Association mapping in structured populations

The use, in association studies, of the forthcoming dense genomewide collection of single-nucleotide polymorphisms (SNPs) has been heralded as a potential breakthrough in the study of the genetic basis of common complex disorders. A serious problem with association mapping is that population structure can lead to spurious associations between a candidate marker and a phenotype. One common solution has been to abandon case-control studies in favor of family-based tests of association, such as the transmission/disequilibrium test (TDT), but this comes at a considerable cost in the need to collect DNA from close relatives of affected individuals. In this article we describe a novel, statistically valid, method for case-control association studies in structured populations. Our method uses a set of unlinked genetic markers to infer details of population structure, and to estimate the ancestry of sampled individuals, before using this information to test for associations within subpopulations. It provides power comparable with the TDT in many settings and may substantially outperform it if there are conflicting associations in different subpopulations.     

The structure of linkage disequilibrium at the DBH locus strongly influences the magnitude of association between diallelic markers and plasma dopamine beta-hydroxylase activity

There is currently a great deal of interest in using linkage disequilibrium (LD) mapping to locate both disease and quantitative-trait loci on a genomewide scale. Recent findings suggest that much of the human genome is organized in discrete "blocks" of low haplotype diversity, but the utility of such blocks in identifying genes influencing complex traits is not yet known and must ultimately be tested empirically through use of real data. We recently identified a putative functional polymorphism (-1021C-->T) in the 5' upstream region of the DBH gene that accounted for 35%-52% of the total phenotypic variance in plasma dopamine beta-hydroxylase (DBH) activity in samples from three distinct populations. In the present study, we genotyped 11 diallelic markers at the DBH locus surrounding -1021C-->T in 386 unrelated individuals of European origin. We identified a single 10-kb block containing -1021C-->T, in which four haplotypes comprised 93% of the observed chromosomes. Only markers within the block were highly associated with phenotype (P< or =2.2 x 10(-10)), with one exception. In general, association with phenotype was strongly correlated with the degree of LD between each marker and -1021C-->T. Of four LD measures assessed, d(2) was the best predictor of this relationship. Had one attempted to map quantitative-trait loci for plasma DBH activity on a genomewide basis without prior knowledge of candidate regions and not included (by chance) markers within this haplotype block, the DBH locus might have been missed entirely. These results provide a direct example of the potential value of constructing a haplotype map of the human genome prior to embarking on large-scale association studies.     

Evidence for a susceptibility gene, SLEV1, on chromosome 17p13 in families with vitiligo-related systemic lupus erythematosus

Both systemic lupus erythematosus (SLE) and vitiligo are autoimmune disorders that have strong evidence of complex genetic contributions to their etiology, but, to date, efforts using genetic linkage to find the susceptibility genes for either phenotype have met with limited success. Since autoimmune diseases are thought to share at least some of their genetic origins, and since only a small minority (16 of 92) of the European-American pedigrees multiplex for SLE in our collection have one or more affected members with vitiligo, we hypothesized that these pedigrees might be more genetically homogeneous at loci important to both SLE and vitiligo and, hence, have increased power for detection of linkage. We therefore evaluated genomewide microsatellite-marker-scan data for markers at an average marker density of approximately 11 cM in these 16 European-American pedigrees and identified a significant linkage at 17p13, where the maximum multipoint parametric LOD score was 3.64 (P<4.3x10(-5)) and the nonparametric linkage score was 4.02 (P<2.8x10(-5)), respectively. The segregation behavior of this linkage suggests a recessive mode of inheritance with a virtually homogeneous genetic effect in these 16 pedigrees. These results support the hypotheses that SLE and vitiligo may share important genetic effects and that sampling on the basis of clinical covariates dramatically improves power to identify genetic effects.     

