Positional cloning of a novel gene influencing asthma from chromosome 2q14.

Asthma is a common disease in children and young adults. Four separate reports have linked asthma and related phenotypes to an ill-defined interval between 2q14 and 2q32 (refs. 1-4), and two mouse genome screens have linked bronchial hyper-responsiveness to the region homologous to 2q14 (refs. 5,6). We found and replicated association between asthma and the D2S308 microsatellite, 800 kb distal to the IL1 cluster on 2q14. We sequenced the surrounding region and constructed a comprehensive, high-density, single-nucleotide polymorphism (SNP) linkage disequilibrium (LD) map. SNP association was limited to the initial exons of a solitary gene of 3.6 kb (DPP10), which extends over 1 Mb of genomic DNA. DPP10 encodes a homolog of dipeptidyl peptidases (DPPs) that cleave terminal dipeptides from cytokines and chemokines, and it presents a potential new target for asthma therapy.

Genome-wide mapping of susceptibility to coronary artery disease identifies a novel replicated locus on chromosome 17.

Coronary artery disease (CAD) is a leading cause of death world-wide, and most cases have a complex, multifactorial aetiology that includes a substantial heritable component. Identification of new genes involved in CAD may inform pathogenesis and provide new therapeutic targets. The PROCARDIS study recruited 2,658 affected sibling pairs (ASPs) with onset of CAD before age 66 y from four European countries to map susceptibility loci for CAD. ASPs were defined as having CAD phenotype if both had CAD, or myocardial infarction (MI) phenotype if both had a MI. In a first study, involving a genome-wide linkage screen, tentative loci were mapped to Chromosomes 3 and 11 with the CAD phenotype (1,464 ASPs), and to Chromosome 17 with the MI phenotype (739 ASPs). In a second study, these loci were examined with a dense panel of grid-tightening markers in an independent set of families (1,194 CAD and 344 MI ASPs). This replication study showed a significant result on Chromosome 17 (MI phenotype; p = 0.009 after adjustment for three independent replication tests). An exclusion analysis suggests that further genes of effect size lambda(sib) > 1.24 are unlikely to exist in these populations of European ancestry. To our knowledge, this is the first genome-wide linkage analysis to map, and replicate, a CAD locus. The region on Chromosome 17 provides a compelling target within which to identify novel genes underlying CAD. Understanding the genetic aetiology of CAD may lead to novel preventative and/or therapeutic strategies.

Targeted mutagenesis of the Hira gene results in gastrulation defects and patterning abnormalities of mesoendodermal derivatives prior to early embryonic lethality.

The Hira gene encodes a nuclear WD40 domain protein homologous to the yeast transcriptional corepressors Hir1p and Hir2p. Using targeted mutagenesis we demonstrate that Hira is essential for murine embryogenesis. Analysis of inbred 129Sv embryos carrying the null mutation revealed an initial requirement during gastrulation, with many mutant embryos having a distorted primitive streak. Mutant embryos recovered at later stages have a range of malformations with axial and paraxial mesendoderm being particularly affected, a finding consistent with the disruption of gastrulation seen earlier in development. This phenotype could be partially rescued by a CD1 genetic background, although the homozygous mutation was always lethal by embryonic day 11, with death probably resulting from abnormal placentation and failure of cardiac morphogenesis.

The association between common vitamin D receptor gene variations and osteoporosis: a participant-level meta-analysis.

BACKGROUND: Polymorphisms of the vitamin D receptor (VDR) gene have been implicated in the genetic regulation of bone mineral density (BMD). However, the clinical impact of these variants remains unclear. OBJECTIVE: To evaluate the relation between VDR polymorphisms, BMD, and fractures. DESIGN: Prospective multicenter large-scale association study. SETTING: The Genetic Markers for Osteoporosis consortium, involving 9 European research teams. PARTICIPANTS: 26,242 participants (18,405 women). MEASUREMENTS: Cdx2 promoter, FokI, BsmI, ApaI, and TaqI polymorphisms; BMD at the femoral neck and the lumbar spine by dual x-ray absorptiometry; and fractures. RESULTS: Comparisons of BMD at the lumbar spine and femoral neck showed nonsignificant differences less than 0.011 g/cm2 for any genotype with or without adjustments. A total of 6067 participants reported a history of fracture, and 2088 had vertebral fractures. For all VDR alleles, odds ratios for fractures were very close to 1.00 (range, 0.98 to 1.02) and collectively the 95% CIs ranged from 0.94 (lowest) to 1.07 (highest). For vertebral fractures, we observed a 9% (95% CI, 0% to 18%; P = 0.039) risk reduction for the Cdx2 A-allele (13% risk reduction in a dominant model). LIMITATIONS: The authors analyzed only selected VDR polymorphisms. Heterogeneity was detected in some analyses and may reflect some differences in collection of fracture data across cohorts. Not all fractures were related to osteoporosis. CONCLUSIONS: The FokI, BsmI, ApaI, and TaqI VDR polymorphisms are not associated with BMD or with fractures, but the Cdx2 polymorphism may be associated with risk for vertebral fractures.

