WO2010033825A2 - Variants génétiques associés à des anévrismes de l'aorte abdominale - Google Patents

Variants génétiques associés à des anévrismes de l'aorte abdominale Download PDF

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WO2010033825A2
WO2010033825A2 PCT/US2009/057511 US2009057511W WO2010033825A2 WO 2010033825 A2 WO2010033825 A2 WO 2010033825A2 US 2009057511 W US2009057511 W US 2009057511W WO 2010033825 A2 WO2010033825 A2 WO 2010033825A2
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aaa
risk
allele
alleles
individual
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WO2010033825A3 (fr
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James R. Elmore
David J. Carey
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Geisinger Clinic
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes

Definitions

  • the present invention relates to methods and kits for identifying individuals who are at risk for developing abdominal aortic aneurysms (AAAs) by detection of genetic variants associated with AAA formation.
  • AAAs abdominal aortic aneurysms
  • the methods of the present invention allow for individuals showing a genetic susceptibility for developing AAAs to undergo monitoring and treatment as necessary.
  • AAAs Abdominal aortic aneurysms
  • 1 2
  • Many patients with AAAs are undiagnosed.
  • the lack of diagnosis increases their risk of death from AAA, since surgical treatments to repair aneurysms before they rupture are safe and effective.
  • the most important risk factors for AAAs are old age, male gender, smoking, and family history of AAA.
  • the underlying causes of aneurysm formation are not known. Investigations into the pathophysiology of AAA have focused on remodeling of the extracellular matrix and inflammation as important mechanisms in AAA formation. (3-8) Family and epidemiologic studies demonstrate a substantial genetic risk for AAA.
  • AAA is a complex disease with risk influenced by an unknown number of genes that act in combination with environmental factors such as smoking. Identifying specific genetic factors that are associated with AAA would be extremely useful in developing improved methods to diagnose and treat the disease. [0006] While a number of candidate gene association studies that examined genes selected on the basis of their predicted role in AAA have been reported, (4,9) none of these studies have conclusively identified any AAA-associated variants. Previously, whole genome linkage analyses using family-based or affected-relative-pair methods identified two AAA-associated genetic loci designated as the AAAl (on 19ql3) and AAA2 (4q31) susceptibility loci. (4,10,11) The specific AAA-associated genetic variants in these regions have not yet been identified.
  • GWAS genome wide association study
  • SNPs single nucleotide polymorphisms
  • the methods of the present invention involve isolating a nucleic acid sample from an individual and determining the presence or absence of an allele associated with AAA risk.
  • a single polymorph of the present invention may be analyzed to determine the individual's risk of developing an AAA. If an individual has at least one allele associated with AAA risk at the polymorphic position analyzed, then the individual is indicated as having a risk of developing an AAA. [0012] In another aspect of the present invention, more than one polymorph may be analyzed to determine the individual's risk. If more than one polymorph is to be analyzed, the number of alleles associated with AAA risk may be determined. If the total number of alleles associated with AAA risk is at least half of the total number of alleles analyzed, then the individual is indicated as having a risk of developing an AAA.
  • Alleles associated with AAA risk in the present invention include a C allele at rsl2039875 and a C allele at rs7635818. Other polymorphs in linkage disequilibrium with these alleles may also be used in determining genetic predisposition to an AAA according to the present invention.
  • Individuals who are indicated as having a risk of developing an AAA by the methods of the present invention may then be further tested using methods well known in the art for determining the presence of an AAA, such as ultrasound scanning. If no AAA is found to be currently present, those individuals indicated as having a risk of developing an AAA may be subsequently monitored to determine if an AAA develops. Further, individuals indicated as having a risk of developing an AAA may be considered candidates for pharmacotherapeutic agents which may prevent or slow the growth of AAAs.
  • kits for determining an individual's risk of developing an AAA may include reagents, nucleic acids and probes as necessary for determining the presence or absence of alleles associated with AAA risk.
  • Figure 1 Flow chart for the pooled DNA genome wide association study (GWAS) of Example 1.
  • GWAS pooled DNA genome wide association study
  • AF allele frequency
  • CV coefficient of variation.
  • Figure 2 Genome wide association identifies a candidate AAA-associated region on chromosome 3pl2.3.
  • Panel A The vertical arrow indicates a region on chromosome 3 that contains SNPs with significant allele frequency differences between AAA cases and controls. The known genes in this region are indicated below the line with their gene names and accession numbers. Vertical lines and numbers above the line indicate chromosomal position (in millions of base pairs).
  • Panel B Transformed P values of case- control allele frequency differences corresponding to the region indicated by the horizontal bar in panel A are shown.
  • FIG. 3 Genotype analysis and haplotype structure of the AAA-associated region on chromosome 3pl2.3. Vertical arrows indicate the physical positions of the 10 SNPs from this region that were individually genotyped in 451 cases and 279 controls from the Geisinger Vascular Clinic (GVC) sample set. The position of the CNTN3 gene is shown. The inverted triangle is the HapMap R LD plot for the CEU population (gray indicates highest R 2 values). The table shows the reference numbers, map positions and X 2 P values for AAA association of the genotyped SNPs. The significantly associated SNPs are outlined by boxes and lie within a CEU haplotype block characterized by strong LD.
  • FIG. 4 LD analysis of AAA-associated SNPs. Top; -1Og 10 transformed P values for AAA association in the Geisinger Clinic population of the 10 SNPs individually genotyped in the chromosome 3pl2.3 region. Bottom: LD correlation R values of these SNPs; the most significantly associated SNP and regions of strong LD with that SNPs (R 2 > 0.95) are indicated in red.
  • FIG. 5 Odds ratios and P values for AAA association of SNP s7635818.
  • the graph shows the calculated odds ratios (with 95% confidence intervals) for all samples combined, the set of all GVC samples, and the GVC samples from subjects with 20 or more years of smoking history. The total sample size and X 2 P values are also shown. These values were calculated using Helix Tree Software, Version 6.4 assuming a dominant genetic model. The dashed vertical lines indicate an odds ratio of 1.
  • Figure 6 Expression of contactin-3 transcripts in aortic tissue.
  • RNA isolated from AAA tissue, control (normal) aortic tissue, aortic occlusive disease tissue (AOD) or peripheral blood mononuclear cells from patients with AAA (PBMC) were used as templates for reverse transcriptase and PCR amplification using contactin-3 specific primers.
  • the products were resolved on an agarose gel and stained with ethidium bromide. The expected position of the contactin-3 -specific product is indicated by the arrow. Two independent samples of each type were analyzed. Identical results were obtained with a different pair of contactin-3 specific PCR primers.
  • Figure 7 Genome wide association identifies a candidate AAA-associated region on chromosome Iq41.
  • Top panel Transformed P values of case-control SNP allele frequency differences on chromosome 1. Each point represents the rolling sum of 5 consecutive -logio P values. The horizontal bar indicates the candidate region in Iq41.
  • Lower panel Transformed P values of case-control SNP allele frequency differences in a region of ⁇ 1.5 million bp corresponding to the horizontal bar in the top panel. The figure above the graph shows known genes in this region.
  • FIG 8 AAA-associated haplotype on chromosome Iq41. Vertical arrows indicate the locations of the 6 SNPs from this region that were individually genotyped in a subset of 451 cases and 279 controls from the Geisinger Vascular Clinic (GVC) sample set. The physical map of the KCNK2 gene is shown. The inverted triangle is the HapMap R 2 LD plot for the CEU population. The table insert shows the reference numbers, map positions and X 2 P values for AAA association of the genotyped SNPs. The significantly associated SNPs are outlined by boxes and overlaps a CEU haplotype block characterized by strong LD.
  • Figure 9 AAA-associated haplotype.
  • FIG. 10 Expression of KCNK2 transcripts in aortic tissue. Total RNA was isolated from AAA tissue, control (normal) aortic tissue, and aortic occlusive disease (AOD) tissue.
  • RNA was copied to cDNA by reverse transcriptase and then used as templates for PCR amplification using primers specific for each of the 3 KCNK2 isoforms and for GAPDH.
  • the PCR products were resolved on an agarose gel and stained with ethidium bromide. The expected positions of the products from the 3 KCNK2 transcripts and GAPDHmQ indicated.
  • the N-terminal sequences of the 3 KCNK2 isoforms are shown at the top (isoform a (SEQ ID NO: 8), isoform b (SEQ ID NO: 9) and isoform c (SEQ ID NO: 10). Each lane represents an independent sample. NTC, no template control.
  • the graph shows KCNK2 expression levels in the tissue samples standardized to GAPDH levels. Horizontal bars indicate mean values for each group. The mean value in normal aortic tissue was set to 1.0.
  • FIG. 1 Additive effect of AAA-associated SNP alleles.
  • AAA cases (938) and controls (1,126) were genotyped for 3 AAA-associated SNPs, rs 10757278 (9p21), rs7635818 (3pl2.3), and rsl2039875 (Iq41).
  • Top panel distribution of total AAA- associated alleles for these 3 SNPs in the case (hatched bars) and control (solid bars) samples. The case and control distributions were significantly different by Chi-squared test for trend (P ⁇ 0.0001).
  • Middle panel Relative odds ratios for AAA disease were calculated for individuals with 0-2, > 3, or 6 AAA risk alleles. Error bars indicate 95% confidence intervals.
  • Lower panel: Linear regression analysis of AAA SNP allele burden vs. AAA disease odds. Filled circles indicate disease odds calculated for each risk allele value (from 0 to 6). The solid line is the regression line (non-zero slope, P ⁇ 0.01 , R 2 0.80). The dashed lines are the 95% confidence intervals.
  • the present invention provides methods and kits for determining an individual's risk of developing an abdominal aortic aneurysm (AAA).