Genomewide scan for anal atresia in swine identifies linkage and association with a chromosome region on Sus scrofa chromosome 1

Anal atresia is a rare and severe disorder in swine occurring with an incidence of 0.1-1.0%. A whole-genome scan based on affected half-sibs was performed to identify susceptibility loci for anal atresia. The analysis included 27 families with a total of 95 animals and 65 affected piglets among them. Animals were genotyped for 126 microsatellite markers distributed across the 18 autosomal porcine chromosomes and the X chromosome, covering an estimated 2080 cM. Single-point and multipoint nonparametric linkage scores were calculated using the computer package ALLEGRO 1.0. Significant linkage results were obtained for chromosomes 1, 3, and 12. Markers on these chromosomes and additionally on chromosomes for which candidate genes have been postulated in previous studies were subjected to the transmission disequilibrium test (TDT). The test statistic exceeded the genomewide significance level for adjacent markers SW1621 (P = 7 x 10(-7)) and SW1902 (P = 3 x 10(-3)) on chromosome 1, supporting the results of the linkage analysis. A specific haplotype associated with anal atresia that could prove useful for selection against the disorder was revealed. Suggestive linkage and association were also found for markers S0081 on chromosome 9 and SW957 on chromosome 12.     

Phosphodiesterase 8B gene variants are associated with serum TSH levels and thyroid function

Thyroid-stimulating hormone (TSH) controls thyroid growth and hormone secretion through binding to its G protein-coupled receptor (TSHR) and production of cyclic AMP (cAMP). Serum TSH is a sensitive indicator of thyroid function, and overt abnormalities in thyroid function lead to common endocrine disorders affecting approximately 10% of individuals over a life span. By genotyping 362,129 SNPs in 4,300 Sardinians, we identified a strong association (p = 1.3 x 10(-11)) between alleles of rs4704397 and circulating TSH levels; each additional copy of the minor A allele was associated with an increase of 0.13 muIU/ml in TSH. The single-nucleotide polymorphism (SNP) is located in intron 1 of PDE8B, encoding a high-affinity cAMP-specific phosphodiesterase. The association was replicated in 4,158 individuals, including additional Sardinians and two genetically distant cohorts from Tuscany and the Old Order Amish (overall p value = 1.9 x 10(-20)). In addition to association of TSH levels with SNPs in PDE8B, our genome scan provided evidence for association with PDE10A and several biologically interesting candidates in a focused analysis of 24 genes. In particular, we found evidence for association of TSH levels with SNPs in the THRB (rs1505287, p = 7.3 x 10(-5)), GNAQ (rs10512065, p = 2.0 x 10(-4)), TG (rs2252696, p = 2.2 x 10(-3)), POU1F1 (rs1976324, p = 3.9 x 10(-3)), PDE4D (rs27178, p = 8.3 x 10(-3)), and TSHR (rs4903957, p = 8.6 x 10(-3)) loci. Overall, the results suggest a primary effect of PDE8B variants on cAMP levels in the thyroid. This would affect production of T4 and T3 and feedback to alter TSH release by the pituitary. PDE8B may thus provide a candidate target for the treatment of thyroid dysfunction.     

Genome-Wide Association Studies in Dogs and Humans Identify ADAMTS20 as a Risk Variant for Cleft Lip and Palate

Cleft lip with or without cleft palate (CL/P) is the most commonly occurring craniofacial birth defect. We provide insight into the genetic etiology of this birth defect by performing genome-wide association studies in two species: dogs and humans. In the dog, a genome-wide association study of 7 CL/P cases and 112 controls from the Nova Scotia Duck Tolling Retriever (NSDTR) breed identified a significantly associated region on canine chromosome 27 (unadjusted p=1.1 x 10^-13; adjusted p= 2.2 x 10^-3). Further analysis in NSDTR families and additional full sibling cases identified a 1.44 Mb homozygous haplotype (chromosome 27: 9.29 – 10.73 Mb) segregating with a more complex phenotype of cleft lip, cleft palate, and syndactyly (CLPS) in 13 cases. Whole-genome sequencing of 3 CLPS cases and 4 controls at 15X coverage led to the discovery of a frameshift mutation within ADAMTS20 (c.1360_1361delAA (p.Lys453Ilefs*3)), which segregated concordant with the phenotype. In a parallel study in humans, a family-based association analysis (DFAM) of 125 CL/P cases, 420 unaffected relatives, and 392 controls from a Guatemalan cohort, identified a suggestive association (rs10785430; p =2.67 x 10^-6) with the same gene, ADAMTS20. Sequencing of cases from the Guatemalan cohort was unable to identify a causative mutation within the coding region of ADAMTS20, but four coding variants were found in additional cases of CL/P. In summary, this study provides genetic evidence for a role of ADAMTS20 in CL/P development in dogs and as a candidate gene for CL/P development in humans.     