Large-scale evidence for the effect of the COLIA1 Sp1 polymorphism on osteoporosis outcomes: the GENOMOS study.

BACKGROUND: Osteoporosis and fracture risk are considered to be under genetic control. Extensive work is being performed to identify the exact genetic variants that determine this risk. Previous work has suggested that a G/T polymorphism affecting an Sp1 binding site in the COLIA1 gene is a genetic marker for low bone mineral density (BMD) and osteoporotic fracture, but there have been no very-large-scale studies of COLIA1 alleles in relation to these phenotypes. METHODS AND FINDINGS: Here we evaluated the role of COLIA1 Sp1 alleles as a predictor of BMD and fracture in a multicenter study involving 20,786 individuals from several European countries. At the femoral neck, the average (95% confidence interval [CI]) BMD values were 25 mg/cm2 (CI, 16 to 34 mg/cm2) lower in TT homozygotes than the other genotype groups (p < 0.001), and a similar difference was observed at the lumbar spine; 21 mg/cm2 (CI, 1 to 42 mg/cm2), (p = 0.039). These associations were unaltered after adjustment for potential confounding factors. There was no association with fracture overall (odds ratio [OR] = 1.01 [CI, 0.95 to 1.08]) in either unadjusted or adjusted analyses, but there was a non-significant trend for association with vertebral fracture and a nominally significant association with incident vertebral fractures in females (OR = 1.33 [CI, 1.00 to 1.77]) that was independent of BMD, and unaltered in adjusted analyses. CONCLUSIONS: Allowing for the inevitable heterogeneity between participating teams, this study-which to our knowledge is the largest ever performed in the field of osteoporosis genetics for a single gene-demonstrates that the COLIA1 Sp1 polymorphism is associated with reduced BMD and could predispose to incident vertebral fractures in women, independent of BMD. The associations we observed were modest however, demonstrating the importance of conducting studies that are adequately powered to detect and quantify the effects of common genetic variants on complex diseases.

Single-point haplotype scores telomeric to human leukocyte antigen-C give a high susceptibility major histocompatibility complex haplotype for psoriasis in a Caucasian population.

Psoriasis is a chronic inflammatory skin disease that affects 0.1%-5% depending on the population. PSORS1 is the major susceptibility locus, accounting for approximately 33%-50% of the genetic component of psoriasis among Caucasians. PSORS1 is located within the major histocompatibility complex (MHC) locus on 6p21.3. Its position has been refined to hundreds of kilobase and the region located at approximately 100-200 kb telomeric to human leukocyte antigen (HLA)-C is a very strong candidate. To determine the MHC psoriasis risk haplotype, we screened the whole 46 kb interval for single-nucleotide polymorphisms (SNP) and identified 138 SNP. We genotyped 29 SNP throughout this region in psoriatic nuclear families. We calculated the frequency of haplotypes generated by the 29 SNP using all genotyped founder individuals and found four common haplotype with frequency >0.10. We then used SNPtagger to derive the best six SNP and fed these into Transmit using 148 nuclear families. We found that CTGGAC haplotype is a single-point score haplotypes telomeric to HLA-C and gives a 1 df, chi2 of 50.27 (p<0.0001). Most importantly the six selected SNP accurately tagged the most common haplotype found in this region. Moreover, using the same program (Transmit) we show that the association with CTGGAC is higher than the one with HLA-Cw6 (chi2=10.53; p=0.0051). Our results give scores as high as the highest single-point scores suggesting that it is unlikely to be able to discriminate the origin of the association on this analysis on strength of association.

Association of the TSHR gene with Graves' disease: the first disease specific locus.