  • AAAs which are typically easily treated, often develop undetected as the patient is not aware of the presence of the aneurysm, hi certain embodiments, the present invention may be used as a general screen to detect individuals at risk for the development of an AAA, allowing for that individual to be monitored and treated as necessary.
  • AAAs are diagnosed using ultrasound scans to detect dilation of the abdominal aorta. Ultrasound scans are usually only performed on persons who fall into the highest risk groups, namely men over 65 years of age who have ever smoked and men and women with a family history of AAAs. Most guidelines recommend repair of AAAs that are larger than 5.5 cm in men and 5 cm in women, which are considered more likely to rupture. Repair of AAAs is typically a straightforward procedure with minimal risk, while rupture of an AAA is associated with approximately an 80% mortality rate. However, as only the most significant risk groups are ever screened and as there are not other reliable tests to determine AAA risk, a significant number of cases of rupture occur in patients who were unaware that they even had an aneurysm.
  • the present invention provides genetic variants associated with risk of developing an AAA that can be used to determine which patients should be further tested for the presence of an AAA, such as through an ultrasound scan.
  • the methods of the present invention allow for the detection of these genetic variants during routine medical visits, in order to determine whether further testing for an AAA should be undertaken.
  • the methods of the present invention may be incorporated into standard visits to a doctor's office, such as for a physical or check-up.
  • the genetic variants of the present invention may be used as part of a general panel of tests done in these types of appointments. Further, the methods and genetic variants of the present invention may be used in direct to consumer genetic testing procedures.
  • nucleic acid sequences are written left to right in a 5' to 3' orientation.
  • Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer or any non-integer fraction within the defined range.
  • all technical and scientific terms used herein have the same meaning as commonly understood by the ordinary person skilled in the art to which the invention pertains.
  • An "allele” refers to the nucleotide sequence of a given locus (position) on a chromosome.
  • a polymorphic marker allele thus refers to the composition (i.e., sequence) of the marker on a chromosome.
  • Genomic DNA from an individual contains two alleles
  • any given polymorphic marker representative of each copy of the marker on each chromosome.
  • a nucleotide position at which more than one sequence is possible in a population is referred to herein as a "polymorphic site”.
  • a "Single Nucleotide Polymorphism” or "SNP” is a DNA sequence variation occurring when a single nucleotide at a specific location in the genome differs between members of a species or between paired chromosomes in an individual. Most SNP polymorphisms have two alleles. Each individual is in this instance either homozygous for one allele of the polymorphism (i.e. both chromosomal copies of the individual have the same nucleotide at the SNP location), or the individual is heterozygous (i.e. the two sister chromosomes of the individual contain different nucleotides).
  • the SNP nomenclature as reported herein refers to the official Reference SNP (rs) ID identification tag as assigned to each unique SNP by the National Center for Biotechnological
  • a "variant”, as described herein, refers to a segment of DNA that differs from the reference DNA.
  • a marker or a polymorphic marker is a variant. Alleles that differ from the reference are referred to as "variant" alleles.
  • haplotype refers to a segment of genomic DNA that is characterized by a specific combination of alleles arranged along the segment.
  • a haplotype comprises one member of the pair of alleles for each polymorphic marker or locus .
  • the haplotype can comprise two or more alleles, three or more alleles, four or more alleles, or five or more alleles.
  • HapMap Positions refer to enumerated positions on the chromosome as are described by the International HapMap project (www.hapmap.org) and as are well known in the art. Each HapMap position number designates a specific nucleotide position on a human chromosome.
  • a "computer-readable medium” is an information storage medium that can be accessed by a computer using a commercially available or custom-made interface.
  • Exemplary compute- readable media include memory (e.g., RAM, ROM, flash memory, etc.), optical storage media (e.g., CD-ROM), magnetic storage media (e.g., computer hard drives, floppy disks, etc.), punch cards, or other commercially available media.
  • Information maybe transferred between a system of interest and a medium, between computers, or between computers and the computer- readable medium for storage or access of stored information. Such transmission can be electrical, or by other available methods, such as IR links, wireless connections, etc.
  • a "nucleic acid sample” is a sample obtained from a individual that contains nucleic acid.
  • the nucleic acid sample comprises genomic DNA.
  • Such a nucleic acid sample can be obtained from any source that contains genomic DNA, including as a blood sample, sample of amniotic fluid, sample of cerebrospinal fluid, or as a tissue sample from skin, muscle, buccal or conjunctival mucosa, placenta, gastrointestinal tract or other organs.
  • a reference sequence may be referred to for a particular polymorphic site.
  • the reference allele may referred to as the "wild-type” allele and it usually is chosen as either the first sequenced allele or as the allele from a "non- affected" individual (e.g., an individual that does not have an increased risk for developing an AAA).
  • Alleles for SNP markers as referred to herein refer to the bases A, C, G or T as they occur at the polymorphic site in the SNP assay employed.
  • the assay employed may either measure the percentage or ratio of the two bases possible, i.e. A and G.
  • the percentage or ratio of the complementary bases T/C can be measured.
  • a reference sequence is referred to for a particular sequence.
  • variants Alleles that differ from the reference are referred to as "variant” alleles.
  • a variant sequence refers to a sequence that differs from the reference sequence but is otherwise substantially similar.
  • the allele that corresponds to the reference sequence e.g. the allele that is not associated with an increased risk of AAA, may be referred to as the "major" allele.
  • a haplotype refers to a segment of DNA that is characterized by a specific combination of alleles arranged along the segment.
  • a haplotype comprises one member of the pair of alleles for each polymorphic marker or locus .
  • the haplotype can comprise two or more alleles, three or more alleles, four or more alleles, or five or more alleles, each allele corresponding to a specific polymorphic marker along the segment.
  • Haplotypes can comprise a combination of various polymorphic markers, e.g., SNPs and microsatellites, having particular alleles at the polymorphic sites. The haplotypes thus comprise a combination of alleles at various genetic markers.
  • Detecting specific polymorphic markers and/or haplotypes can be accomplished by methods known in the art for detecting sequences at polymorphic sites. For example, standard techniques for genotyping for the presence of SNPs and/or microsatellite markers can be used, such as fluorescence-based techniques (e.g., Chen, X. et al., Genome Res. 9(5): 492-98 (1999); Kutyavin et al., Nucleic Acid Res. 34:el28 (2006)), utilizing PCR, LCR, Nested PCR and other techniques for nucleic acid amplification.
  • fluorescence-based techniques e.g., Chen, X. et al., Genome Res. 9(5): 492-98 (1999); Kutyavin et al., Nucleic Acid Res. 34:el28 (2006)
  • SNP genotyping include, but are not limited to, TaqMan genotyping assays and SNPIex platforms (Applied Biosystems), mass spectrometry (e.g., MassARRAY system from Sequenom), minisequencing methods, real-time PCR, Bio-Plex system (BioRad), CEQ and SNPstream systems (Beckman), Molecular Inversion Probe array technology (e.g., Affymetrix GeneChip), and BeadArray Technologies (e.g., Illumina GoldenGate and Infinium assays).
  • Applied Biosystems mass spectrometry
  • MassARRAY system from Sequenom minisequencing methods
  • real-time PCR Real-time PCR
  • Bio-Plex system BioRad
  • CEQ and SNPstream systems Beckman
  • Molecular Inversion Probe array technology e.g., Affymetrix GeneChip
  • BeadArray Technologies e.g., Illumina GoldenGate and Infinium assays
  • the present invention provides SNPs which are for the first time associated with risk of developing an AAA, as wells as polymorphisms in LD with these SNPs.
  • the primary SNPs of the present invention which are associated with AAA risk are: [0051] rs7635818 - where C is the allele associated with AAA risk.
  • This SNP is located on chromosome 3pl2.3, has HapMap position 74866166, and is represented by SEQ ID NO: 1 , where position 27 of SEQ ID NO: 1 is the site of the SNP.
  • This SNP is located upstream of the CNTN3 gene, which encodes contactin-3.
  • rsl 2039875 where C is the allele associated with AAA risk.
  • SNPs of the present invention associated with AAA risk include those in the same haplotype block as rs7635818 (HapMap Positions 74762904-74998753), as shown in Table 1.
  • SNPs of the present invention associated with AAA risk also include those in the same haplotype block as rsl2039875 (HapMap Positions 213267336 - 213447659), as shown in Table 2.
  • an individual who is at an increased susceptibility (i.e., at risk) for developing an AAA is an individual in whom at least one specific allele at one or more polymorphic site or haplotype conferring increased susceptibility for developing an AAA is identified (i.e., at-risk marker alleles or haplotypes).
  • the at-risk marker or haplotype is one that confers a significant increased risk (or susceptibility) of developing an AAA.
  • the significance associated with a marker or haplotype is measured by a relative risk (RR),
  • significance associated with a marker or haplotype is measured by an odds ratio (OR).
  • the significance is measured by a percentage.
  • a significant increased risk is measured as a risk (relative risk and/or odds ratio) of at least 1.2, including but not limited to: at least 1.2, at least 1.3, at least 1.4, at least 1.5, at least 1.6, at least 1.7, 1.8, at least 1.9, at least 2.0, at least 2.5, at least 3.0, at least 4.0, and at least 5.0.
  • a risk (relative risk and/or odds ratio) of at least 1.2 is significant.
  • a risk of at least 1.3 is significant.
  • a risk of at least 1.4 is significant.
  • a relative risk of at least 1.5 is significant.
  • a significant increase in risk is at least 1.7 is significant.
  • other cutoffs are also contemplated, e.g., at least 1.15, 1.25, 1.35, and so on, and such cutoffs are also within scope of the present invention.
  • a significant increase in risk is at least about 20%, including but not limited to about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, and 500%.
  • a significant increase in risk is at least 20%.
  • a significant increase in risk is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and at least 100%.
