesizer such as are commercially available from Biosearch (Novato, CA); Applied Biosystems (Foster City, CA) and other methods
  • phosphorothioate oligonucleotides can be synthesized by the method of Stein et al. (1 988, Nucl. Acids Res. 1 6:3209)
  • methylphosphonate oligonucleotides for example, can be prepared by use of controlled pore glass polymer supports (Sarin et al. , 1 988, Proc. Natl. Acad. Sci. U.S.A. 85:7448- 7451 ).
  • Probes and primers used in the methods of detecting allelic variants in human AKAP10 genes are of sufficient length to specifically hybridize to portions of AKAP10 gene at polymorphic sites. Typically such lengths depend upon the complexity of the source organism genome. For humans such lengths are at least 14-1 6 nucleotides, and typically can be 20, 30, 50, 100 or more nucleotides.
  • probes and primers include the following:
  • nucleic acid molecule includes at least 5 contiguous nucleotides from nucleotide 2069 to nucleotide 2077 of SEQ. ID. NO: 3; (2) at least 14 or 1 6 contiguous nucleotides of the AKAP10 allele or complement thereof, wherein the nucleic acid includes the nucleotide at position 2073 of SEQ ID No. 1 replaced with G, C or T.
  • nucleic acid molecule includes at least 5 contiguous nucleotides from nucleotide 1 29556 to nucleotide 1 29604 of SEQ. ID. NO: 1 4;
  • nucleic acid includes the nucleotide at position 1 29600 of SEQ ID No. 1 7 replaced with A, C or T; (5) at least 14 or 1 6 contiguous nucleotides of the AKAP1 0 allele or complement thereof, wherein the nucleic acid molecule includes at least 5 contiguous nucleotides from nucleotide 83583 to nucleotide 83591 of SEQ. ID. NO: 1 3;
  • nucleic acid includes the nucleotide at position 83587 of SEQ ID No. 1 7 replaced with G, A or T;
  • nucleic acid molecule includes at least 5 contiguous nucleotides from nucleotide 1 56,273 to nucleotide 1 56281 of SEQ. ID. NO: 1 8;
  • nucleic acid includes the nucleotide at position 156277 of SEQ ID No. 17 replaced with C, A or G;
  • probes and primers they have fewer nucleotides than the sequence of nucleotides 1 38 to 21 26 of SEQ. ID. NO: 1 or fewer nucleotides than the sequence of nucleotides 83,580 to 1 56,577 of SEQ ID NO: 1 7.
  • Antisense compounds can be conveniently and routinely made through the well-known technique of solid phase synthesis.
  • Antisense compounds are typically 8 to 30 nucleotides in length complementary to a targeted to a nucleic acid molecule and modulates its expression.
  • the targeted nucleic acid molecule represents the coding strand.
  • an antisense compound is an antisense oligonucleotide which comprises the complement of at least an 8 nucleotide segment of SEQ ID NO: 3 including the nucleotide at position 2073 of SEQ ID NO: 3.
  • An antisense compound can contain at least one modified nucleotide which can confer nuclease resistance or increase the binding of the antisense compound with the target nucleotide.
  • the antisense compound can containing at least one internucleoside linkage wherein the modified internucleoside linkage of the antisense oligonucleotide can be a phosphorothioate linkage, a morpholino linkage or a peptide-nucleic acid linkage.
  • Typical modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • oligonucleosides include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH 2 component parts.
  • Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. :
  • An antisense compound can contain at least one least one modified sugar moiety wherein the modified sugar moiety of the antisense oligonucleotide is a 2'-0-methoxyethyl sugar moiety or a 2'- dimethylaminooxyethoxy sugar moiety.
  • Modified oligonucleotides can also contain one or more substituted sugar moieties.
  • Typical oligonucleotides comprise one of the following at the 2' position: OH; F; 0-, S ⁇ , or N-alkyl; 0-, S-, or N-alkenyl; 0-, S-- or N-alkynyl; or 0-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C-, to C 10 alkyl or C 2 to C 10 alkenyl and alkynyl.
  • Exemplary oligonucleotides contain are 0[(CH 2 ) n 0] m CH 3 , 0(CH 2 ) n OCH 3 , 0(CH 2 ) n NH 2 , 0(CH 2 ) n CH 3 , 0(CH 2 ) n NH 2 , and 0(CH 2 ) n ON[(CH 2 ) n CH 3 )] 2 , where n and m are from 1 to about 10.
  • oligonucleotides comprise one of the following at the 2' position: C-, to C 10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O- aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , S0 2 CH 3 , ON0 2 , N0 2 , N 3 , NH 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties.
  • a typical modification includes an alkoxyalkoxy group, 2'-methoxyethoxy (2'-0-CH 2 CH 2 OCH 3 , also known as 2'-0-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1 995, 78, 486-504) .
  • Another exemplary modification includes 2'- dimethylaminooxyethoxy, i.e., a 0(CH 2 ) 2 ON(CH 3 ) 2 group, also known as 2'-DMAOE.
  • modifications include 2'-methoxy (2'-0-CH 3 ), 2'- aminopropoxy (2'-OCH 2 CH 2 CH 2 NH 2 ) and 2'-fluoro (2'-F) . Similar modifications can also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligonucleotides can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the reparation of such modified sugar structures include, but are not limited to, U.S. Pat.
  • An antisense compound can contain at least one modified nucleobase.
  • Oligonucleotides can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U) .
  • Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6- methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyI uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4- thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8- substituted adenines and guanines, 5-haIo particularly 5-bromo, 5- trifluoromethyl and other 5-substit
  • nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1 990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1 991 , 30, 613, and those disclosed by Sanghvi, Y. S., Crooke, S. T., and Lebleu, B. eds., Antisense Research and Applications, CRC Press, Boca Raton, 1 993, pp. 289-302. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention.
  • the antisense compound can be a chimeric oligonucleotide.
  • Chimeric antisense compounds can be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos.
  • electrocardiographs Devices that can be used to measure an electrocardiogram are referred to as electrocardiographs.
  • electrocardiographs A variety of electrocardiographs are well known in the art and include, for example, those disclosed is U.S. Pat. Nos. 4,377,81 3, 4,483,346, 4,98,479, 4,840, 1 83, 4,974599. Any known electrocardiograph can be used in the methods provided herein. Methods of using electrocardiographs to determine P-R interval and Q-T interval also are well known in the art, and any such method can be used in the methods provided herein. F. Association of AKAP10 Allelic Variants with Morbidity or Increased Mortality
  • Polymorphisms of the genome can lead to altered gene function, protein function or mRNA instability. To identify those polymorphisms that have clinical relevance is the goal of a world-wide scientific effort.
  • a morbidity susceptibility gene could be a gene that is expressed in many different cell types or tissues (housekeeping gene) and its altered function can facilitate the expression of a clinical phenotype caused by a disease-specific susceptibility gene that is involved in a pathway specific for this disorder.
  • morbidity susceptibility genes might predispose people to develop a distinct disease according to their genetic make-up for this disease.
  • Candidates for these genes can involve basic cellular processes such as: transcription, translation, heat-shock proteins, protein trafficking, DNA repair, assembly systems for subcellular structures (e.g. , mitochondria, peroxysomes and other cellular microbodies), receptor signaling cascades, immunology, etc. Those pathways control the quality of life at the cellular level as well as for the entire organism.
  • Mutations/polymorphisms located in genes encoding proteins for those pathways can reduce the fitness of cells and make the organism more susceptible to express the clinical phenotype caused by the action of a disease-specific susceptibility gene. Therefore, these morbidity susceptibility genes can be potentially involved in a whole variety of different complex diseases if not in all.
  • AKAP A kinase anchoring protein
  • AKAPs have a PKA binding region located in their COOH-terminal portion.
  • Polymorphic AKAP genes such as those provided herein, serve as markers for detecting predisposition to disease and various conditions.
  • the AKAP alleles and gene products, especially the AKAP10-5 gene product should be suitable pharmaceutical targets and gene therapy targets.
  • designated AKAP10-7 contains a single nucleotide polymorphism (SNP), a G-to-A transition, at nucleotide position 1 29,600 of the human chromosome 1 7 sequence (also referred to herein as SNP "In10").