The development of autoimmune thyroid disease (AITD) is associated with autoantibodies directed against the thyroid stimulating hormone receptor (TSHR). Previous studies have failed to demonstrate a consistent association between the TSHR and AITD, or any of its sub-phenotypes. In the present study, we analysed the linkage disequilibrium (LD) structure encompassing the TSHR, to identify LD 'blocks' and SNPs, which capture the majority of intra-block haplotype diversity. The haplotype tagging SNPs, plus all common SNPs reported in previous studies were genotyped in 1,059 AITD Caucasian cases and 971 Caucasian controls. A haplotype, across two LD blocks, showed association (P<1 x 10(-6), OR 1.7) with Graves' disease (GD) but not autoimmune hypothyroidism (AIH). We replicated these findings by genotyping the most associated GD SNP, rs2268458, in a separate UK Caucasian cohort of 1,366 AITD cases and 1,061 controls (GD, P=2 x 10(-6), OR 1.3; AIH, P=NS). These results in two independent Caucasian data sets suggest that the TSHR is the first replicated GD-specific locus meriting further fine mapping and functional analysis to identify the aetiological variants.

Fine mapping of the IBD1 locus did not identify Crohn disease-associated NOD2 variants: implications for complex disease genetics.

Crohn disease (CD) is a chronic relapsing inflammatory condition of the gastrointestinal tract. Recently, polymorphisms in NOD2 (CARD15), a gene mapping to the chromosome 16 IBD1 susceptibility locus, have been associated with susceptibility to CD. One group identified the gene by using classic positional cloning methods. Here, we report linkage and fine mapping analyses using 27 microsatellite markers encompassing the IBD1 susceptibility locus in 131 CD affected sibling pairs, and a simplex family cohort. No evidence for linkage was observed, and microsatellite markers close to NOD2 did not show association. However, significant association was confirmed in 294 CD trios for the NOD2 variants Arg702Trp and Leu1007fsinsC. Our fine mapping study of the IBD1 locus did not enable us to identify NOD2 as a CD gene, despite the presence of association with disease-causing alleles. This study illustrates the difficulties facing microsatellite linkage and linkage disequilibrium mapping methods for identifying disease genes in complex traits.

Differential genetic effects of ESR1 gene polymorphisms on osteoporosis outcomes.

CONTEXT: Both bone mineral density (BMD) and fracture risk have a strong genetic component. Estrogen receptor alpha (ESR1) is a candidate gene for osteoporosis, but previous studies of ESR1 polymorphisms in this field were hampered by small sample size, lack of standardization, and inconclusive results. OBJECTIVE: To generate large-scale evidence on whether 3 common ESR1 polymorphisms (intron 1 polymorphisms XbaI [dbSNP: rs9340799] and PvuII [dbSNP: rs2234693] and promoter TA repeats microsatellite) and haplotypes thereof are associated with BMD and fractures. DESIGN AND SETTING: Meta-analysis of individual-level data involving standardized genotyping of 18 917 individuals in 8 European centers. MAIN OUTCOME MEASURES: BMD of femoral neck and lumbar spine; all fractures and vertebral fractures by genotype. RESULTS: No between-center heterogeneity was observed for any outcome in any genetic contrast. None of the 3 polymorphisms or haplotypes had any statistically significant effect on BMD in adjusted or unadjusted analyses, and estimated differences between genetic contrasts were 0.01 g/cm2 or less. Conversely, we found significant reductions in fracture risk. In women homozygous for the absence of an XbaI recognition site, the adjusted odds of all fractures were reduced by 19% (odds ratio, 0.81 [95% CI, 0.71-0.93]; P = .002) and vertebral fractures by 35% (odds ratio, 0.65 [95% CI, 0.49-0.87]; P = .003). Effects on fractures were independent of BMD and unaltered in adjusted analyses. No significant effects on fracture risk were seen for PvuII and TA repeats. CONCLUSIONS: ESR1 is a susceptibility gene for fractures, and XbaI determines fracture risk by mechanisms independent of BMD. Our study demonstrates the value of adequately powered studies with standardized genotyping and clinical outcomes in defining effects of common genetic variants on complex diseases.

LPIN2 is associated with type 2 diabetes, glucose metabolism, and body composition.