  • Other cutoffs or ranges as deemed suitable by the person skilled in the art to characterize the invention are however also contemplated, and those are also within scope of the present invention.
  • a significant increase in risk is characterized by a p- value, such as a p- value of less than 0.05, less than 0.01, less than 0.001, less than 0.0001 , less than 0.00001, less than 0.000001, less than 0.0000001, less than 0.00000001 , or less than 0.000000001.
  • An at-risk polymorphic marker or haplotype of the present invention is one where at least one allele of at least one marker or haplotype is more frequently present in an individual at risk for developing an AAA (affected), compared to the frequency of its presence in a healthy individual (control), and wherein the presence of the marker or haplotype is indicative of susceptibility to developing an AAA.
  • the control group may in one embodiment be a population sample, i.e. a random sample from the general population.
  • the control group is represented by a group of individuals who are determined to not have AAAs.
  • Such AAA free control may in one embodiment be characterized by the absence of one or more specific clinical signs of AAAs.
  • the AAA free control group is characterized by the absence of one or more AAA specific risk factors, such as age, gender, race and a history of smoking.
  • a simple test for correlation would be a Fisher-exact test on a two by two table.
  • the two by two table is constructed out of the number of chromosomes that include both of the markers or haplotypes, one of the markers or haplotypes but not the other and neither of the markers or haplotypes.
  • Other statistical tests of association known to the skilled person are also contemplated and are also within scope of the invention.
  • Linkage Disequilibrium refers to a non-random assortment of two genetic elements. For example, if a particular genetic element (e.g., "alleles" of a polymorphic marker) occurs in a population at a frequency of 0.50 (50%) and another occurs at a frequency of 0.50 (50%), then the predicted occurrence of a person's having both elements is 0.25 (25%), assuming a random distribution of the elements.
  • a particular genetic element e.g., "alleles" of a polymorphic marker
  • Allele or haplotype frequencies can be determined in a population by genotyping individuals in a population and determining the frequency of the occurrence of each allele or haplotype in the population. For populations of diploids, e.g., human populations, individuals will typically have two alleles or allelic combinations for each genetic element (e.g., a marker, haplotype or gene).
  • that is ⁇ 1 indicates that historical recombination may have occurred between two sites (recurrent mutation can also cause
  • the measure r 2 represents the statistical correlation between two sites, and takes the value of 1 if only two haplotypes are present. [0066]
  • the r 2 measure is arguably the most relevant measure for association mapping, because there is a simple inverse relationship between r 2 and the sample size required to detect association between susceptibility loci and SNPs.
  • a significant r 2 value can be at least 0.1 such as at least 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, or at least 0.99.
  • the significant r 2 value can be at least 0.2.
  • linkage disequilibrium refers to linkage disequilibrium characterized by values of
  • linkage disequilibrium represents a correlation between alleles of distinct markers. It is measured by correlation coefficient or
  • linkage disequilibrium is defined in terms of values for both the r 2 and
  • a significant linkage disequilibrium is defined as r 2 > 0.1 and
  • Linkage disequilibrium can be determined in a single human population, as defined herein, or it can be determined in a collection of samples comprising individuals from more than one human population. In one embodiment of the invention, LD is determined in a sample from one or more of the HapMap populations (Caucasian, African, Japanese, Chinese), as defined (http://www.hapmap.org).
  • LD is determined in the CEU population of the HapMap samples. In another embodiment, LD is determined in the YRI population. In yet another embodiment, LD is determined in samples from the Icelandic population. If all polymorphisms in the genome were independent at the population level (i.e., no LD), then every single one of them would need to be investigated in association studies, to assess all the different polymorphic states. However, due to linkage disequilibrium between polymorphisms, tightly linked polymorphisms are strongly correlated, which reduces the number of polymorphisms that need to be investigated in an association study to observe a significant association. Another consequence of LD is that many polymorphisms may give an association signal due to the fact that these polymorphisms are strongly correlated.
  • Genomic LD maps have been generated across the genome, and such LD maps have been proposed to serve as framework for mapping disease-genes (Risch, N. & Merkiangas, K, Science 273: 1516-1517 (1996); Maniatis, N., et al., Proc Natl Acad Sci USA 99:2228-2233 (2002); Reich, DE et al, Nature 411: 199-204 (2001)). It is also now established that many portions of the human genome can be broken into series of discrete haplotype blocks containing a few common haplotypes; for these blocks, linkage disequilibrium data provides little evidence indicating recombination (see, e.g., Wall., J. D.
  • blocks can be defined as regions of DNA that have limited haplotype diversity (see, e.g., Daly, M. et al., Nature Genet.
  • haplotype block or “LD block” includes blocks defined by any of the above described characteristics, or other alternative methods used by the person skilled in the art to define such regions.
  • Haplotype blocks can be used to map associations between phenotype and haplotype status, using single markers or haplotypes comprising a plurality of markers. The main haplotypes can be identified in each haplotype block, and then a set of "tagging" SNPs or markers (the smallest set of SNPs or markers needed to distinguish among the haplotypes) can then be identified.
  • tagging SNPs or markers can then be used in assessment of samples from groups of individuals, in order to identify association between phenotype and haplotype. If desired, neighboring haplotype blocks can be assessed concurrently, as there may also exist linkage disequilibrium among the haplotype blocks.
  • markers used to detect association thus in a sense represent "tags" for a genomic region (i.e., a haplotype block or LD block) that is associating with a given disease or trait, and as such are useful for use in the methods and kits of the present invention.
  • One or more causative (functional) variants or mutations may reside within the region found to be associating to the disease or trait.
  • Such variants may confer a higher relative risk (RR) or odds ratio (OR) than observed for the tagging markers used to detect the association.
  • the present invention thus refers to the markers used for detecting association to the disease, as described herein, as well as markers in linkage disequilibrium with the markers.
  • markers that are in LD with the markers and/or haplotypes of the invention, as described herein maybe used as surrogate markers.
  • the surrogate markers have in one embodiment relative risk (RR) and/or odds ratio (OR) values smaller than for the markers or haplotypes initially found to be associating with the disease, as described herein.
  • the surrogate markers have RR or OR values greater than those initially determined for the markers initially found to be associating with the disease, as described herein.
  • An example of such an embodiment would be a rare, or relatively rare (such as ⁇ 10% allelic population frequency) variant in LD with a more common variant (> 10% population frequency) initially found to be associating with the disease, such as the variants described herein. Identifying and using such markers for detecting the association discovered by the inventors as described herein can be performed by routine methods well known to the person skilled in the art, and are therefore within the scope of the present invention. [0071 ] Determination of haplotype frequency
  • the frequencies of haplotypes in patient and control groups can be estimated using an expectation-maximization algorithm (Dempster A. et al., J. R. Stat. Soc. B, 39: 1-38 (1977)). An implementation of this algorithm that can handle missing genotypes and uncertainty with the phase can be used. Under the null hypothesis, the patients and the controls are assumed to have identical frequencies. Using a likelihood approach, an alternative hypothesis is tested, where a candidate at-risk-haplotype, which can include the markers described herein, is allowed to have a higher frequency in patients than controls, while the ratios of the frequencies of other haplotypes are assumed to be the same in both groups.
  • One general approach to haplotype analysis involves using likelihood-based inference applied to NEsted MOdels (Gretarsdottir S., et al., Nat. Genet. 35: 131-38 (2003)).
  • the method is implemented in the program NEMO, which allows for many polymorphic markers, SNPs and micros atellites.
  • the method and software are specifically designed for case-control studies where the purpose is to identify haplotype groups that confer different risks. It is also a tool for studying LD structures.
  • maximum likelihood estimates, likelihood ratios and p-values are calculated directly, with the aid of the EM algorithm, for the observed data treating it as a missing-data problem.
  • haplotype counts of the affected and controls each have multinomial distributions, but with different haplotype frequencies under the alternative hypothesis.
  • haplotypes h, and hj
  • HSk(Aj)ZnSk(Z?,) WP ⁇ )/(f j ZP ] )
  • f and p denote, respectively, frequencies in the affected population and in the control population. While there is some power loss if the true model is not multiplicative, the loss tends to be mild except for extreme cases. Most importantly, p- values are always valid since they are computed with respect to null hypothesis.
  • LD between pairs of markers can be calculated using the standard definition of D' and r 2 (Lewontin, R., Genetics 49:49-67 (1964); Hill, W. G. & Robertson, A. Theor. Appl. Genet. 22:226- 231 (1968)).
  • D' and r 2 Lewontin, R., Genetics 49:49-67 (1964); Hill, W. G. & Robertson, A. Theor. Appl. Genet. 22:226- 231 (1968)).
  • NEMO frequencies of the two marker allele combinations are estimated by maximum likelihood and deviation from linkage equilibrium is evaluated by a likelihood ratio test.
  • the definitions of D' and r 2 are extended to include microsatellites by averaging over the values for all possible allele combination of the two markers weighted by the marginal allele probabilities.
  • an absolute risk of developing a disease or trait defined as the chance of a person developing the specific disease or trait over a specified time- period.
  • a woman's lifetime absolute risk of breast cancer is one in nine. That is to say, one woman in every nine will develop breast cancer at some point in their lives.
  • Risk is typically measured by looking at very large numbers of people, rather than at a particular individual, Risk is often presented in terms of Absolute Risk (AR) and Relative Risk (RR).
  • AR Absolute Risk
  • RR Relative Risk
  • Relative Risk is used to compare risks associating with two variants or the risks of two different groups of people. For example, it can be used to compare a group of people with a certain genotype with another group having a different genotype.
  • a relative risk of 2 means that one group has twice the chance of developing a disease as the other group.
  • certain polymorphic markers and haplotypes comprising such markers are found to be useful for risk assessment of AAA.
  • Risk assessment can involve the use of the markers for diagnosing a susceptibility to developing an AAA.