Genome-wide meta-analysis identifies regions on 7p21 (AHR) and 15q24 (CYP1A2) as determinants of habitual caffeine consumption

We report the first genome-wide association study of habitual caffeine intake. We included 47,341 individuals of European descent based on five population-based studies within the United States. In a meta-analysis adjusted for age, sex, smoking, and eigenvectors of population variation, two loci achieved genome-wide significance: 7p21 (P = 2.4 × 10(-19)), near AHR, and 15q24 (P = 5.2 × 10(-14)), between CYP1A1 and CYP1A2. Both the AHR and CYP1A2 genes are biologically plausible candidates as CYP1A2 metabolizes caffeine and AHR regulates CYP1A2.     

Family-based association tests for genomewide association scans

With millions of single-nucleotide polymorphisms (SNPs) identified and characterized, genomewide association studies have begun to identify susceptibility genes for complex traits and diseases. These studies involve the characterization and analysis of very-high-resolution SNP genotype data for hundreds or thousands of individuals. We describe a computationally efficient approach to testing association between SNPs and quantitative phenotypes, which can be applied to whole-genome association scans. In addition to observed genotypes, our approach allows estimation of missing genotypes, resulting in substantial increases in power when genotyping resources are limited. We estimate missing genotypes probabilistically using the Lander-Green or Elston-Stewart algorithms and combine high-resolution SNP genotypes for a subset of individuals in each pedigree with sparser marker data for the remaining individuals. We show that power is increased whenever phenotype information for ungenotyped individuals is included in analyses and that high-density genotyping of just three carefully selected individuals in a nuclear family can recover >90% of the information available if every individual were genotyped, for a fraction of the cost and experimental effort. To aid in study design, we evaluate the power of strategies that genotype different subsets of individuals in each pedigree and make recommendations about which individuals should be genotyped at a high density. To illustrate our method, we performed genomewide association analysis for 27 gene-expression phenotypes in 3-generation families (Centre d'Etude du Polymorphisme Humain pedigrees), in which genotypes for ~860,000 SNPs in 90 grandparents and parents are complemented by genotypes for ~6,700 SNPs in a total of 168 individuals. In addition to increasing the evidence of association at 15 previously identified cis-acting associated alleles, our genotype-inference algorithm allowed us to identify associated alleles at 4 cis-acting loci that were missed when analysis was restricted to individuals with the high-density SNP data. Our genotype-inference algorithm and the proposed association tests are implemented in software that is available for free.     

Localization of a susceptibility gene for type 2 diabetes to chromosome 5q34-q35.2

We report a genomewide linkage study of type 2 diabetes (T2D [MIM 125853]) in the Icelandic population. A list of type 2 diabetics was cross-matched with a computerized genealogical database clustering 763 type 2 diabetics into 227 families. The diabetic patients and their relatives were genotyped with 906 microsatellite markers. A nonparametric multipoint linkage analysis yielded linkage to 5q34-q35.2 (LOD = 2.90, P=1.29 x 10(-4)) in all diabetics. Since obesity, here defined as body mass index (BMI) > or =30 kg/m(2), is a key risk factor for the development of T2D, we studied the data either independently of BMI or by stratifying the patient group as obese (BMI > or =30) or nonobese (BMI <30). A nonparametric multipoint linkage analysis yielded linkage to 5q34-q35.2 (LOD = 3.64, P=2.12 x (10)-5) in the nonobese diabetics. No linkage was observed in this region for the obese diabetics. Linkage analysis conditioning on maternal transmission to the nonobese diabetics resulted in a LOD score of 3.48 (P=3.12 x 10(-5)) in the same region, whereas conditioning on paternal transmission led to a substantial drop in the LOD score. Finally, we observed potential interactions between the 5q locus and two T2D susceptibility loci, previously mapped in other populations.     