  • This SNP is located four bases 3' to the exon 10/intron 10 boundary of AKAP10 gene.
  • Another identified SNP, AKAP10-1 is an allelic variant with a T to C transversion at nucleotide position 1 56,277 of the AKAP10 genomic clone which is located in the 3' untranslated region of the gene (also referred to herein as SNP "3' UTR").
  • SNP "3' UTR" the frequency of occurrence of two allelic variants of the AKAP10 gene, AKAP10-5 and AKAP10-1 , in such a population were found to decrease with age.
  • the AKAP1 0-5 and AKAP10-1 alleles are useful markers for predicting susceptibility to morbidity and/or increased or early mortality.
  • the methods provided herein can be used for predicting susceptibility to morbidity, increased or early mortality, or morbidity and increased mortality, by detecting the presence of the various AKAP1 0 allelic variants known in the art or dislcosed herein, individually, or in combination with other AKAP10 allelic variants, in an organism, particularly an animal and particularly a human.
  • AKAP1 0-5 and other allelic variants of the AKAP10 gene known in the art or dislcosed herein are potential functional variants of a morbidity susceptibility gene and/or of a gene involved in increased mortality and/or a gene related to an alteration in signal transduction and associated disorders and thus is useful for screening for potential therapeutics.
  • allelic variants of AKAP10 gene expression can be determined according to methods known in the art.
  • Allelic variants of AKAP genes can be assayed individually or in combination.
  • RNA Analysis a. Northern Blot Detection of RNA
  • the northern blot technique is used to identify a RNA fragment of a specific size from a complex population of RNA using gel electrophoresis and nucleic acid hybridization.
  • Northern blotting is a well- known technique in the art. Northern blot analysis is commonly used to detect specific RNA transcripts expressed in a variety of biological samples and have been described in Sambrook, J. et al. (Molecular Cloning, 3 rd Edition, Cold Spring Harbor Press) . Briefly, total RNA is isolated from any biological sample by the method of Chomczynski and Sacchi (Anal. Biochem. (1 987) 1 62, 1 56- 1 59) .
  • Poly-adenylated mRNA is purified from total RNA using mini-oligo (dT) cellulose spin column kit with methods as outlined by the suppliers (Invitrogen, Carlsbad CA.). Denatured RNA is electrophoresed through a denaturing 1 .5% agarose gel and transferred onto a nitrocellulose or nylon based matrix. The mRNAs are detected by hybridization of a radiolabeled or biotinylated oligonucleotide probe specific to the polymorphic regions as disclosed herein. b. Dot Blot/Slot Blot Specific RNA transcripts can be detected using dot and slot blot assays to evaluate the presence of a specific nucleic acid sequence in a complex mix of nucleic acids.
  • RNA transcripts can be detected by adding the RNA mixture to a prepared nitrocellulose or nylon membrane. RNA is detected by the hybridization of a radiolabeled or biotinylated oligonucleotide probe complementary to the AKAP sequences as disclosed herein. c. RT-PCR
  • the RT-PCR reaction can be performed, as described by K.-Q. Hu et al., Virology 1 81 :721 -726 (1 991 ), as follows: the extracted mRNA is transcribed in a reaction mixture 1 micromolar antisense primer, and 25 U AMV (avian myeloblastosis virus) or MMLV (Moloney murine leukemia virus) reverse transcriptase. Reverse transcription is performed and the cDNA is amplified in a PCR reaction volume with Taq polymerase. Optimal conditions for cDNA synthesis and thermal cycling can be readily determined by those skilled in the art.
  • AKAP10 proteins can be expressed in a recombinantly engineered cell such as bacteria, yeast, insect, mammalian, or plant cells. Those of skill in the art are knowledgeable in the numerous expression systems available for expression of a nucleic acid encoding proteins such as polymorphic human AKAP1 0 proteins. b. Expression of AKAP Protein
  • the isolated nucleic acid encoding a full-length polymorphic human AKAP10 protein, or a portion thereof, such as a fragment containing the site of the polymorphism, can be introduced into a vector for transfer into host cells. Fragments of the polymorphic human AKAP10 proteins can be produced by those skilled in the art, without undue experimentation, by eliminating portions of the coding sequence from the isolated nucleic acids encoding the full-length proteins.
  • Expression vectors are used expression of the protein in the host cell is desired.
  • An expression vector includes vectors capable of expressing nucleic acids that are operatively linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such nucleic acids.
  • an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the cloned DNA.
  • Appropriate expression vectors are well known to those of skill in the art and include those that are replicable in eukaryotic cells and/or prokaryotic cells and those that remain episomal or those which integrate into the host cell genome.
  • Such plasmids for expression of polymorphic human AKAPI O-encoding nucleic acids in eukaryotic host cells, particularly mammalian cells include cytomegalovirus (CMV) promoter-containing vectors, such as pCMV5, the pSV2dr.fr expression vectors, which contain the SV40 early promoter, mouse dhfr gene, SV40 polyadenylation and splice sites and sequences necessary for maintaining the vector in bacteria, and MMTV promoter-based vectors.
  • CMV cytomegalovirus
  • the nucleic acids encoding polymorphic human AKAP10 proteins, and vectors and cells containing the nucleic acids as provided herein permit production of the polymorphic proteins, as well as antibodies to the proteins.
  • This provides a means to prepare synthetic or recombinant polymorphic human AKAP10 proteins and fragments thereof that are substantially free of contamination from other AKAPs and proteins in general, the presence of which can interfere with analysis of the polymorphic proteins.
  • the polymorphic proteins can be expressed in combination with selected other proteins that AKAP1 0 can associate with in cells. The ability to selectively express the polymorphic AKAP1 0 proteins alone or in combination with other selected proteins makes it possible to observe the functioning of the recombinant polymorphic proteins within the environment of a cell.
  • isolated nucleic acids encoding an AKAP protein will typically be achieved by operably linking, for example, the DNA or cDNA to a promoter (which is either constitutive or regulatable), followed by incorporation into an expression vector.
  • the vectors can be suitable for replication and integration in either prokaryotes or eukaryotes.
  • Typical expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the DNA encoding a protein.
  • it is desirable to construct expression vectors which contain, a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription/translation terminator.
  • modifications can be made to an AKAP10 protein without diminishing its biological activity. Some modifications can be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications are well known to those of skill in the art and include, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids (e.g., poly His) placed on either terminus to create conveniently located purification sequences. Restriction sites or termination codons can also be introduced. There are expression vectors that specifically allow the expression of functional proteins. One such vector, Plasmid 577, described in U.S. Pat. No.
  • This plasmid contains the following DNA segments: (a) a fragment of pBR322 containing bacterial beta-lactamase and origin of DNA replication; (b) a cassette directing expression of a neomycin resistance gene under control of HSV-1 thymidine kinase promoter and poly-A addition signals; (c) a cassette directing expression of a dihydrofolate reductase gene under the control of a SV-40 promoter and poly-A addition signals; (d) cassette directing expression of a rabbit immunoglobulin heavy chain signal sequence fused to a modified hepatitis C virus (HCV) E2 protein under the control of the Simian Virus 40 T-Ag promoter and transcription enhancer, the hepatitis B virus surface antigen (HBsAg) enhancer I followed by a fragment of Herpes Simplex Virus-1 (HS)
  • Plasmids for the expression of secreted AKAP proteins can be constructed by replacing the hepatitis C virus E2 protein coding sequence in plasmid 577 with a AKAP sequence of SEQ ID NO: 3 or a fragment thereof.
  • the resulting plasmid is transfected into CHO/dhfr-cells (DXB- 1 1 1 ) (Uriacio, et al., PNAS 77, 4451 -4466; 1 980); these cells are available from the A.T.C.C, 1 2301 Parklawn Drive, Rockville, Md. 20852, under Accession No.