  • Particular alleles of polymorphic markers are found more frequently in individuals with an AAA, compared with individuals without diagnosis of an AAA. Therefore, these marker alleles have predictive value for determining AAA risk.
  • Tagging markers within haplotype blocks or LD blocks comprising at-risk markers, such as the markers of the present invention can be used as surrogates for other markers and/or haplotypes within the haplotype block or LD block. Markers with values of r 2 equal to 1 are perfect surrogates for the at-risk variants, i.e.
  • markers with smaller values of r 2 than 1 can also be surrogates for the at-risk variant, or alternatively represent variants with relative risk values as high as or possibly even higher than the at-risk variant.
  • the at-risk variant identified may not be the functional variant itself, but is in this instance in linkage disequilibrium with the true functional variant.
  • the present invention encompasses the assessment of such surrogate markers for the markers as disclosed herein. Such markers are annotated, mapped and listed in public databases, as well known to the skilled person, or can alternatively be readily identified by sequencing the region or a part of the region identified by the markers of the present invention in a group of individuals, and identify polymorphisms in the resulting group of sequences.
  • the tagging or surrogate markers in LD with the at-risk variants detected also have predictive value for determining risk of developing AAAs.
  • These tagging or surrogate markers that are in LD with the markers of the present invention can also include other markers that distinguish among haplotypes, as these similarly have predictive value for detecting susceptibility to developing an AAA.
  • the present invention can in certain embodiments be practiced by assessing a sample comprising genomic DNA from an individual for the presence of variants described herein to be associated with AAA risk. Such assessment includes steps of detecting the presence or absence of at least one allele of at least one polymorphic marker, using methods well known to the skilled person and further described herein, and based on the outcome of such assessment, determine whether the individual from whom the sample is derived is at increased or decreased risk (increased or decreased susceptibility) of developing an AAA.
  • the invention can be practiced utilizing a dataset comprising information about the genotype status of at least one polymorphic marker described herein to be associated with AAA (or markers in linkage disequilibrium with at least one marker shown herein to be associated with AAA).
  • a dataset containing information about such genetic status for example in the form of genotype counts at a certain polymorphic marker, or a plurality of markers (e.g., an indication of the presence or absence of certain at-risk alleles), or actual genotypes for one or more markers, can be queried for the presence or absence of certain at-risk alleles at certain polymorphic markers shown by the present inventors to be associated with AAA.
  • a positive result for a variant (e.g., marker allele) associated with AAA, as shown herein, is indicative of the individual from which the dataset is derived is at increased risk of developing AAA.
  • the haplotype block structure of the human genome has the effect that a large number of variants (markers and/or haplotypes) in linkage disequilibrium with the variant originally associated with a disease or trait may be used as surrogate markers for assessing association to the disease or trait.
  • the number of such surrogate markers will depend on factors such as the historical recombination rate in the region, the mutational frequency in the region (i.e., the number of polymorphic sites or markers in the region), and the extent of LD (size of the LD block) in the region.
  • markers are usually located within the physical boundaries of the LD block or haplotype block in question as defined using the methods described herein, or by other methods known to the person skilled in the art.
  • markers and haplotypes in LD typically characterized by r 2 greater than 0.1, such as r 2 greater than 0.2, including r 2 greater than 0.3, also including r 2 greater than 0,4
  • markers and haplotypes of the present invention are also within the scope of the invention, even if they are physically located beyond the boundaries of the haplotype block as defined.
  • This may include markers that are in strong LD (e.g., characterized by r 2 greater than 0.1 or 0.2 and/or
  • the methods and kits of the invention can be utilized from samples containing nucleic acid material (DNA or RNA) from any source and from any individual.
  • the individual is a human individual.
  • the individual can be an adult, child, or fetus.
  • the nucleic acid source may be any sample comprising nucleic acid material, including biological samples, or a sample comprising nucleic acid material derived therefrom.
  • the present invention also provides for assessing markers and/or haplotypes in individuals who are members of a target population.
  • a target population is in one embodiment a population or group of individuals at risk of developing an AAA, such as those having the most common risk factors, old age, male gender, smoking, and a family history of AAA.
  • kits for assaying a sample from a individual to detect susceptibility to developing an AAA are also encompassed by the invention.
  • methods are provided for determining the presence or absence of a variant allele at certain polymorphic sites and correlating the presence of a variant allele with increased risk of developing an AAA.
  • a nucleic acid sample as described above, may be taken from an individual to be tested.
  • the sample may be analyzed to determine the nucleotide present at the SNP position of interest. This determination may be done by standard sequencing methods well known in the art, as well as other methods, such as those described below.
  • the individual is analyzed to determine the nucleotide at SNP rs7635818 (position 27 of SEQ ID NO: 1). If a C allele is found to be present at rs7635818, then the individual may be considered as having an increased risk of developing an AAA, and may be recommended for ultrasound scan and subsequent follow up to determine if an AAA is present or may develop. The presence of one C allele at rs7635818 of the individual may be enough to indicate an ultrasound scan, with the presence of two C alleles at this position indicating an even greater risk.
  • the odds ratio for association of the presence of a C allele at rs7635818 with the risk of developing an AAA in a general sample set maybe considered as having an odds ratio of 1.33.
  • the presence of a C allele at rs7635818 may be considered as having an odds ratio of 1.8.
  • the individual is analyzed to determine the nucleotide at SNP rsl2039875 (position 27 of SEQ ID NO: 2). If a C allele is found to be present at rs 12039875, then the individual may be considered as having an increased risk of developing an AAA, and may be recommended for an ultrasound scan and subsequent follow up to determine if an AAA is present or may develop. The presence of one C allele at rsl2039875 of the individual may be enough to indicate an ultrasound scan, with the presence of two C alleles at this position indicating an even greater risk.
  • the odds ratio for association of the presence of a C allele at rs 12039875 with the risk of developing an AAA in a general sample set may be considered as having an odds ratio of 1.18.
  • more than one SNP may be analyzed to determine AAA risk.
  • the nucleotides at both rs7635818 and rsl2039875 may be analyzed to give a total of four alleles (two for each SNP).
  • the total number of variant alleles, the presence of a C for both rs7635818 and rs!2039875, can then be used to determine the risk of developing an AAA, with the presence of 2 or more variant alleles indicating that the individual should have an ultrasound scan and subsequent follow up to determine if an AAA is present or may develop.
  • the presence of 3 or 4 alleles indicates an even greater risk of developing an AAA, and the individual should also be monitored for development of an AAA as described.
  • a panel of three SNPs for determining the risk of developing an AAA is provided.
  • the panel of three SNPs may be made up of rs7635818, rsl2039875 and rsl 0757278, as is described in International Publication Number WO2008/102380.
  • the G allele is associated with AAA risk for rsl 0757278.
  • This panel of three SNPs provides six total alleles. The presence of three or more variant alleles indicates that the individual should have an ultrasound scan and subsequent follow up to determine if an AAA is present or may develop. The presence of 4, 5 or 6 alleles indicates an even greater risk of developing an AAA, and the individual should also be monitored for development of an AAA as described.
  • the odds ratio for association of the presence of three or more variant alleles with the risk of developing an AAA may be considered as having an odds ratio of 2.4, while the presence of six variant alleles may be considered as having an odds ratio of 3.5.
  • polymorphisms in linkage disequilibrium with rs7635818, rsl 2039875 and rsl 0757278 may be done in combination with one, two or all of these three SNPs.
  • polymorphisms that may be used include those in Tables 1 and 2 above, as well as other polymorphisms in the same linkage disequilibrium block as the SNPs in Tables 1 and 2. The presence of variant alleles at these other polymorpisms suggests a further increased risk of developing an AAA.
  • the analysis of the polymorphs of the present invention may be combined with other risk factors to further refine the risk of developing an AAA.
  • This risk factors include old age, male gender, history of smoking, and family history of AAAs.
  • the significance of association of the at least one marker allele or haplotype is characterized by a p value ⁇ 0.05. In other embodiments, the significance of association is characterized by smaller p-values, such as ⁇ 0.01, ⁇ 0.001, ⁇ 0.0001, O.00001, O.000001 , O.0000001, 0.00000001 or ⁇ 0.000000001.
  • the presence of the at least one marker allele or haplotype is indicative of a susceptibility to developing an AAA. These diagnostic methods involve detecting the presence or absence of at least one marker allele or haplotype that is associated with AAA risk.
  • the haplotypes described herein include combinations of alleles at various genetic markers (e.g., SNPs, microsatellites).
  • the detection of the particular genetic marker alleles that make up the particular haplotypes can be performed by a variety of methods described herein and/or known in the art.
  • the marker alleles or haplotypes of the present invention correspond to fragments of a genomic DNA sequence associated with development of AAAs. Such fragments encompass the DNA sequence of the polymorphic marker or haplotype in question, but may also include DNA segments in strong LD (linkage disequilibrium) with the marker or haplotype (e.g., as determined by particular values of r 2 and/or
  • diagnosis of a susceptibility to developing an AAA can be accomplished using hybridization methods, including, but not limited to, Southern analysis, Northern analysis, and/or In situ hybridizations (see Current Protocols in Molecular Biology, Ausubel, F. et al., eds., John Wiley & Sons, including all supplements).
  • the presence of a specific marker allele can be indicated by sequence- specific hybridization of a nucleic acid probe specific for the particular allele.
  • the presence of more than one specific marker allele or a specific haplotype can be indicated by using several sequence- specific nucleic acid probes, each being specific for a particular allele.
  • a haplotype can be indicated by a single nucleic acid probe that is specific for the specific haplotype (i.e., hybridizes specifically to a DNA strand comprising the specific marker alleles characteristic of the haplotype).
  • a sequence- specific probe can be directed to hybridize to genomic DNA, RNA, or cDNA.