Genome-wide scan on total serum IgE levels identifies FCER1A as novel susceptibility locus

High levels of serum IgE are considered markers of parasite and helminth exposure. In addition, they are associated with allergic disorders, play a key role in anti-tumoral defence, and are crucial mediators of autoimmune diseases. Total IgE is a strongly heritable trait. In a genome-wide association study (GWAS), we tested 353,569 SNPs for association with serum IgE levels in 1,530 individuals from the population-based KORA S3/F3 study. Replication was performed in four independent population-based study samples (total n = 9,769 individuals). Functional variants in the gene encoding the alpha chain of the high affinity receptor for IgE (FCER1A) on chromosome 1q23 (rs2251746 and rs2427837) were strongly associated with total IgE levels in all cohorts with P values of 1.85 x 10(-20) and 7.08 x 10(-19) in a combined analysis, and in a post-hoc analysis showed additional associations with allergic sensitization (P = 7.78 x 10(-4) and P = 1.95 x 10(-3)). The "top" SNP significantly influenced the cell surface expression of FCER1A on basophils, and genome-wide expression profiles indicated an interesting novel regulatory mechanism of FCER1A expression via GATA-2. Polymorphisms within the RAD50 gene on chromosome 5q31 were consistently associated with IgE levels (P values 6.28 x 10(-7)-4.46 x 10(-8)) and increased the risk for atopic eczema and asthma. Furthermore, STAT6 was confirmed as susceptibility locus modulating IgE levels. In this first GWAS on total IgE FCER1A was identified and replicated as new susceptibility locus at which common genetic variation influences serum IgE levels. In addition, variants within the RAD50 gene might represent additional factors within cytokine gene cluster on chromosome 5q31, emphasizing the need for further investigations in this intriguing region. Our data furthermore confirm association of STAT6 variation with serum IgE levels.     

A missense single-nucleotide polymorphism in a gene encoding a protein tyrosine phosphatase (PTPN22) is associated with rheumatoid arthritis

Rheumatoid arthritis (RA) is the most common systemic autoimmune disease, affecting approximately 1% of the adult population worldwide, with an estimated heritability of 60%. To identify genes involved in RA susceptibility, we investigated the association between putative functional single-nucleotide polymorphisms (SNPs) and RA among white individuals by use of a case-control study design; a second sample was tested for replication. Here we report the association of RA susceptibility with the minor allele of a missense SNP in PTPN22 (discovery-study allelic P=6.6 x 10(-4); replication-study allelic P=5.6 x 10(-8)), which encodes a hematopoietic-specific protein tyrosine phosphatase also known as "Lyp." We show that the risk allele, which is present in approximately 17% of white individuals from the general population and in approximately 28% of white individuals with RA, disrupts the P1 proline-rich motif that is important for interaction with Csk, potentially altering these proteins' normal function as negative regulators of T-cell activation. The minor allele of this SNP recently was implicated in type 1 diabetes, suggesting that the variant phosphatase may increase overall reactivity of the immune system and may heighten an individual carrier's risk for autoimmune disease.     