  • Heterologous nucleic acid can be introduced into host cells by any method known to those of skill in the art, such as transfection with a vector encoding the heterologous nucleic acid by CaP0 4 precipitation (see, e.g. , Wigler et al. (1 979) Proc. Natl. Acad. Sci. USA 75: 1 373-1 376) or lipofectamine (GIBCO BRL #1 8324-01 2) . Recombinant cells can then be cultured under conditions whereby the polymorphic human AKAP10 protein encoded by the nucleic acid is expressed. Suitable host cells include mammalian cells (e.g., HEK293, including but are not limited to, those described in U.S. Patent No.
  • Xenopus o ⁇ cytes can also be used for expression of in vitro RNA transcripts of the DNA.
  • Heterologous nucleic acid can be stably incorporated into cells or can be transiently expressed using methods known in the art.
  • Stably transfected mammalian cells can be prepared by transfecting cells with an expression vector having a selectable marker gene (such as, for example, the gene for thymidine kinase, dihydrofolate reductase, neomycin resistance, and the like), and growing the transfected cells under conditions selective for cells expressing the marker gene.
  • a reporter gene such as the E. coli ⁇ -galactosidase gene
  • Heterologous nucleic acid can be maintained in the cell as an episomal element or can be integrated into chromosomal DNA of the cell.
  • the resulting recombinant cells can then be cultured or subcultured (or passaged, in the case of mammalian cells) from such a culture or a subculture thereof. Methods for transfection, injection and culturing recombinant cells are known to the skilled artisan.
  • the polymorphic human AKAP1 0 proteins or fragments thereof can be purified using protein purification methods known to those of skill in the art. For example, antibodies or other ligands that specifically bind to the proteins can be used for affinity purification and immunoprecipitation of the proteins.
  • the AKAP10 proteins can be purified by standard techniques well known to those of skill in the art. Recombinantly produced proteins can be directly expressed or expressed as a fusion protein. The recombinant protein is purified by a combination of cell lysis (e.g., sonication, French press) and affinity chromatography. The proteins, recombinant or synthetic, can be purified to substantial purity by standard techniques well known in the art, including detergent solubilization, selective precipitation with such substances as ammonium sulfate, column chromatography, immunopurification methods, and others. (See, for example, R.
  • the AKAP proteins when presented as an immunogen, should elicit production of a specifically reactive antibody.
  • Immunoassays for determining binding are well known to those of skill in the art, as are methods of making and assaying for antibody binding specificity/affinity.
  • Exemplary immunoassay formats include ELISA, competitive immunoassays, radioimmunoassays, Western blots, indirect immunofluorescent assays, in vivo expression or immunization protocols with purified protein preparations.
  • the detection of immunocomplex formation is well known in the art and can be achieved by methods generally based upon the detection of a label or marker, such as any of the radioactive, fluorescent, biological or enzymatic tags.
  • antibodies are raised to these proteins in either their native configurations or in non-native configurations. Anti-idiotypic antibodies can also be generated.
  • a variety of analytic methods are available to generate a hydrophilicity profile of proteins. Such methods can be used to guide the artisan in the selection of peptides for use in the generation or selection of antibodies which are specifically reactive, under immunogenic conditions. See, e.g., J. Janin, Nature, 277 (1 979) 491 - 492; Wolfenden, et al., Biochemistry 20(1 981 ) 849-855; Kyte and Doolite, J. Mol. Biol. 1 57 (1982) 105-132; Rose, et al., Science 229 ( 1 985) 834-838.
  • immunogens can be used to produce antibodies specifically reactive with AKAP proteins.
  • Isolated recombinant, synthetic, or native polypeptides are typical immunogens (antigen) for the production of monoclonal or polyclonal antibodies.
  • Polypeptides are typically denatured, and optionally reduced, prior to formation of antibodies for screening expression libraries or other assays in which a putative AKAP protein is expressed or denatured in a non-native secondary, tertiary, or quartenary structure.
  • the AKAP protein (SEQ ID NO: 4, or a portion thereof) is injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies can be generated for subsequent use in immunoassays to measure the presence and quantity of the protein.
  • an immunogen typically a purified protein, a protein coupled to an appropriate carrier (e.g., GST, keyhole limpet hemanocyanin, etc.), or a protein incorporated into an immunization vector such as a recombinant vaccinia virus (see, U.S. Pat. No.
  • the filter is placed in a heat-sealable plastic bag containing a solution of 5% nonfat dried milk in PBS with a 1 : 1 00 to 1 :2000 dilution of affinity purified anti-AKAP peptide antibodies, incubated at 4 degrees Celsius for 2 hours, followed by three 1 0 min washes in PBS.
  • An alkaline phosphatase conjugated secondary antibody i.e., anti-mouse/rabbit IgG
  • the bands are visualized upon the addition and development of a chromogenic substrate such as 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT).
  • a chromogenic substrate such as 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT).
  • the filter is incubated in the solution at room temperature until the bands develop to the desired intensity.
  • Molecular mass determination is made based upon the mobility of pre-stained molecular weight standards (Rainbow markers, Amersham, Arlington Heights, III.).
  • MMIA Microparticle Enzyme Immunoassay
  • AKAP10 proteins and peptides are detected using a standard commercialized antigen competition EIA assay or polyclonal antibody sandwich EIA assay on the IMx.RTM Analyzer (Abbott Laboratories,
  • Samples containing the AKAP1 0 protein are incubated in the presence of anti-AKAP10 coated microparticles .
  • the microparticles are washed and secondary polyclonal anti-AKAP10 antibodies conjugated with detectable entities (i.e., alkaline phosphatase) are added and incubated with the microparticles.
  • the microparticles are washed and the bound antibody/antigen/antibody complexes are detected by adding a substrate (i.e. 4-methyl umbelliferyl phosphate) (MUP) that will react with the secondary conjugated antibody to generate a detectable signal.
  • a substrate i.e. 4-methyl umbelliferyl phosphate
  • Intracellular localization of the AKAP10 protein can be determined by a variety of in situ hybridization techniques. In one method cells are fixed with fixed in 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS; pH7.4) for 5 min., rinsed in PBS for 2 min., dilapidated and dehydrated in an ethanol series (50, 70 and 95%) (5 min. each and stored in 95% ethanol at 4 degrees Celsius) .
  • PBS phosphate buffered saline
  • the cells are stained with the primary anti-AKAP1 0 antibody and a mixture of secondary antibodies used for detection. Laser-scanning confocal microscopy is performed to localize the AKAP1 0 protein. 4. Binding Assays
  • Assays to measure the interaction between AKAP10 and the regulatory subunits Rl and/or Rll of the Protein Kinase A holoenzyme include immobilized binding assays, solution binding assays and the like. In some instances, it can be desirable to monitor binding between
  • AKAP10 and PKA can be desirable to specifically monitor the binding between AKAP10 and a cellular component (other than PKA) to which it binds.
  • Assays can be performed in a variety of formats, including cell-based assays, such as di-hybrid screening or complementation assays as described in U.S. Pat. No. 5,283, 1 73 and Patent Cooperation Treaty (PCT) Publication No. WO 91 /1 6457, respectively. Assays of this type are particularly useful for assessing intracellular efficacy of test compounds.
  • Non-cell-based assays include scintillation proximity assays, cAMP competition assays, ELISA assays, radioimmunoassays, chemiluminescent assays, and the like.
  • assay procedures are well known in the art and generally described, e.g., in Boudet et al., J. Immunol. Meth., 142:73-82 (1 991 ); Ngai et al., J. Immunol. Meth., 1 58:267-276 (1 993); Pruslin et al., J. Immunol. Meth., 1 37:27-35 (1 991 ); Udenfriend et al., Proc. Natl. Acad. Sci. USA,
  • PKA proteins were visualized by Coomassie Staining.
  • PKA proteins can be radiolabeled or labeled with a flurophore to allow detection.
  • PKA phosphorylation of protein substrate Cyclic AMP-dependent protein kinase (PKA) catalyzes the transfer of gamma phosphate from adenosine triphosphate (ATP) to a serine or threonine residue in a protein substrate.
  • PKA Cyclic AMP-dependent protein kinase
  • a short synthetic peptide (Leucine-Arg-Arg-Alanine-Serine-Leucine-Glycine or LRRASLG) is used as a substrate to assay the specific type of PKA activity as described in Pearson et. al., Methods of Enzymology 200, 62-81 (1 991 ) .