  • a "nucleic acid probe”, as used herein, can be a DNA probe or an RNA probe that hybridizes to a complementary sequence. One of skill in the art would know how to design such a probe so that sequence specific hybridization will occur only if a particular allele is present in a genomic sequence from a test sample.
  • a method utilizing a detection oligonucleotide probe comprising a fluorescent moiety or group at its 3' terminus and a quencher at its 5' terminus, and an enhancer oligonucleotide, is employed, as described by Kutyavin et al. (Nucleic Acid Res. 34:el28 (2006)).
  • the fluorescent moiety can be Gig Harbor Green or Yakima Yellow, or other suitable fluorescent moieties,
  • the detection probe is designed to hybridize to a short nucleotide sequence that includes the SNP polymorphism to be detected.
  • the SNP is anywhere from the terminal residue to -6 residues from the 3' end of the detection probe.
  • the enhancer is a short oligonucleotide probe which hybridizes to the DNA template 3' relative to the detection probe.
  • the probes are designed such that a single nucleotide gap exists between the detection probe and the enhancer nucleotide probe when both are bound to the template.
  • the gap creates a synthetic abasic site that is recognized by an endonuclease, such as Endonuclease IV.
  • the enzyme cleaves the dye off the fully complementary detection probe, but cannot cleave a detection probe containing a mismatch.
  • assessment of the presence of a particular allele defined by nucleotide sequence of the detection probe can be performed.
  • the detection probe can be of any suitable size, although preferably the probe is relatively short. In one embodiment, the probe is from 5-100 nucleotides in length. In another embodiment, the probe is from 10-50 nucleotides in length, and in another embodiment, the probe is from 12-30 nucleotides in length. Other lengths of the probe are possible and within scope of the skill of the average person skilled in the art.
  • the DNA template containing the SNP polymorphism is amplified by Polymerase Chain Reaction (PCR) prior to detection. In such an embodiment, the amplified DNA serves as the template for the detection probe and the enhancer probe,
  • Certain embodiments of the detection probe, the enhancer probe, and/or the primers used for amplification of the template by PCR include the use of modified bases, including modified A and modified G.
  • modified bases can be useful for adjusting the melting temperature of the nucleotide molecule (probe and/or primer) to the template DNA, for example for increasing the melting temperature in regions containing a low percentage of G or C bases, in which modified A with the capability of forming three hydrogen bonds to its complementary T can be used, or for decreasing the melting temperature in regions containing a high percentage of G or C bases, for example by using modified G bases that form only two hydrogen bonds to their complementary C base in a double stranded DNA molecule.
  • modified bases are used in the design of the detection nucleotide probe. Any modified base known to the skilled person can be selected in these methods, and the selection of suitable bases is well within the scope of the skilled person based on the teachings herein and known bases available from commercial sources as known to the skilled person.
  • Northern analysis see Current Protocols in Molecular Biology, Ausubel, F. et al., eds., John Wiley & Sons, supra) is used to identify the presence of a polymorphism associated with AAA risk.
  • a test sample of RNA is obtained from the individual by appropriate means.
  • RNA from the individual is indicative of a particular allele complementary to the probe.
  • nucleic acid probes see, for example, U.S. Patent Nos. 5,288,611 and 4,851,330.
  • PNA peptide nucleic acid
  • a PNA is a DNA mimic having a peptide-like, inorganic backbone, such as N-(2- aminoethyl)glycine units, with an organic base (A, G, C, T or U) attached to the glycine nitrogen via a methylene carbonyl linker (see, for example, Nielsen, P., et al., Bioconjug. Chem. 5:3-7 (1994)).
  • the PNA probe can be designed to specifically hybridize to a molecule in a sample suspected of containing one or more of the marker alleles or haplotypes that are associated with risk of developing an AAA. Hybridization of the PNA probe is thus diagnostic for AAA risk.
  • a test sample containing genomic DNA obtained from the individual is collected and the polymerase chain reaction (PCR) is used to amplify a fragment comprising one ore more markers or haplotypes of the present invention.
  • PCR polymerase chain reaction
  • identification of a particular marker allele or haplotype associated with AAA risk can be accomplished using a variety of methods ⁇ e.g., sequence analysis, analysis by restriction digestion, specific hybridization, single stranded conformation polymorphism assays (SSCP), electrophoretic analysis, etc.).
  • diagnosis is accomplished by expression analysis using quantitative PCR (kinetic thermal cycling). This technique can, for example, utilize commercially available technologies, such as TaqMan ® (Applied Biosystems, Foster City, CA) .
  • the technique can assess the presence of an alteration in the expression or composition of a polypeptide or splicing variant(s) that is encoded by a nucleic acid associated with an increased risk of AAA. Further, the expression of the variant(s) can be quantified as physically or functionally different.
  • analysis by restriction digestion can be used to detect a particular allele if the allele results in the creation or elimination of a restriction site relative to a reference sequence.
  • a test sample containing genomic DNA is obtained from the individual.
  • PCR can be used to amplify particular regions that are associated with risk of AAA development (e.g. the polymorphic markers recited herein) nucleic acid in the test sample from the test individual.
  • Restriction fragment length polymorphism (RFLP) analysis can be conducted, e.g., as described in Current Protocols in Molecular Biology, supra. The digestion pattern of the relevant DNA fragment indicates the presence or absence of the particular allele in the sample.
  • Sequence analysis can also be used to detect specific alleles at polymorphic sites associated with risk of AAA development. Therefore, in one embodiment, determination of the presence or absence of a particular marker alleles or haplotypes comprises sequence analysis. For example, a test sample of DNA or RNA can be obtained from the test individual. PCR or other appropriate methods can be used to amplify a portion of a nucleic acid associated with AAA risk, and the presence of a specific allele can then be detected directly by sequencing the polymorphic site (or multiple polymorphic sites) of the genomic DNA in the sample.
  • arrays of oligonucleotide probes that are complementary to target nucleic acid sequence segments from a individual can be used to identify polymorphisms in a nucleic acid associated with AAA risk.
  • an oligonucleotide array can be used.
  • Oligonucleotide arrays typically comprise a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations. These arrays can generally be produced using mechanical synthesis methods or light directed synthesis methods that incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis methods, or by other methods known to the person skilled in the art (see, e.g., Fodor, S.
  • linear arrays can be utilized. Additional descriptions of use of oligonucleotide arrays for detection of polymorphisms can be found, for example, in U.S. Patent Nos. 5,858,659 and 5,837,832, the entire teachings of both of which are incorporated by reference herein.
  • nucleic acid analysis can be used to detect a particular allele at a polymorphic site associated with risk of developing an AAA.
  • Representative methods include, for example, direct manual sequencing (Church and Gilbert, Proc. Natl. Acad. Sci. USA, 81 : 1991-1995 (1988); Sanger, F., et al., Proc. Natl. Acad. Sci. USA, 74:5463-5467 (1977); Beavis, et al., U.S. Patent No.
  • CMC chemical mismatch cleavage
  • RN as e protection assays Myers, R., et al., Science, 230: 1242- 1246 (1985); use of polypeptides that recognize nucleotide mismatches, such as E. coli mutS protein; and allele-specific PCR.
  • the variants (markers or haplotypes) of the invention showing association to cardiovascular disease affect the expression of a nearby gene (e.g., CNTN3 for rs7635818 or KCNK2 for rsl2039875).
  • a nearby gene e.g., CNTN3 for rs7635818 or KCNK2 for rsl2039875.
  • regulatory element affecting gene expression may be located far away, even as far as tenths or hundreds of kilobases away, from the promoter region of a gene.
  • the detection of the markers or haplotypes of the present invention can be used for assessing expression for one or more of the CNTN3 and/or KCNK2 genes.
  • a variety of methods can be used for detecting protein expression levels, including enzyme linked immunosorbent assays (ELISA), Western blots, immunoprecipitations and immunofluorescence.
  • ELISA enzyme linked immunosorbent assays
  • Western blots immunoprecipitations
  • immunofluorescence immunofluorescence.
  • Kits useful in the methods of the invention comprise components useful in any of the methods described herein, including for example, primers for nucleic acid amplification, hybridization probes, restriction enzymes (e.g., for RFLP analysis), allele- specific oligonucleotides, a genomic segment comprising at least one polymorphic marker and/or haplotype of the present invention) or to a non-altered (native) polypeptide encoded by a nucleic acid associated with the risk of developing an AAA, means for amplification of a nucleic acid associated with AAA risk, means for analyzing the nucleic acid sequence of a nucleic acid associated with AAA risk, means for analyzing the amino acid sequence of a polypeptide encoded by a nucleic acid associated with AAA risk, etc.
  • kits can for example include necessary buffers, nucleic acid primers for amplifying nucleic acids of the invention (e.g., a nucleic acid segment comprising one or more of the polymorphic markers as described herein), and reagents for allele-specific detection of the fragments amplified using such primers and necessary enzymes (e.g., DNA polymerase). Additionally, kits can provide reagents for assays to be used in combination with the methods of the present invention, e.g., reagents for use with other diagnostic assays as described herein.
  • nucleic acid primers for amplifying nucleic acids of the invention e.g., a nucleic acid segment comprising one or more of the polymorphic markers as described herein
  • reagents for allele-specific detection of the fragments amplified using such primers and necessary enzymes e.g., DNA polymerase.
  • kits can provide reagents for assays to be used in combination with the methods of the present invention, e
  • the invention pertains to a kit for assaying a sample from a individual to detect the risk of developing an AAA, wherein the kit comprises reagents necessary for selectively detecting at least one allele of at least one polymorphism of the present invention in the genome of the individual.
  • the reagents comprise at least one contiguous oligonucleotide that hybridizes to a fragment of the genome of the individual comprising at least one polymorphism of the present invention.