Quantitative trait locus on chromosome 12q14.1 influences variation in plasma plasminogen levels in the San Antonio Family Heart Study

Plasminogen is a hemostasis-related phenotype and is commonly implicated in thrombotic and bleeding disorders. In the San Antonio Family Heart Study (SAFHS), we performed to our knowledge the first genomewide linkage scan for quantitative trait loci (QTLs) that influence the level of plasminogen. The subset of the SAFHS population used for this study consists of 629 individuals distributed across 26 extended Mexican American families. Pedigree-based variance component linkage analyses were performed using SOLAR. The mean plasminogen level was 114.94% +/- 17.8 (range, 42-195). The heritability (h2) of plasminogen was 0.43 +/- 0.08 (p < 6.3 x 10(-13)). One region on chromosome 12 (12q14.1) showed suggestive evidence of linkage (LOD = 2.73, nominal p < 0.0002, genomewide p = 0.0786) near marker D12S1609. Because plasminogen has important effects in many human health problems, such as cancer and atherosclerosis, the role of this putative QTL in the regulation of plasminogen variability needs to be studied further.     

Genomewide-linkage and haplotype-association studies map intracranial aneurysm to chromosome 7q11

Rupture of intracranial aneurysms (IAs) causes subarachnoid hemorrhage, a devastating condition with high morbidity and mortality. Angiographic and autopsy studies show that IA is a common disorder, with a prevalence of 3%-6%. Although IA has a substantial genetic component, little attention has been given to the genetic determinants. We report here a genomewide linkage study of IA in 104 Japanese affected sib pairs in which positive evidence of linkage on chromosomes 5q22-31 (maximum LOD score [MLS] 2.24), 7q11 (MLS 3.22), and 14q22 (MLS 2.31) were found. The best evidence of linkage is detected at D7S2472, in the vicinity of the elastin gene (ELN), a candidate gene for IA. Fourteen distinct single-nucleotide polymorphisms (SNPs) were identified in ELN, and no obvious allelic association between IA and each SNP was observed. The haplotype between the intron-20/intron-23 polymorphism of ELN is strongly associated with IA (P=3.81x10-6), and homozygous patients are at high risk (P=.002), with an odds ratio of 4.39. These findings suggest that a genetic locus for IA lies within or close to the ELN locus on chromosome 7.     

An x chromosome association scan of the Norfolk Island genetic isolate provides evidence for a novel migraine susceptibility locus at Xq12

Migraine is a common and debilitating neurovascular disorder with a complex envirogenomic aetiology. Numerous studies have demonstrated a preponderance of women affected with migraine and previous pedigree linkage studies in our laboratory have identified susceptibility loci on chromosome Xq24-Xq28. In this study we have used the genetic isolate of Norfolk Island to further analyse the X chromosome for migraine susceptibility loci.An association approach was employed to analyse 14,124 SNPs spanning the entire X chromosome. Genotype data from 288 individuals comprising a large core-pedigree, of which 76 were affected with migraine, were analysed. Although no SNP reached chromosome-wide significance (empirical α = 1 × 10(-5)) ranking by P-value revealed two primary clusters of SNPs in the top 25. A 10 SNP cluster represents a novel migraine susceptibility locus at Xq12 whilst a 11 SNP cluster represents a previously identified migraine susceptibility locus at Xq27. The strongest association at Xq12 was seen for rs599958 (OR = 1.75, P = 8.92 × 10(-4)), whilst at Xq27 the strongest association was for rs6525667 (OR = 1.53, P = 1.65 × 10(-4)). Further analysis of SNPs at these loci was performed in 5,122 migraineurs from the Women's Genome Health Study and provided additional evidence for association at the novel Xq12 locus (P<0.05).Overall, this study provides evidence for a novel migraine susceptibility locus on Xq12. The strongest effect SNP (rs102834, joint P = 1.63 × 10(-5)) is located within the 5'UTR of the HEPH gene, which is involved in iron homeostasis in the brain and may represent a novel pathway for involvement in migraine pathogenesis.     