  • the PKA assay is typically carried out in a reaction of the enzyme with a peptide substrate and gamma 32P-ATP followed by separation of the 32P-peptide product from the unreacted gamma 32P-ATP on a phosphocellulose membrane.
  • This method requires at least one basic amino acid residue in the peptide substrate.
  • the peptide substrate can be tagged with a biotin group so that the biotinylated 32P-peptide product consistently binds to a streptavidin membrane in a manner independent of the peptide sequence as described in Goueli et al Analytical Biochemistry 225, 1 0-1 7, (1 995) .
  • the separation of the 32P-peptide product from the free gamma 32P-ATP using affinity binding and ultrafiltration separation to analyze a mixture sample as described in U.S. Patent No. 5,869,275.
  • the effect of the mutation can be determined, e.g. , by producing transgenic animals in which the allelic variant has been introduced and in which the wild-type gene or predominant allele can have been knocked out. Comparison of the level of expression of the protein in the mice transgenic for the allelic variant with mice transgenic for the predominant allele will reveal whether the mutation results in increased or decreased synthesis of the associated protein and/or aberrant tissue distribution or intracellular localization of the associated protein. Such analysis could also be performed in cultured cells, in which the human variant allele gene is introduced and, e.g. , replaces the endogenous gene in the cell. For mutant AKAP proteins binding to signaling enzymes such as PKA is also examined.
  • a specific treatment can be administered to a subject having such a mutation.
  • the subject can be treated by administration of a compound which increases synthesis, such as by increasing AKAP gene expression, and wherein the compound acts at a regulatory element different from the one which is mutated.
  • the subject can be treated by administration of a compound which reduces protein production, e.g. , by reducing AKAP gene expression or a compound which inhibits or reduces the activity of AKAP protein.
  • an individual allelic variant that associates with morbidity and/or mortality and/or an alteration in signal transduction will not be used in isolation as a prognosticator.
  • An allelic variant typically will be one of a plurality of indicators that are used. The other indicators can be the manifestation of other risk factors for morbidity and/or mortality and other evidence of altered signal transduction.
  • allelic variants of the AKAP10 gene can be determined. Variants can be assayed individually or assayed simultaneously using multiplexing methods as described above or any other labelling method that allows different variants to be identified. In particular, variants of the AKAP1 0 gene can be assayed using kits (see below) or any of a variety microarrays known to those in the art. For example, oligonucleotide probes comprising the polymorphic regions surrounding any polymorphism in the AKAP1 0 gene can be designed and fabricated using methods such as those described in U.S. Patent Nos.
  • exemplary steps include detecting the presence or absence of an allele of the human AKAP10 containing other than an A at position 2073 of the coding sequence of the AKAP10 gene; wherein the presence of an allele containing other than an A at position 2073 is indicative of increased susceptibility to morbidity, increased or early mortality, or morbidity and increased or early mortality as compared to the susceptibility of a human being who does not comprise an allele containing other than an A at position 2073 of the AKAP1 0 gene coding sequence.
  • a healthy patient database see, U.S.
  • nucleic acid sequences encoding polymorphic human AKAP1 0 proteins are represented by nucleotides which encode the amino acid sequence as set forth in SEQ. ID. NO: 3.
  • Such polymorphic nucleotide sequences can encode variant amino acid sequences, such as the sequence set forth in SEQ. ID. NO: 4 in which amino acid 646 has been replaced with a valine; other amino acid sequence variants at amino acid 646 include leucine or phenylalanine.
  • nucleic acid sequences represent allelic variants of the AKAP10 gene which are not located in protein coding regions. Such as set forth in nucleotide position 83,580 to position 1 56,577 of SEQ ID NO: 1 3, 1 4 and 1 8.
  • Nucleic acid encoding polymorphic human AKAP10 proteins and genes provided herein can be isolated by screening suitable human cDNA or human genomic libraries under suitable hybridization conditions with nucleic acids such as those provided in SEQ. ID. NOS: 1 , 3, 1 3, 14, 1 7 and 1 8.
  • Suitable libraries can be prepared from human tissue and cell samples.
  • cDNA encoding a polymorphic human AKAP10 libraries prepared from different tissues can be screend since the allele can not be expressed in all tissues or at similar levels in different tissues.
  • the library can be screened with a portion of DNA including substantially the entire human AKAP10 or polymorphic AKAP10 protein-encoding sequence as set forth in SEQ. ID. NOS.
  • the library can be screened with a suitable probe.
  • positive clones are identified by detecting a hybridization signal; the identified clones are characterized by restriction enzyme mapping and/or DNA sequence analysis, and then examined, by comparison with the sequences set forth herein to ascertain whether they include DNA encoding a complete polymorphic human AKAP10 protein (i.e., if they include translation initiation and termination codons) . If the selected clones are incomplete, they can be used to rescreen the same or a different library to obtain overlapping clones. If the library is genomic, then the overlapping clones can include exons and introns. If the library is a cDNA library, then the overlapping clones will include an open reading frame. In both instances, clones can be identified by comparison with the DNA and encoded proteins provided herein.
  • oligonucleotides based on the human AKAP10 or polymorphic AKAP10 protein-encoding sequence as set forth in SEQ. ID. NOS. 1 , 3, 1 3, 14, 1 7 and 1 8, can be used to amplify fragments of the protein coding region of the AKAP10 gene from human cDNA or genomic sequence.
  • vector or plasmid refers to discrete elements that are used to introduce heterologous DNA into cells for either expression or replication thereof. Selection and use of such vehicles are well within the skill of the artisan.
  • K. Transgenic Animals Methods for making transgenic animals using a variety of transgenes have been described in Wagner et al. , Proc. Nat. Acad. Sc. U.S.A., Vol. 78, p. 5016, 1981 ; Stewart et al. , Science, Vol. 21 7, p. 1046, 1 982; Constantini et al. , Nature, Vol. 294, p.
  • transgene is used herein to describe genetic material that has been or is about to be artificially inserted into the genome of a mammalian cell, particularly a mammalian cell of a living animal.
  • the transgene is used to transform a cell, meaning that a permanent or transient genetic change, typically a permanent genetic change, is induced in a cell following incorporation of exogenous DNA.
  • a permanent genetic change is generally achieved by introduction of the DNA into the genome of the cell.
  • Vectors for stable integration include, but are not limited to, plasmids, retroviruses and other animal viruses and YACS.
  • transgenic mammals including, but are not limited to, cows, pigs, goats, horses and others, and particularly rodents, including rats and mice.
  • the transgenic-animals are mice.
  • Transgenic animals contain an exogenous nucleic acid sequence present as an extrachromosomal element or stably integrated in all or a portion of its cells, especially germ cells. Unless otherwise indicated, it will be assumed that a transgenic animal comprises stable changes to the germline sequence.
  • "chimeras” or “chimeric animals” are generated, in which only a subset of cells have the altered genome. Chimeras are primarily used for breeding purposes in order to generate the desired transgenic animal. Animals having a heterozygous alteration are generated by breeding of chimeras. Male and female heterozygotes are typically bred to generate homozygous animals.
  • the exogenous gene is usually either from a different species than the animal host, or is otherwise altered in its coding or non-coding sequence.
  • the introduced gene can be a wild-type gene, naturally occurring polymorphism or a genetically manipulated sequence, for example having deletions, substitutions or insertions in the coding or non-coding regions.
  • the introduced gene is a coding sequence, it is usually operably linked to a promoter, which can be constitutive or inducible, and other regulatory sequences required for expression in the host animal.
  • Transgenic animals can comprise other genetic alterations in addition to the presence of alleles of AKAP genes.
  • the genome can be altered to affect the function of the endogenous genes, contain marker genes, or contain other genetic alterations (e.g. , alleles of other genes associated with cardiovascular disease).
  • a “knock-out” of a gene means an alteration in the sequence of the gene that results in a decrease of function of the target gene, typically such that target gene expression is undetectable or insignificant.
  • a knock-out of an endogenous AKAP gene means that function of the gene has been substantially decreased so that expression is not detectable or only present at insignificant levels.