  • the reagents comprise at least one pair of oligonucleotides that hybridize to opposite strands of a genomic segment obtained from a individual, wherein each oligonucleotide primer pair is designed to selectively amplify a fragment of the genome of the individual that includes one polymorphism, wherein the polymorphism is selected from the group consisting of the polymorphisms listed above, and polymorphic markers in linkage disequilibrium therewith.
  • the fragment is at least 20 base pairs in size.
  • oligonucleotides or nucleic acids can be designed using portions of the nucleic acids flanking polymorphisms (e.g., SNPs or microsatellites) that are indicative of a risk of developing an AAA.
  • the kit comprises one or more labeled nucleic acids capable of detecting one or more specific polymorphic markers or haplotypes associated with AAA risk, and reagents for detection of the label.
  • Suitable labels include, e.g., a radioisotope, a fluorescent label, an enzyme label, an enzyme co-factor label, a magnetic label, a spin label, an epitope label.
  • the polymorphic marker or haplotype to be detected by the reagents of the kit comprises one or more markers, two or more markers, or three or more markers, selected from the group consisting of the rs7635818, rsl2039875 and rsl0757278, and markers in linkage disequilibrium therewith.
  • the fluorescent moiety can be Gig Harbor Green or Yakima Yellow, or other suitable fluorescent moieties.
  • the detection probe is designed to hybridize to a short nucleotide sequence that includes the SNP polymorphism to be detected.
  • the SNP is anywhere from the terminal residue to -6 residues from the 3' end of the detection probe.
  • the enhancer is a short oligonucleotide probe which hybridizes to the DNA template 3' relative to the detection probe.
  • the probes are designed such that a single nucleotide gap exists between the detection probe and the enhancer nucleotide probe when both are bound to the template.
  • the gap creates a synthetic abasic site that is recognized by an endonuclease, such as Endonuclease IV.
  • the enzyme cleaves the dye off the fully complementary detection probe, but cannot cleave a detection probe containing a mismatch.
  • assessment of the presence of a particular allele defined by nucleotide sequence of the detection probe can be performed.
  • the detection probe can be of any suitable size, although preferably the probe is relatively short. In one embodiment, the probe is from 5-100 nucleotides in length. In another embodiment, the probe is from 10-50 nucleotides in length, and in another embodiment, the probe is from 12-30 nucleotides in length. Other lengths of the probe are possible and within scope of the skill of the average person skilled in the art.
  • the DNA template containing the SNP polymorphism is amplified by Polymerase Chain Reaction (PCR) prior to detection, and primers for such amplification are included in the reagent kit. In such an embodiment, the amplified DNA serves as the template for the detection probe and the enhancer probe.
  • PCR Polymerase Chain Reaction
  • Certain embodiments of the detection probe, the enhancer probe, and/or the primers used for amplification of the template by PCR include the use of modified bases, including modified A and modified G.
  • modified bases can be useful for adjusting the melting temperature of the nucleotide molecule (probe and/or primer) to the template DNA, for example for increasing the melting temperature in regions containing a low percentage of G or C bases, in which modified A with the capability of forming three hydrogen bonds to its complementary T can be used, or for decreasing the melting temperature in regions containing a high percentage of G or C bases, for example by using modified G bases that form only two hydrogen bonds to their complementary C base in a double stranded DNA molecule.
  • modified bases are used in the design of the detection nucleotide probe. Any modified base known to the skilled person can be selected in these methods, and the selection of suitable bases is well within the scope of the skilled person based on the teachings herein and known bases available from commercial sources as known to the skilled person.
  • the presence of the marker or haplotype is indicative of an increased risk of developing an AAA.
  • the presence of the marker or haplotype is indicative of response to a therapeutic agent for treatment of an AAA.
  • the presence of the marker or haplotype is indicative of prognosis of an AAA.
  • the presence of the marker or haplotype may indicate that the AAA should be repaired even if it is smaller than aneurysms that are typically repaired, e.g. it is smaller than 5.5 cm for men and 5 cm for women.
  • the kit further comprises a set of instructions for using the reagents comprising the kit.
  • the present invention also relates to computer-implemented applications of the polymorphic markers and haplotypes described herein to be associated with AAA risk.
  • Such applications can be useful for storing, manipulating or otherwise analyzing genotype data that is useful in the methods of the invention.
  • One example pertains to storing genotype information derived from an individual on readable media, so as to be able to provide the genotype information to a third party (e.g., the individual), or for deriving information from the genotype data, e.g., by comparing the genotype data to information about genetic risk factors contributing to increased susceptibility to developing an AAA, and reporting results based on such comparison.
  • One such aspect relates to computer-readable media.
  • such medium has capabilities of storing (i) identifier information for at least one polymorphic marker or a haplotye; (ii) an indicator of the frequency of at least one allele of said at least one marker, or the frequency of a haplotype, in individuals with increased AAA risk, and an indicator of the frequency of at least one allele of said at least one marker, or the frequency of a haplotype, in a reference population.
  • the reference population can be a population of individuals without clinical evidence of an AAA.
  • the reference population is a random sample from the general population, and is thus representative of the population at large.
  • the frequency indicator may be a calculated frequency, a count of alleles and/or haplotype copies, or normalized or otherwise manipulated values of the actual frequencies that are suitable for the particular medium.
  • Additional information about the individual can be stored on the medium, such as ancestry information, information about sex, physical attributes or characteristics (including height and weight), biochemical measurements (such as blood pressure, blood lipid levels, lipid levels, such as cholesterol levels) or other useful information that is desirable to store or manipulate in the context of the genotype status of a particular individual.
  • the invention furthermore relates to an apparatus that is suitable for determination or manipulation of genetic data useful for determining a susceptibility to develop an AAA in a human individual.
  • Such an apparatus can include a computer- readable memory, a routine for manipulating data stored on the computer-readable memory, and a routine for generating an output that includes a measure of the genetic data.
  • Such measure can include values such as allelic or haplotype frequencies, genotype counts, sex, age, phenotype information, values for odds ratio (OR) or relative risk (RR), population attributable risk (PAR), or other useful information that is either a direct statistic of the original genotype data or based on calculations based on the genetic data.
  • markers and haplotypes shown herein to be associated with increased risk of developing an AAA are in certain embodiments useful for interpretation and/or analysis of genotype data.
  • an identification of an at-risk allele for AAA, as shown herein, or an allele at a polymorphic marker in LD with any one of the markers shown herein to be associated with AAA risk indicates the increased risk of the individual to developing an AAA.
  • genotype data is generated for at least one polymorphic marker shown herein to be associated with AAA risk, or a marker in linkage disequilibrium therewith.
  • the genotype data is subsequently made available to the individual from whom the data originates, for example via a user interface accessible over the internet, together with an interpretation of the genotype data, e.g., in the form of a risk measure (such as an absolute risk (AR), risk ratio (RR) or odds ration (OR)) for the development of an AAA.
  • a risk measure such as an absolute risk (AR), risk ratio (RR) or odds ration (OR)
  • AR absolute risk
  • RR risk ratio
  • OR odds ration
  • results of such risk assessment can be reported in numeric form (e.g., by risk values, such as absolute risk, relative risk, and/or an odds ratio, or by a percentage increase in risk compared with a reference), by graphical means, or by other means suitable to illustrate the risk to the individual from whom the genotype data is derived.
  • the results of risk assessment is made available to a third party, e.g., a physician, other healthcare worker or genetic counselor.
  • nucleic acids and polypeptides described herein can be used in methods and kits of the present invention.
  • An "isolated" nucleic acid molecule is one that is separated from nucleic acids that normally flank the gene or nucleotide sequence (as in genomic sequences) and/or has been completely or partially purified from other transcribed sequences (e.g., as in an RNA library).
  • an isolated nucleic acid of the invention can be substantially isolated with respect to the complex cellular milieu in which it naturally occurs, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
  • the isolated material will form part of a composition (for example, a crude extract containing other substances), buffer system or reagent mix.
  • the material can be purified to essential homogeneity, for example as determined by polyacrylamide gel electrophoresis (PAGE) or column chromatography (e.g., HPLC).
  • An isolated nucleic acid molecule of the invention can comprise at least about 50%, at least about 80% or at least about 90% (on a molar basis) of all macromolecular species present.
  • genomic DNA the term "isolated" also can refer to nucleic acid molecules that are separated from the chromosome with which the genomic DNA is naturally associated.
  • the isolated nucleic acid molecule can contain less than about 250 kb, 200 kb, 150 kb, 100 kb, 75 kb, 50 kb, 25 kb, 10 kb, 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of the nucleotides that flank the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid molecule is derived.
  • the nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated.
  • recombinant DNA contained in a vector is included in the definition of "isolated” as used herein.
  • isolated nucleic acid molecules include recombinant DNA molecules in heterologous host cells or heterologous organisms, as well as partially or substantially purified DNA molecules in solution.
  • isolated nucleic acid molecules also encompass in vivo and in vitro RNA transcripts of the DNA molecules of the present invention.
  • An isolated nucleic acid molecule or nucleotide sequence can include a nucleic acid molecule or nucleotide sequence that is synthesized chemically or by recombinant means.
  • Such isolated nucleotide sequences are useful, for example, in the manufacture of the encoded polypeptide, as probes for isolating homologous sequences (e.g., from other mammalian species), for gene mapping (e.g., by in situ hybridization with chromosomes), or for detecting expression of the gene in tissue (e.g., human tissue), such as by Northern blot analysis or other hybridization techniques.
  • homologous sequences e.g., from other mammalian species
  • gene mapping e.g., by in situ hybridization with chromosomes
  • tissue e.g., human tissue
  • the invention also pertains to nucleic acid molecules that hybridize under high stringency hybridization conditions, such as for selective hybridization, to a nucleotide sequence described herein (e.g., nucleic acid molecules that specifically hybridize to a nucleotide sequence containing a polymorphic site associated with a haplotype described herein).