An autosome-wide search using longitudinal data for loci linked to type 2 diabetes progression

A genome-wide screen was conducted for type 2 diabetes progression genes using measures of elevated fasting glucose levels as quantitative traits from the offspring enrolled in the Framingham Heart Study. We analyzed young (20-34 years) and old (>or= 35 years) subjects separately, using single-point and multipoint sibpair analysis, because of the possible differential impact of progression on the groups of interest. We observed significant linkage with change in fasting glucose levels on 1q25-32 (p = 5.21 x 10(-8)), 3p26.3-21.31 (p = 1 x 10(-11)), 8q23.1-24.13 (p = 2.94 x 10(-6)), 9p24.1-21.3 (p = 7 x 10(-7)), and 18p11.31-q22.1 (p < 10(-11)). The evidence for linkage on chromosomes 8 and 18 was consistent for the subset of study participants aged 43 through 55 years.     

A large-scale genetic association study confirms IL12B and leads to the identification of IL23R as psoriasis-risk genes

We performed a multitiered, case-control association study of psoriasis in three independent sample sets of white North American individuals (1,446 cases and 1,432 controls) with 25,215 genecentric single-nucleotide polymorphisms (SNPs) and found a highly significant association with an IL12B 3'-untranslated-region SNP (rs3212227), confirming the results of a small Japanese study. This SNP was significant in all three sample sets (odds ratio [OR](common) 0.64, combined P [Pcomb]=7.85x10(-10)). A Monte Carlo simulation to address multiple testing suggests that this association is not a type I error. The coding regions of IL12B were resequenced in 96 individuals with psoriasis, and 30 additional IL12B-region SNPs were genotyped. Haplotypes were estimated, and genotype-conditioned analyses identified a second risk allele (rs6887695) located approximately 60 kb upstream of the IL12B coding region that exhibited association with psoriasis after adjustment for rs3212227. Together, these two SNPs mark a common IL12B risk haplotype (OR(common) 1.40, Pcomb=8.11x10(-9)) and a less frequent protective haplotype (OR(common) 0.58, Pcomb=5.65x10(-12)), which were statistically significant in all three studies. Since IL12B encodes the common IL-12p40 subunit of IL-12 and IL-23, we individually genotyped 17 SNPs in the genes encoding the other chains of these cytokines (IL12A and IL23A) and their receptors (IL12RB1, IL12RB2, and IL23R). Haplotype analyses identified two IL23R missense SNPs that together mark a common psoriasis-associated haplotype in all three studies (OR(common) 1.44, Pcomb=3.13x10(-6)). Individuals homozygous for both the IL12B and the IL23R predisposing haplotypes have an increased risk of disease (OR(common) 1.66, Pcomb=1.33x10(-8)). These data, and the previous observation that administration of an antibody specific for the IL-12p40 subunit to patients with psoriasis is highly efficacious, suggest that these genes play a fundamental role in psoriasis pathogenesis.     

Genome-wide association study of allergic diseases in Russians of Western Siberia

Genome-wide association studies are currently considered as one of the most powerful tools to establishing the genetic basis of complex diseases. A number of such studies were carried out for allergic diseases; however, in Russian population this analysis has not been performed so far. For the first time, we performed genome-wide association study of allergic diseases in Russian inhabitants of Western Siberia. Two new loci associated with childhood bronchial asthma were identified (20q13.12, rs2425656, P = 1.99 x 10(-7); 1q32.1, rs3817222, rs12734001, P = 2.18 x 10(-7) and 2.79 x 10(-7), respectively) as well as one locus, associated with allergic rhinitis (2q36.1, rs1597167, P = 3.69 x 10(-7)). Genes located in the loci, YWHAB and PPP1R12B for asthma and KCNE4 for allergic rhinitis, are new genes for these diseases. It was found that BAT1 (6p21.33), MAGI2 (7q21.11) and ACPL2 (3q23) genes are, likely, common (syntropic) genes of allergic disease and a topic sensitisation. It was shown that RIT2 (18q12.3) and (5q31.1) genes can be involved in the control of lung function. The results of the study enlarge the body of data on genetic factors of allergy and expand the list of genes underlying these diseases.