  • "Knock-out" transgenics can be transgenic animals having a heterozygous knock-out of an AKAP gene or a homozygous knock-out.
  • “Knock-outs” also include conditional knock-outs, where alteration of the target gene can occur upon, for example, exposure of the animal to a substance that promotes target gene alteration, introduction of an enzyme that promotes recombination at the target gene site (e.g. , Cre in the Cre-lox system), or other method for directing the target gene alteration postnatally.
  • a "knock-in" of a target gene means an alteration in a host cell genome that results in altered expression (e.g. , increased (including ectopic)) of the target gene, e.g. , by introduction of an additional copy of the target gene, or by operatively inserting a regulatory sequence that provides for enhanced expression of an endogenous copy of the target gene.
  • "Knock-in" transgenics of interest can be transgenic animals having a knock-in of an AKAP gene. Such transgenics can be heterozygous or homozygous for the knock-in gene. "Knock-ins" also encompass conditional knock-ins.
  • a construct is suitable for use in the generation of transgenic animals if it allows the desired level of expression of an AKAP encoding sequence or the encoding sequence of another gene associated with cardiovascular disease.
  • Methods of isolating and cloning a desired sequence, as well as suitable constructs for expression of a selected sequence in a host animal, are well known in the art and are described below.
  • a transgenic non-human mammal showing high expression of the desired gene can be created by microinjecting a vector ligated with the gene into a fertilized egg of the subject non-human mammal (e.g. , rat fertilized egg) downstream of various promoters capable of expressing the protein and/or the corresponding protein derived from various mammals (rabbits, dogs, cats, guinea pigs, hamsters, rats, mice etc. , typicall rats etc.)
  • Useful vectors include Escherichia coli-derived plasmids, Bacillus subtilis-derived plasmids, yeast-derived plasmids, bacteriophages such as lambda, phage, retroviruses such as Moloney leukemia virus, and animal viruses such as vaccinia virus or baculovirus.
  • Useful promoters for such gene expression regulation include, for example, promoters for genes derived from viruses (cytomegalovirus, Moloney leukemia virus, JC virus, breast cancer virus etc. ), and promoters for genes derived from various mammals (humans, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice etc.) and birds (chickens etc.) (e.g.
  • the above-mentioned vectors have a sequence for terminating the transcription of the desired messenger RNA in the transgenic animal (generally referred to as terminator); for example, gene expression can be manipulated using a sequence with such function contained in various genes derived from viruses, mammals and birds.
  • terminator a sequence for terminating the transcription of the desired messenger RNA in the transgenic animal
  • gene expression can be manipulated using a sequence with such function contained in various genes derived from viruses, mammals and birds.
  • the simian virus SV40 terminator etc. are commonly used.
  • a portion of the intron of a eukaryotic organism gene can be ligated 5' upstream of the promoter region, or between the promoter region and the translational region, or 3' downstream of the translational region as desired.
  • a translational region for a protein of interest can be obtained using the entire or portion of genomic DNA of blood, kidney or fibroblast origin from various mammals (humans, rabbits, dogs, cats, guinea pigs, hamsters, rats, mice etc.) or of various commercially available genomic DNA libraries, as a starting material, or using complementary DNA prepared by a known method from RNA of blood, kidney or fibroblast origin as a starting material. Also, an exogenous gene can be obtained using complementary DNA prepared by a known method from RNA of human fibroblast origin as a starting material. All these translational regions can be used in transgenic animals.
  • DNA constructs for random integration need not include regions of homology to mediate recombination. Where homologous recombination is desired, the DNA constructs will comprise at least a portion of the target gene with the desired genetic modification, and will include regions of homology to the target locus. Conveniently, markers for positive and negative selection are included. Methods for generating cells having targeted gene modifications through homologous recombination are known in the art. For various techniques for transfecting mammalian cells, see Keown et al. (1 990) Methods in Enzymology 1 85:527-537.
  • the transgenic animal can be created by introducing an AKAP gene construct into, for example, an unfertilized egg, a fertilized egg, a spermatozoon or a germinal cell containing a primordial germinal cell thereof, typically in the embryogenic stage in the development of a non- human mammal (more typically in the single-cell or fertilized cell stage and generally before the 8-cell phase), by standard means, such as the calcium phosphate method, the electric pulse method, the lipofection method, the agglutination method, the microinjection method, the particle gun method, the DEAE-dextran method and other such method.
  • standard means such as the calcium phosphate method, the electric pulse method, the lipofection method, the agglutination method, the microinjection method, the particle gun method, the DEAE-dextran method and other such method.
  • a desired AKAP gene into a somatic cell, a living organ, a tissue cell or other cell, by gene transformation methods, and use it for cell culture, tissue culture and any other method of propagation.
  • these cells can be fused with the above- described germinal cell by a commonly known cell fusion method to create a transgenic animal.
  • an ES cell line can be employed, or embryonic cells can be obtained freshly from a host, e.g. mouse, rat, guinea pig, etc. Such cells are grown on an appropriate fibroblast-feeder layer or grown in the presence of appropriate growth factors, such as leukemia inhibiting factor (LIF) .
  • LIF leukemia inhibiting factor
  • ES cells When ES cells have been transformed, they can be used to produce transgenic animals. After transformation, the cells are plated onto a feeder layer in an appropriate medium. Cells containing the construct can be detected by employing a selective medium. After sufficient time for colonies to grow, they are picked and analyzed for the occurrence of homologous recombination or integration of the construct.
  • Blastocysts are obtained from 4 to 6 week old superovulated females.
  • the ES cells are trypsinized, and the modified cells are injected into the blastocoel of the blastocyst. After injection, the blastocysts are returned to each uterine horn of pseudopregnant females. Females are then allowed to go to term and the resulting litters screened for mutant cells having the construct.
  • chimeric progeny can be readily detected.
  • the chimeric animals are screened for the presence of the modified gene and males and females having the modification are mated to produce homozygous progeny. If the gene alterations cause lethality at some point in development, tissues or organs can be maintained as allogeneic or congenic grafts or transplants, or in in vitro culture.
  • Animals containing more than one transgene, such as allelic variants of AKAP genes and/or other genes associated with morbidity and/or mortality can be made by sequentially introducing individual alleles into an animal in order to produce the desired phenotype (manifestation of morbidity and/or predisposition to early mortality) .
  • allelic variants of AKAP genes and/or other genes associated with morbidity and/or mortality can be made by sequentially introducing individual alleles into an animal in order to produce the desired phenotype (manifestation of morbidity and/or predisposition to early mortality) .
  • Modulators of AKAP10 biological activities can be identified by using any of the disclosed methods related to AKAP10 binding to PKA, AKAP1 0 localization in the mitochondria, binding to other signaling enzymes and phosphorylation by PKA.
  • a variant protein such as AKAP10-5
  • PKA protein can then be isolated and quantitated or phosphorylation can be determined using the disclosed PKA assay.
  • An increase in the amount of PKA protein in the mitochondria or the quantity of test peptide phosphorylated by mitochondrial isolated PKA would indicate a positive effect of the test molecule.
  • Binding of AKAP1 0 protein and PKA could be directly assessed using an in vitro binding assay, or other disclosed binding assays, or by immunoassays such as immunoprecipitation.
  • a ribozyme targets the RNA genome and RNA transcripts and copies thereof.
  • Each ribozyme molecule contains a catalytically active segment capable of cleaving the plus or minus strand of RNA, and further comprises flanking sequences having a nucleotide sequence complementary to portions of the target RNA.
  • the flanking sequences serve to anneal the ribozyme to the RNA in a site-specific manner. Absolute complementarity of the flanking sequences to the target sequence is not necessary, however, as only an amount of complementarity sufficient to form a duplex with the target RNA and to allow the catalytically active segment of the ribozyme to cleave at the target sites is necessary.
  • the enzymatic RNA molecule is formed in a hammerhead motif but the ribozyme can also be formed in the motif of a hairpin, hepatitis delta virus, group I intron or RNAse P RNA (in association with an RNA guide sequence) .