  • the invention includes variants that hybridize under high stringency hybridization and wash conditions (e.g., for selective hybridization) to a nucleotide sequence that comprises the nucleotide sequence of SEQ ID Nos: 1, 2, or 3, or a fragment thereof, wherein the nucleotide sequence comprises at least one at-risk allele of at least one polymorphic marker, or at least one haplotype, as described herein.
  • the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%, of the length of the reference sequence.
  • probes or primers are oligonucleotides that hybridize in abase- specific manner to a complementary strand of a nucleic acid molecule.
  • probes and primers include polypeptide nucleic acids (PNA), as described in Nielsen, P. et al., Science 254: 1497-1500 (1991).
  • a probe or primer comprises a region of nucleotide sequence that hybridizes to at least about 15, typically about 20-25, and in certain embodiments about 40, 50 or 75, consecutive nucleotides of a nucleic acid molecule comprising a contiguous nucleotide sequence from a haplotype block of the invention, and comprising at least one allele of at least one polymorphic marker or at least one haplotype described herein, or the complement thereof.
  • a probe or primer can comprise 100 or fewer nucleotides; for example, in certain embodiments from 6 to 50 nucleotides, or, for example, from 12 to 30 nucleotides.
  • the probe or primer is at least 70% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical, to the contiguous nucleotide sequence or to the complement of the contiguous nucleotide sequence.
  • the probe or primer is capable of selectively hybridizing to the contiguous nucleotide sequence or to the complement of the contiguous nucleotide sequence.
  • the probe or primer further comprises a label, e.g., a radioisotope, a fluorescent label, an enzyme label, an enzyme co-factor label, a magnetic label, a spin label, an epitope label.
  • nucleic acid molecules of the invention can be identified and isolated using standard molecular biology techniques and the sequence information provided by the nucleotide sequence of SEQ ID Nos: 1, 2, and 3. See generally PCR Technology: Principles and Applications for DNA Amplification (ed. H. A. Erlich, Freeman Press, NY, NY, 1992); PCR Protocols: A Guide to Methods and Applications (Eds. Innis, et al., Academic Press, San Diego, CA, 1990); Mattila, P. et al., Nucleic Acids Res., 19:4967- 4973 (1991); Eckert, K.
  • the isolated nucleic acid sequences of the invention can be used as molecular weight markers on Southern gels, and as chromosome markers that are labeled to map related gene positions.
  • the nucleic acid sequences can also be used to compare with endogenous DNA sequences in patients to identify a susceptibility to development of an AAA, and as probes, such as to hybridize and discover related DNA sequences or to subtract out known sequences from a sample (e.g., subtractive hybridization).
  • the nucleic acid sequences can further be used to derive primers for genetic fingerprinting, to raise anti-polyp eptide antibodies using immunization techniques, and/or as an antigen to raise anti-DNA antibodies or elicit immune responses.
  • the following Examples are meant to illustrate specific embodiments of the present invention, and are not intended to limit the scope of the invention as described above and as claimed herein.
  • GVC Geisinger vascular clinic
  • Geisinger MyCode samples A secondary control group was obtained through the Geisinger MyCode Project, a population cohort of Geisinger Clinic primary care patients recruited for genomic studies. Enrolled participants agreed to provide blood samples for genomic research and permission to extract clinical and demographic data from their EMR. For this study, DNA samples from 442 MyCode participants were randomly selected from a collection of DNA samples from more than 5000 patients and were matched for age distribution and gender to the GVC cases, and excluded patients with a diagnosis of AAA in their EMR. Characteristics of the MyCode individuals are provided in Table III. [00143] Replication sample set. These samples consisted of 453 AAA cases and 418 controls that were recruited from Belgium, Canada, and the USA. These samples have been used in other genetic studies of AAA, and have been described previously. (12) Appropriate institutional approvals were obtained for use of human individuals in this research. Characteristics of the replication sample set individuals are provided in Table III.
  • DNA was purified from EDTA anti- coagulated blood using a Qiagen BioRobot M48 Workstation and MagAttract DNA Blood Midi M48 kit (Qiagen, Valencia, Calif). The yield and purity of DNA was determined by measuring the absorbance at 260 and 280 nm using a Nanodrop spectrophotometer (ThermoFisher Scientific, Waltham, Mass).
  • Genome wide association study We carried out an initial GWAS of AAA cases and controls using a DNA pooling strategy similar to that used in previous studies. 13-16 A flow-chart that summarizes the GWAS strategy is in Fig 1. Three identical DNA pools were generated independently from DNA samples of 123 cases or 112 controls (a total of 6 pools) from the GVC samples. Cases and controls were matched for male/female ratio and age; all had a smoking history of >20 years. The characteristics of the individuals used for the GWAS are shown in Table IV. To create the DNA pools, 100 ng of DNA from each individual was combined and then precipitated at 20 0 C by adding 0.1 volume of 3 M sodium acetate, pH 5.2, and 2.5 volumes of ethanol. The DNA was collected by centrifugation, washed with 70% ethanol, and air-dried. The dried pellets were dissolved in water at a final concentration of 50 ng/mL. Table IV
  • the six DNA pools were hybridized separately to Affymetrix SNP arrays using the GeneChip Mapping 500K Manual Protocol (Affymetrix, Inc, Santa Clara, Calif). Pooled DNA (250 ng/array) was digested with Nsp I or Sty I and ligated to adapters that recognize overhangs generated by the restriction enzymes. Oligonucleotides that recognize the adapter sequences were used as primers to amplify the adapter-ligated DNA fragments. The amplified DNA was fragmented, labeled, and hybridized to GeneChip Human Mapping 250K Nsp and 250K Sty arrays (Affymetrix, Inc) for 18 hours at 47 0 C.
  • GCOS GeneChip Operating Software
  • GTYPE GeneChip Genotyping Analysis Software
  • the relative frequencies of the two alleles for each SNP were determined from the hybridization signals. Only the perfect match probe signals were used in the allele frequency calculation. To identify SNPs with a high measurement variance, the intra- group coefficient of variation (CV) of the allele frequency for each SNP was calculated. SNPs with a CV >0,l in either the case or control group were discarded. For the remaining 306,330 SNPs, the difference in mean allele frequency between cases and controls was calculated. The / test P values for the allele frequency difference between cases and controls for each SNP were also calculated.
  • CV intra- group coefficient of variation
  • Candidate SNPs for follow-up studies were chosen from genomic regions that contained multiple SNPs with highest case-control allele frequency differences. This was based on the assumption that the high density of SNPs on the arrays (median distance between SNPs on the arrays is 2.5 kb) would reveal multiple SNPs in LD with the genetic variants contributing to the disease. To identify such regions, the SNP allele frequency difference data were sorted by physical position on each autosomal chromosome. Rolling sums of the relative allele frequency differences of five adjacent SNPs or sums of the - logio transformed P values of the SNP allele frequency differences of five adjacent SNPs along each chromosome were calculated. [00150] Individual genotype analysis.
  • PCR Fast Real-Time polymerase chain reaction
  • Assay conditions were as follows: pre-read hold at 60°C for 30 seconds, fluorescence recording; 1 cycle at 95°C for 10 minutes to activate AmpliTaq Gold DNA Polymerase (Affymetrix, Inc); 40 cycles of 95°C for 15 seconds and 60°C for 1 minute followed by a fluorescence recording after each cycle; and a hold at 6O 0 C for 30 seconds followed by an endpoint fluorescence recording. Data were analyzed using the 7500 Fast Sequence Detection System Software version 1.4 (Applied Biosystems), with background fluorescence subtraction and automatic allele calling. For SNP rs7635818, the genotype call rates were 99.9% with the Geisinger Clinic DNA samples and 98.8% with the replication samples. [00152] Contactin-3 transcript assay.
  • Total RNA was used as a template for cDNA synthesis in a reaction that contained the following: 2 ⁇ g RNA, 4 ⁇ l of 1OX Mg free buffer (Promega, Madison, Wis), 5 mM MgC12 (Promega, Madison, Wis), 5 ⁇ M random hexamers, 1 mM dNTPs, 2 ⁇ l Superscript II RT (Invitrogen), and PCR grade water to a final volume of 40 ⁇ l. Reaction conditions were 25°C for 10 minutes, 50°C for 50 minutes, 70 0 C for 15 minutes followed by a 4°C hold.
  • cDNAs were used as templates for PCR amplification of CNTN3 transcripts using JumpStart Taq DNA Polymerase (Sigma, St Louis, Mo). The standard protocol was used, but with the addition of MgCl2 to a final concentration of 15 mM. Primers for the CNTN3 gene were designed using PrimerQuest (Integrated DNA Technologies, Coralville, Iowa).
  • Primer pairs used were: forward 5' - TGCCCTTGGAAATCCCATACCTCA (SEQ ID NO:4) - 3' and reverse 5' - TGTCCTCCACGGCTATTTCCACAT - 3' (SEQ ID NO:5) (predicted product size of 251 bp); and forward 5' - ACAGACACAACAGCCCAACTCTCT - 3' (SEQ ID NO:6) and reverse 5' - GGCGGAAAGCAACAACATACCCAA - 3' (SEQ ID NO;7) (predicted product size of 397 bp).
  • PCR amplification conditions were 1 minute at 94°C followed by 35 cycles of 94 0 C for 1 minute, 55°C for 1 minute, and 72°C for 30 seconds, followed by a final extension at 72°C for 5 minutes and a 4°C hold. Products were analyzed on 1 % agarose gels and stained with ethidium bromide. [00156] Statistical analyses.