  • hammerhead motifs are described by Rossi et al., AIDS Res. Hum. Retrovir. 8: 1 83 ( 1 992), hairpin motifs are described by Hampel et al., Biochem. 28:4929 ( 1 989) and Hampel et al., Nucl. Acids Res.
  • hepatitis delta virus motif is exemplified in Perrotta and Been, Biochem. 31 : 1 6 (1 992)
  • an RNAseP motif is described in Gueerier- Takada et al., Cell 35:849 (1 983)
  • examples of the group I intron motif are described in Cech et al., U.S. Pat. No. 4,987,071 , each of the foregoing disclosures being incorporated herein by reference.
  • Ribozymes can be prepared by chemical synthesis or produced by recombinant vectors according to methods established for the synthesis of RNA molecules. See, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1 989), incorporated herein by reference.
  • the ribozyme sequence can be synthesized, for example, using RNA polymerases such as T7 or SP6.
  • the ribozymes can be prepared from a corresponding DNA sequence (DNA which on transcription yields a ribozyme) operably linked to an RNA polymerase promoter such as the promoter for T7 RNA polymerase or SP6 RNA polymerase.
  • a DNA sequence corresponding to a ribozyme can be ligated in to a DNA vector, such as a plasmid, bacteriophage or other virus.
  • a DNA vector such as a plasmid, bacteriophage or other virus.
  • the transfer vector contains an RNA polymerase promoter operably linked to DNA corresponding to a ribozyme
  • the ribozyme can be conveniently produced upon incubation with an RNA polymerase. Ribozymes can therefore be produced in vitro by incubation of RNA polymerase with an RNA polymerase promoter operably linked to DNA corresponding to a ribozyme, in the presence of ribonucleotides.
  • procaryotic or eucaryotic cells can be transfected with an appropriate vector containing genetic material corresponding to a ribozyme, operably linked to an RNA polymerase promoter such that the ribozyme is transcribed in the host cell.
  • Ribozymes can be directly transcribed in vivo from a transfer vector, or alternatively, can be transcribed as part of a larger RNA molecule.
  • DNA corresponding to ribozyme sequence can be ligated into the 3' end of a carrier gene, for example, after a translation stop signal. Larger RNA molecules can help to stabilize the ribozyme molecules against nuclease digestion within the cells.
  • the carrier gene can give rise to a protein, whose presence can be directly assayed if desired, for example, by enzymatic reaction when the carrier gene encodes an enzyme.
  • Those of skill in the art based on the above description and the sequences disclosed herein can design ribozymes to target RNA representing the allelic variants of the AKAP10 gene.
  • the sequence of anti-AKAP10-5 hammerhead ribozyme is 5- U G C A C U G A N G A G C C U G G A C G A A A A C U - 3' (SEQ ID NO: 25).
  • the sequence UGCA is complementary to target RNA with C hybridizing to the G nucleotide at position 2073 of SEQ ID NO: 3 of the AKAP10-5 allelic variant.
  • the simplest hammerhead ribozyme must have UG at the 5' end of the substrate binding site. N. Kits
  • Kits can be used to indicate whether a subject is at risk of increased susceptibility to morbidity and/or predisposition for premature or increased or early mortality.
  • the kits can also be used to determine if a subject has a genetic predisposition to a disorder related to signal transduction. This information could be used, e.g. , to optimize treatment of such individuals as a particular genotype can be associated with drug response.
  • kits comprise a probe or primer which is capable of hybridizing adjacent to or at a polymorphic region of AKAP10 and thereby identifying whether the AKAP10 gene contains an allelic variant which is associated with increased susceptibility to morbidity and/or predisposition for premature or increased or early mortality or a genetic predisposition to a disorder related to signal transduction and/or protein phosphorylation.
  • kits further comprise instructions for use in carrying out assays, interpreting results and diagnosing a subject as having increased susceptibility to morbidity and/or predisposition for premature or increased or early mortality or a genetic predisposition to a disorder related to signal transduction and/or protein phosphorylation.
  • Kits for amplifying a region of AKAP10 gene or other genes associated with morbidity and/or mortality and/or signal transduction comprise two primers which flank a polymorphic region of the gene of interest.
  • primers can comprise the sequences of SEQ ID NOs.:5, 6, 7, 10, 12 and 16.
  • primers or probes hybridize to a polymorphic region or 5' or 3' to a polymorphic region depending on which strand of the target nucleic acid is used.
  • specific probes and primers comprise sequences designated as SEQ ID NOs: 8, 1 5, 1 9 and 20.
  • Those of skill in the art can synthesize primers and probes which hybridize adjacent to or at the polymorphic regions described herein and other SNPs in genes associated with morbidity and/or mortality and/or signal transduction
  • kits comprise at least one reagent necessary to perform an assay.
  • the kit can comprise an enzyme, such as a nucleic acid polymerase.
  • the kit can comprise a buffer or any other necessary reagent.
  • kits comprise microarrays of probes to detect allelic variants of the AKAP10 gene.
  • the kits further comprise instructions for their use and interpreting the results.
  • the following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.
  • the practice of methods and development of the products provided herein employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed.
  • EXAMPLE 1 Isolation of DNA from blood samples of a healthy donor population The results are provided of a screen comparing allele frequencies of 6,500 SNPs located in approximately 5,000 genes between a sample of young and elderly healthy individuals. This resulted in the identification of a gene encoding a functional variant with an impact on morbidity that can be involved in the etiology of cardiac dysfunction. All subjects involved in the studies signed a written informed consent and the institutional ethics committees of participating institutions approved the experimental protocols. Subjects for the disease susceptibility genome screen were part of a sample that was recruited during a routine blood donation from private blood collection centers in San Bernardino and Collinso Mirage, California, USA.
  • the staff of the blood collection agencies invited all healthy blood donors to participate, and helped the subjects fill out a consent form and a simple personal data collection form prior to sample collection.
  • the data collection form included information about age, sex, body size, personal and family disease history, and ethnic background of both parents.
  • Table 4 Composition of age-, gender-, and ethnicity-stratified groups.
  • CA Caucasian-American
  • HI Hispanic-American
  • AF African-American
  • AS Asian- American
  • YF Young Female
  • YM Young Male
  • OF Old Female
  • OM Old Male
  • S.D. Standard Deviation.
  • a healthy population represents an unbiased population of sufficient health to donate blood according to blood bank criteria, and not further selected for any disease state. Typically such individuals are not taking any medications.
  • Blood was obtained from a donor by venous puncture and preserved with 1 mM EDTA pH 8.0. Ten milliliters of whole blood from each donor was centrifuged at 2000x g.
  • One milliliter of the buffy coat was added to 9 milliliters of 155mM NH 4 CI, 10mM KHC0 3 , and 0.1 mM Na 2 EDTA, incubated 10 minutes at room temperature and centrifuged for 1 0 minutes at 2000x g. The supernatant was removed and the white cell pellet was washed in 1 55mM NH 4 CI, 1 0mM KHC0 3 , and 0.1 mM Na 2 EDTA and resuspended in 4.5 milliliters of 50mM Tris, 5mM EDTA, and 1 % SDS.
  • Proteins were precipitated from the cell lysate by 6M ammonium acetate pH 7.3 and separated from the nucleic acid by centrifugation at 3000x g.
  • the nucleic acid was recovered from the supernatant by the addition of an equal volume of 100% isopropanol and centrifugation at 2000x g.
  • the dried nucleic acid pellet was hydrated in lOmM Tris pH 7.6 and 1 mM Na 2 EDTA and stored at 4°C.
  • Statistical Analysis Estimates of allele frequencies derived from pooled DNA were based on independent mass spectrometry measurements of four analyte aliquots derived from a single PCR reaction. The median standard deviation for these values was approximately 0.01 .
  • SNPs were identified that had p-values less than 0.05 among all measured SNPs, followed by a second, independent measurement of all significant SNPs based on three separate PCRs of each DNA pool. The results of the second round of measurement were analyzed in a manner similar to the first round, and were compared for consistency. SNPs that showed statistically significant differences between young and old groups from pooled DNA analyses were individually genotyped for final validation.
  • the SNPs found to be associated with age were further analyzed for association with disease-related quantitative traits in the twin collection.
  • the analysis was conducted using a quantitative transmission-disequilibrium test (QTDT) as described by Abecasis et al., 2000, Am J Hum Genet. , 66:279-292 to take advantage of the twin- based sample and to control for admixture and other non-genetic sources of variation.
  • QTDT quantitative transmission-disequilibrium test
  • the form of the test was implemented that does not require the estimation of variance components. Formal statistical procedures to account for multiple testing were not use, but the distribution of the resulting p-values is reported.
  • More than 50 markers were identified out of the 6,500 tested markers that show a reproducibly significant allele frequency change between the two age groups in at least one gender (P ⁇ 0.05) .
  • the SNP that demonstrated the strongest association with age in both genders is located within the D-AKAP2 gene.
  • D-AKAP2 codes for dual-specific A-kinase anchor protein 2, which is part of a family of scaffold proteins known as A-kinase anchoring proteins (AKAPs) .
  • AKAPs bind the regulatory subunit of cAMP-dependent Protein Kinase (PKA), and target the kinase to various intracellular locations, localizing cAMP-mediated activation of the kinase.
  • PKA is a broad specificity kinase and phosphorylates numerous proteins that function in many essential cellular processes such as metabolism, gene transcription, cell division, and neuronal transmission. In the inactive state, PKA is a tetramer consisting of two catalytic (C) and two regulatory (R) subunits.
  • C catalytic
  • R regulatory
  • the Bayesian, coalescent theory-based method (Stephens et al., 2001 , Am J Hum Genet. 68:978-989) was applied to construct haplotypes at these three tightly linked sites for each subject.
  • the changes in genotype frequencies between age groups for the three sites showed a similar level of statistical significance as the changes in allele frequencies.
  • GG homozygotes were reduced and AA homozygotes increased in the older sample population of both genders. This further supports the hypothesis that the G allele, which determines the Val allele at I646V, is associated with a negative health impact.
  • genotype means in the subset of 207 informative twin pairs were 1 57 ⁇ 23.4, 1 52 ⁇ 26.9, and 146 ⁇ 25.4 (mean ⁇ standard deviation) for genotypes AA, GA, and GG, respectively at a position corresponding to nucleotide 2073 of SEQ ID NO: 1 .
  • AKAP10-1 is an allele of the AKAP10 gene with a single nucleotide polymorphism at nucleotide number 1 56277 (based on the sequence of a genomic clone of the AKAP10 gene, GenBank Accession No. AC005730) .
  • the single nucleotide polymorphism is a T to C transversion located in the 3'non-translated region of the gene encoding AKAP1 0.
  • PCR primers were synthesized by OPERON (Alameda, CA) using phosphoramidite chemistry.
  • Amplification of the AKAP10 target sequence was carried out in single 50 -/I PCR reaction with 25ng of human genomic DNA obtained from samples as described in Example 1 .
  • biotinylated universal primer complementary to the 5' end of the PCR amplicon 5'-AGCGGATAACAATTTCACACAGG-3' (SEQ ID NO: 7) .
  • the biotinylated universal primer could be 5'-GGCGCACGCCTCCACG-3' (SEQ ID NO: 1 6) . After an initial round of amplification of the target with the specific forward and reverse primer, the 5' biotinylated universal primer was hybridized and acted as a reverse primer thereby introducing a 3' biotin capture moiety into the molecule.
  • the amplification protocol resulted in a 5'-biotinylated double stranded DNA amplicon, which dramatically reduces the cost of high throughput genotyping by eliminating the need to 5' biotin label each forward primer used in a genotyping.
  • Thermal cycling was performed in 0.2mL tubes or 96 well plate using an MJ Research Thermal Cycler (Waltham, MA) (calculated temperature) with the following cycling parameters: 94°C for 5 min; 45 cycles: 94°C for 20 sec, 56°C for 30 sec, 72°C for 60 sec; 72°C 3 min.
  • the 50 ⁇ PCR reaction was added to 25 ⁇ l of streptavidin coated magnetic bead (Dynal) prewashed three times and resuspended in 1 M NH 4 CI, 0.06M NH 4 OH.
  • the PCR amplicons were allowed to bind to the beads for 1 5 minutes at room temperature.
  • the beads were then collected with a magnet and the supernatant containing unbound DNA was removed.
  • the unbound strand was release from the double stranded amplicons by incubation in 1 00mM NaOH and washing of the beads three times with 10mM Tris pH 8.0.
  • Genotyping was carried out using the MassEXTENDTM assay and MALDI-TOF.
  • the SNP identified at position 1 56277 of AKAP10 in the GenBank sequence is represented as a T to C transversion.
  • the MassEXTENDTM assay detected the sequence of the complementary strand at the polymorphic position, thus the primer extension product incorporated either a T or a C.
  • the DNA coated magnetic beads were resuspended in 26 mM Tris-HCL pH 9.5, 6.5 mM MgCI 2 and 50 mM each of dTTPs and 50 mM each of ddCTP, ddATP, ddGTP, 2.5U of a thermostable DNA polymerase (Amersham Pharmacia Biotech, Piscataway, NJ) and 20 pmoles of a template specific oligonucleotide primer 5'-CTGGCGCCCACGTGGTCAA-3' (SEQ ID NO: 8) (Operon, Alameda, CA) . Primer extension occurs with three cycles of oligonucleotide primer was hybridization and extension.
  • the extension products were analyzed after denaturation from the template with 50 mM NH 4 CI and transfer of 1 50 nl each sample to a silicon chip preloaded with 1 50 nl of H3PA (3-hydroxy picolinic acid) (Sigma Aldrich, St. Louis, MO) matrix material.
  • the sample material was allowed to crystallize and analyzed by MALDI-TOF (Bruker Daltonics, Billerica, MA, PerSeptive, Foster City, CA) .
  • the mass of the primer used in the MassEXTENDTM reaction was 5500.6 daltons.
  • the allelic variant results in the addition of ddC to the primer to produce an extension product having a mass of 5773.8 daltons.
  • the predominant allele is extended by the addition of dT and ddG to the primer to produce an extension product having a mass of 61 01 daltons.
  • SNP that is present in AKAP1 0-1 is a T to C transversion at nucleotide number 1 56277 of the sequence of a genomic clone of the AKAP10 gene (GenBank Accession No. AC005730) (SEQ ID NO: 1 7) .
  • SEQ ID NO: 1 7 represents the nucleotide sequence of human chromosome 1 7, which contains the genomic nucleotide sequence of the human AKAP10 gene at approximately nucleotide 83,580 to nucleotide 1 56,577.
  • SEQ ID NO: 1 8 represents the nucleotide sequence of human chromosome 1 7, which contains the genomic nucleotide sequence of the human AKAP10-1 allele.
  • the polymorphism is localized in the non-translated 3'-region of the gene encoding the human protein kinase A anchoring protein (AKAP1 0) .
  • the gene is located on chromosome 1 7. Its structure includes 1 5 exons and 1 4 intervening sequences (introns) .
  • the encoded protein is responsible for the sub-cellular localization of the cAMP- dependent protein kinase and, therefore, plays a key role in the G-protein mediated receptor-signaling pathway (Huang et al. PNAS (1007) 94: 1 1 1 84-1 1 1 89) .
  • Genomic DNA was isolated from blood (see Example 1 ) of seventeen (1 7) individuals with a genotype CC at the AKAP1 0-1 gene locus and a single heterozygous individual (CT) (as described in Example 2) .
  • a target sequence in the AKAP1 0-1 gene which encodes the C- terminal PKA binding domain was amplified using the polymerase chain reaction.
  • PCR primers were synthesized by OPERON (Alameda, CA) using phosphoramidite chemistry. Amplification of the AKAP10-1 target sequence was carried out in individual 50 ⁇ l PCR reaction with 25ng of human genomic DNA templates.
  • Thermal cycling was performed in 0.2 mL tubes or 96 well plate using an MJ Research Thermal Cycler (MJ Research, Waltham, MA) (calculated temperature) with the following cycling parameters: 94°C for 5 min; 45 cycles; 94°C for 20 sec, 56°C for 30 sec, 72°C for 60 sec; 72°C 3min. After amplification the amplicons were purified by chromatography (Mo Bio Laboratories (Solana Beach, CA) .