  • Genotype data were analyzed using Helix Tree Software, version 6.4 (Golden Helix, Inc, Bozeman, Mont). Analyses included calculations of deviation from Hardy- Weinberg equilibrium, linkage disequilibrium, and genetic association. P values for genetic association were calculated using recursive partitioning with case/ control selected as a categorical dependent variable and genotype as the independent variable. The data were also analyzed assuming a dominant model of genetic association, which tests the association of having at least one minor allele vs having no copies of the minor allele. Both approaches yielded essentially identical results. Uncorrected P values are reported.
  • GWAS identifies a variant on 3pl2.3 associated with AAA.
  • Relative SNP allele frequencies in triplicate pools of 123 case and 112 control samples were determined using Affymetrix 500K SNP arrays. (See Fig. 1 for additional details on the GWAS.) Criteria for prioritizing candidate SNPs included a combination of absolute case-control SNP allele frequency differences, t test P values of SNP allele frequency differences, and physical clustering of SNPs with allele frequency differences.
  • SNPs in this region were genotyped in a total of 502 GVC cases and 296 control samples.
  • AAA-associated SNPs were contained within a haplotype block in the HapMap CEU (Caucasians of European descent from Utah) population (Fig. 3) and were in strong LD in the GVC case and control samples (D' >0.98) (Fig. 4).
  • the AAA-associated allele was C (with G as the major allele) and its frequency was 0.48 in cases and 0.42 in controls.
  • SNP Single nucleotide polymorphism
  • AAA alxiominal aortic aneurysm
  • N number.
  • CNTN3 is expressed in AAA and other vascular tissues. SNP rs7635818 is upstream of the CNTN3 gene, which encodes a lipid-anchored cell adhesion protein (contactin-3). (18) The CNTN3 gene has not been reported previously to be expressed in vascular tissue.
  • RNA extracted from AAA tissue, control aortic tissue, aortic-occlusive disease (AOD) tissue, and peripheral blood mononuclear cells from AAA patients was used to generate cDNAs that were used as templates for PCR amplification using CNTN3- specific primers. Products derived from CNTN3 transcripts were detected in all the tissues sampled (Fig. 6), demonstrating that the gene is actively expressed in aortic tissue, including AAA specimens. [00170] Discussion
  • SNP rs7635818 is contained within a haplotype block that shows strong LD in the HapMap CEU population and in the case and control groups used for this study.
  • Our genetic mapping studies showed the AAA associated haplotype block that contains SNP rs7635818 to span approximately 30 kb of genomic DNA sequence. This region does not overlap any known protein coding genes, but is approximately 200 kb upstream of the transcription start site for the CNTN3 gene. (The next nearest genes are PDZRN3 and ROBO2, which are approximately 1.3 and 2.2 million bp away, respectively.)
  • the functional genetic variant has a regulatory function. Because it lies upstream of the CNTN3 gene, where sequence motifs that regulate CNTN3 gene activity are expected to be located, our working hypothesis is that the functional variant is in the upstream regulatory region of the CNTN3 gene.
  • the product of the CNTN3 gene is a lipid-anchored cell adhesion protein that had been previously shown to be expressed in the central nervous system.
  • the CNTN3 transcript is readily detected in aortic tissue, including both normal and AAA tissue. Although this genomic region contains consensus DNA binding sites for known transcription factors, no studies to date have examined the effects of sequence variants in the AAA-associated haplotype block marked by SNP rs7635818 on CNTN3 expression.
  • a dominant genetic model yielded the most significant association between SNP rs7635818 genotype and AAA. This would be consistent with the existence of a genetic variant in the CNTN3 promoter that affects contactin-3 expression, either constitutively or in response to regulatory signals. Regulation of contactin-3 expression in vascular tissue has not been investigated.
  • the genetic variant reported here has a moderate but statistically significant association with AAA disease.
  • This variant and the previously reported SNP on chromosome 9p21 account for only a portion of the genetic risk for AAA.
  • a long-term goal of this research is to use genetic information to quantify AAA disease risk, probably through the use of a panel of AAA-associated genetic variants. This would enable high-risk individuals to be identified for closer monitoring to detect undiagnosed AAAs, so that appropriate treatment can be provided in a timely manner.
  • Knowledge of disease- associated genetic variants can also provide new insight into disease mechanisms and provide targets for development of novel therapies. (19) At the present time, there are no medical treatments for AAAs.
  • AAA-associated genetic variant On chromosome Iq41
  • Individuals for a genomic study of AAA were recruited from Geisinger Clinic
  • AAA-associated genetic variants As the first step in a multi-stage process to identify AAA- associated genetic variants we carried out a GWAS using pooled DNA samples from 123 AAA case and 1 12 control individuals recruited through the Geisinger Clinic Department of Vascular Surgery. Controls were matched to the cases for age, gender, and smoking history. Relative SNP allele frequencies were analyzed to identify candidate AAA-associated SNPs for subsequent validation.
  • chromosome Iq41 One genomic region that was selected for further study was within chromosome Iq41. This region contained a cluster of SNPs with statistically significant allele frequency differences between the case and control samples in the pooled DNA GWAS (Fig. 7). As an initial validation step, six SNPs within this region were individually genotyped in a subset of 451 cases and 279 controls from the Geisinger vascular surgery clinic (Fig. 8). Four of these SNPs had genotypes that were significantly associated with AAA (P values ranging from 0.001 to 0.014). These AAA-associated SNPs are in a region of strong LD in the HapMap CEU population (Fig. 8), and were in strong LD in the genotyped samples from the Geisinger vascular clinic (Fig. 9).
  • SNP rsl 2039875 was genotyped in additional case and control samples from the Geisinger Vascular Clinic and from the Geisinger MyCode Project.
  • the genotype of SNP rsl 2039875 was significantly associated with AAA in the combined Geisinger Clinic sample set (P - 0.0085, 690 cases, 792 controls) (Table VIII).
  • KCNK2 is expressed in AAA tissue
  • the AAA-associated haplotype overlies the KCNK2 gene, which encodes a 2- pore-domain membrane potassium channel.
  • KCNK2 transcripts were assayed by PCR amplification of cDNAs generated by reverse transcription of total RNA isolated from AAA tissue, normal aortic tissue, and tissue from aortic occlusive lesions. GAPDH was used as a positive control.
  • Three KCNK2 isoforms are produced from this gene that differ in their 5 '-untranslated and N-terminal amino acid sequences (isoform a (SEQ ID NO: 8), isoform b (SEQ ID NO: 9) and isoform c (SEQ ID NO: 10).
  • This report describes a haplotype on chromosome Iq41 that is associated with AAA disease.
  • the haplotype is identified by several SNP variants that are in strong LD, SNP rsl2039875 was used as a marker for this haplotype to establish its association with AAA in 2 independent populations.
  • the AAA-associated haplotype overlaps the KCNK2, which encodes a 2 -pore membrane potassium channel.
  • the KCNK2 gene is expressed in AAA tissue at levels that are higher than normal aorta.
  • AAA-associated genetic loci each with a significant but relatively small contribution to disease risk, is consistent with the concept of AAA as a complex disease that is affected by an unknown number of genetic factors as well as environmental risk factors.
  • the other AAA-associated genetic loci are SNP rsl0757278 on chromosome 9p21 , (12) and SNP rs7635818 on chromosome 3pl2.3 that was identified by GWAS of pooled DNA samples in our laboratory.
  • the AAA-associated haplotype on chromosome 1 q41 overlies the KCNK2 gene, which encodes a membrane potassium channel.
  • SNP rsl2039875 lies within the first intron of the 2 KCNK2 isoforms that we show are expressed in AAA tissue.
  • the steady state levels of KCNK2 transcripts are significantly higher in AAA tissue than normal aorta.
  • the fact that KCNK2 transcripts were not detected in peripheral blood mononuclear cells of AAA patients suggests that the gene is expressed by cells of the vascular wall and not by infiltrating inflammatory or immune cells, although this needs to be confirmed by additional studies. While not previously reported in AAA tissue, the KCNK2 potassium channel has been shown to be expressed in subsets of vascular smooth muscle cells, where it has been suggested to play a role in regulation of blood pressure in response to lipids. (23)
  • AAA-associated haplotype No common genetic variants that alter the protein coding information of KCNK2 are present in the public databases. Therefore, the functional genetic variant in this AAA-associated haplotype is not known.
  • the lack of known non-synonymous SNPs in this gene and data showing increased expression of KCNK ' 2 in AAA tissue support the hypothesis that the functional genetic variant influences AAA risk by altering KCNK2 expression.
  • One of the limitations of studying human AAA is that the main source of AAA tissue is patients undergoing open surgical repair of their aneurysm. In nearly all cases, however, AAA repair is limited to aneurysmal lesions with a diameter of 5 cm or greater.
  • AAA-screening by ultrasonography is recommended only for male smokers between the ages of 65 and 75 years.
  • Butcher LM Davis OS, Craig IW, Plomin R. Genome- wide quantitative trait locus association scan of general cognitive ability using pooled DNA and 500K single nucleotide polymorphism microarrays. Genes Brain Behav 2008;7:435-46.
  • Boddy AM Lenk GM, Lillvis JH, Nischan J, Kyo Y, Kuivaniemi H. Basic research studies to understand aneurysm disease. Drug News Perspect 2008;21 : 142-8.
  • 1166 polymorphism is associated with abdominal aortic aneurysm in three independent cohorts. Arterioscler Thromb Vase Biol. 2008; 28:764-770.

Abstract

L'invention concerne des procédés et des nécessaires permettant de déterminer le risque de développement d'un anévrisme de l'aorte abdominale. Les procédés de la présente invention impliquent l'analyse d'échantillons d'acide nucléique prélevés sur un individu afin de déterminer la présence d'un ou plusieurs polymorphismes associés aux anévrismes de l'aorte abdominale.
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