  • the sequence of the 1 8 amplicons, representing the target region was determined using a standard Sanger cycle sequencing method with 25 nmoles of the PCR amplicon, 3.2 ⁇ M DNA sequencing primer 5'-CCC ACA GCA GTT AAT CCT TC-3' (SEQ ID NO: 1 1 ) and chain terminating dRhodamine labeled 2', 3' dideoxynucleotides (PE Biosystems, Foster City, CA) using the following cycling parameters: 96°C for 1 5 sec, 25 cycles: 55 °C for 1 5 sec, 60°C for 4 min.
  • the sequencing products were precipitated by 0.3M NaOAc and ethanol, the precipitate was centrifuged and dried. The pellets were resuspended in deionized formamide and separated on a on a 5% polyacrylamide gel. The sequence was determined using the "Sequencher” software (Gene Codes, Ann Arbor, Ml) .
  • the sequence of all 1 7 of the amplicons which are homozygous for the AKAP10-1 SNP revealed a polymorphism at nucleotide position 1 521 71 (numbering for GenBank Accession No. AC005730 for AKAP1 0 genomic clone) with A replaced by G.
  • This SNP can also be designated as located at nucleotide 2073 of a cDNA clone of the wildtype AKAP1 0 (SEQ ID NO: 1 ) (GenBank Accession No. AF037439) .
  • This single nucleotide polymorphism was designated as AKAP10-5 (SEQ ID NO:3) and results in a substitution of a valine for an isoleucine residue at amino acid position 646 (SEQ ID NO:4) .
  • a healthy population stratified by age is a very efficient and a universal screening tool for morbidity associated genes by allowing for the detection of changes of allelic frequencies in the young compared to the old population. Individual samples of this healthy population base can be pooled to further increase the throughput.
  • Healthy samples were obtained through the blood bank of San Bernardino, CA. Both parents of the blood donors were of Caucasian origin. Practically a healthy subject, when human, is defined as human donor who passes blood bank criteria to donate blood for eventual use in the general population. These criteria are as follows: free of detectable viral, bacterial, mycoplasma, and parasitic infections; not anemic; and then further selected based upon a questionnaire regarding history. Thus, a healthy population represents an unbiased population of sufficient health to donate blood according to blood bank criteria, and not further selected for any disease state. Typically such individuals are not taking any medications.
  • PCR primers were synthesized by OPERON (Alameda, CA) using phosphoramidite chemistry. Amplification of the AKAP10 target sequence was carried out in single 50 ⁇ l PCR reaction with 1 00ng- 1 ug of pooled human genomic DNAs in a 50 /I PCR reaction. Individual DNA concentrations within the pooled samples were present in equal concentration with the final concentration ranging from 1 -25ng. Each reaction contained 1 X PCR buffer (Qiagen, Valencia, CA), 200 /M dNTPs, 1 U Hotstar Taq polymerase (Qiagen, Valencia, CA), 4 mM MgCI 2 , and 25 pmols of the forward primer containing the universal primer sequence and the target specific sequence
  • Thermal cycling was performed in 0.2 mL tubes or 96 well plate using an MJ Research Thermal Cycler (Waltham, MA) (calculated temperature) with the following cycling parameters: 94°C for 5 min; 45 cycles: 94°C for 20 sec, 56°C for 30 sec; 72°C for 60 sec; 72°C 3 min.
  • AKAP 10-5 was determined by using the MassEXTENDTM assay and MALDI-TOF (see, U.S. Patent No. 6,043,031 ) .
  • the MassEXTENDTM assay is a primer extension assay that utilizes a primer that hybridizes adjacent to the polymorphic region and which is extended in the presence of one or more ddNTPs. Extension is stopped by the incorporation of a dideoxy nucleotide. At a polymorphic site the different alleles produce different length extension products, which are distinguishable by mass spectrometry.
  • the MassEXTENDTM assay detected the sequence of the sense strand and resulted in the incorporation of either T or C into the extension product.
  • the DNA coated magnetic beads were suspended in 26mM Tris-HCL pH 9.5; 6.5 mM, MgCI 2 and 50mM each of dTTPs and 50mM each of ddCTP, ddATP, ddGTP, 2.5U of a thermostable DNA polymerase (Amersham Pharmacia Biotech, Piscataway NJ) and 20 pmoles of a template specific oligonucleotide primer 5'-ACTGAGCCTGCTGCATAA-3' (SEQ ID NO: 1 5) (Operon) (Alameda, CA) .
  • Primer extension occurs with three cycles of oligonucleotide primer hybridization and extension.
  • the extension products were analyzed after denaturation from the template with 50 mM NH 4 CI and transfer of 1 50 nl each sample to a silicon chip preloaded with 1 50 nl of H3PA (3-hydroxy picolinic acid) (Sigma Aldrich, St. Louis, MO) matrix material.
  • H3PA 3-hydroxy picolinic acid
  • the sample material was allowed to crystallize and analyzed by MALDI-TOF (Bruker Daltonics, Billerica, MA, PerSeptive, Foster City, CA) .
  • the primer had a mass of 5483.6 daltons.
  • allelic variant resulted in the addition of a ddC to the primer to produce an extension product having a mass of 5756.8 daltons.
  • the predominant allele resulted in the addition a T and ddG to the primer giving an extension product with a mass of 6101 daltons.
  • Genomic DNA isolation, amplification of the target regions and sequencing of amplicons was carried out as in Example 3.
  • chromosome 1 7 was BLAST searched to identify the number of exons. Sanger sequencing of the regions around and containing the exons was performed and resulted in the discovery of AKAP1 0-7 polymorphic region.
  • the forward sequencing primer was CACTGCACCCAGCCTTATG (SEQ ID NO: 23) and the reverse sequencing primer was CTGGGATGTGAAGGAAAGGA (SEQ ID NO: 24).
  • Example 4 Samples are obtained and amplified as in Example 4.
  • the identity of the nucleotide present at the polymorphic site of AKAP 10-7 is determined by using the MassEXTENDTM assay and MALDI- TOF (see, U.S. Patent No. 6,043,031 ).
  • the MassEXTENDTM assay detects the sequence of the complementary strand and resulted in the incorporation of either G or A into the extension product. Reactions are carried out as in Example 4.
  • the template specific oligonucleotide primer 5'-CTCTGCGTCTCAGGTATT-3' SEQ ID NO: 20
  • the primer has a mass of 5456.6 daltons.
  • allelic variant results in the addition of a ddA to the primen to produce an extension product having a mass of 5753.6 daltons.
  • the predominant allele results in the addition a G and ddA to the primer giving an extension product with a mass of 6083.0 daltons.

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Abstract

La présente invention a trait à des protéines polymorphes d'ancrage de la kinase A et des acides nucléiques codant pour ces protéines. L'invention a également trait à des procédés de détection de protéines polymorphes d'ancrage de la kinase A et des acides nucléiques codant pour les protéines polymorphes d'ancrage de la kinase A, et des trousses destinés à être utilisées dans les procédés de détection. L'invention a trait en outre à des procédés d'identification de sujets atteints ou à risque de développer des maladies ou troubles, tels que ceux liés à la transduction de signal et/ou de maladie cardio-vasculaire. Enfin, l'invention a trait à des procédés de détermination de la susceptibilité à la morbidité et/ou la mortalité accrue ou prématurée.
EP04718045A 2003-03-07 2004-03-05 Association de proteines polymorphes d'ancrage de kinase a des phenotypes cardiaques et procedes correspondants Withdrawn EP1601972A2 (fr)

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EP2107129B1 (fr) 2003-07-31 2012-08-22 Sequenom, Inc. Procédé de réactions de polymérase en chaîne multiplexe de haut niveau et réactions d'extension de masse homogène pour le génotypage des polymorphismes
WO2011053134A2 (fr) * 2009-10-26 2011-05-05 Academisch Medisch Centrum Bij De Universiteit Van Amsterdam Détermination du risque génétique de fibrillation ventriculaire
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