OBJECTIVE: To identify the type 2 diabetes gene located at chromosome 18p11. RESEARCH DESIGN AND METHODS: We investigated the region in a young genetically isolated population by genotyping 34 single nucleotide polymorphisms (SNPs) in 78 case subjects and 101 control subjects. Two SNPs were selected and followed up in two cohorts. The first cohort came from a general Dutch population. In this cohort, association with type 2 diabetes was investigated using 616 type 2 diabetic case subjects and 2,890 control subjects; association with oral glucose tolerance test data was performed in 361 normoglycemic people. Association with fat distribution was studied in the second replication cohort, consisting of 836 people from the genetically isolated population. RESULTS: At the initial step, we found that the common C allele of SNP rs3745012 was associated with type 2 diabetes (odds ratio 2.01, P = 0.03). This SNP is located at the 3' untranslated region of the LPIN2 gene, which is a plausible candidate for type 2 diabetes and obesity. In the cohort from the general Dutch population, we demonstrated that rs3745012 interacts with BMI in determination of type 2 diabetes: whereas in subjects with high BMI, the common C allele is associated with type 2 diabetes, the same allele exhibits a neutral or protective effect in lean subjects (P = 0.05 overall effect, P = 0.02 interaction). Most remarkably, rs3745012 strongly affected composite insulin sensitivity index (P = 0.006 for overall effect, P = 0.004 for interaction). In the second replication cohort, we found that the allele C of rs3745012 increases trunk-to-legs fat mass ratio (P = 0.001) and may affect other fat-related measurements. CONCLUSIONS: rs3745012 SNP of the LPIN2 gene is associated with type 2 diabetes and fat distribution.

Association between a complex insertion/deletion polymorphism in NOD1 (CARD4) and susceptibility to inflammatory bowel disease.

The identification of the role of genetic variants within NOD2 (CARD15) in Crohn's disease and ulcerative colitis susceptibility highlight the role of the innate immune system in inflammatory bowel disease (IBD) pathogenesis. NOD1 (CARD4) is located on chromosome 7p14.3, in a region of known linkage to IBD and encodes an intracellular bacterial pathogen-associated molecular pattern receptor that is closely related to NOD2. We have identified strong association between haplotypes in the terminal exons of NOD1 and IBD (multi-allelic P = 0.0000003) in a panel of 556 IBD trios. The deletion allele of a complex functional NOD1 indel polymorphism (ND(1) + 32656*1) was significantly associated with early-onset IBD (P = 0.0003) in unrelated cases and controls. ND1 + 32656*1 was also associated with extra-intestinal manifestations of IBD (P = 0.04). These findings in two independent populations provide strong evidence for a role for NOD1 variants in IBD susceptibility and reinforce the role of the innate immune system in IBD pathogenesis.

Genomewide linkage study in 1,176 affected sister pair families identifies a significant susceptibility locus for endometriosis on chromosome 10q26.

Endometriosis is a common gynecological disease that affects up to 10% of women in their reproductive years. It causes pelvic pain, severe dysmenorrhea, and subfertility. The disease is defined as the presence of tissue resembling endometrium in sites outside the uterus. Its cause remains uncertain despite >50 years of hypothesis-driven research, and thus the therapeutic options are limited. Disease predisposition is inherited as a complex genetic trait, which provides an alternative route to understanding the disease. We seek to identify susceptibility loci, using a positional-cloning approach that starts with linkage analysis to identify genomic regions likely to harbor these genes. We conducted a linkage study of 1,176 families (931 from an Australian group and 245 from a U.K. group), each with at least two members--mainly affected sister pairs--with surgically diagnosed disease. We have identified a region of significant linkage on chromosome 10q26 (maximum LOD score [MLS] of 3.09; genomewide P = .047) and another region of suggestive linkage on chromosome 20p13 (MLS = 2.09). Minor peaks (with MLS > 1.0) were found on chromosomes 2, 6, 7, 8, 12, 14, 15, and 17. This is the first report of linkage to a major locus for endometriosis. The findings will facilitate discovery of novel positional genetic variants that influence the risk of developing this debilitating disease. Greater understanding of the aberrant cellular and molecular mechanisms involved in the etiology and pathophysiology of endometriosis should lead to better diagnostic methods and targeted treatments.

Inflammatory bowel disease is associated with a TNF polymorphism that affects an interaction between the OCT1 and NF(-kappa)B transcription factors.

Tumour necrosis factor-alpha (TNF) expression is increased in inflammatory bowel disease (IBD), and TNF maps to the IBD3 susceptibility locus. Transmission disequilibrium and case-control analyses, in two independent Caucasian cohorts, showed a novel association of the TNF(-857C) promoter polymorphism with IBD (overall P=0.001 in 587 IBD families). Further genetic associations of TNF(-857C) with IBD sub-phenotypes were seen for ulcerative colitis and for Crohn's disease, but only in patients not carrying common NOD2 mutations. The genetic data suggest a recessive model of inheritance, and we observed ex vivo lipopolysaccharide-stimulated whole-blood TNF production to be higher in healthy TNF(-857C) homozygotes. We show the transcription factor OCT1 binds TNF(-857T) but not TNF(-857C), and interacts in vitro and in vivo with the pro-inflammatory NF(-kappa)B transcription factor p65 subunit at an adjacent binding site. Detailed functional analyses of these interactions in gut macrophages, in addition to further genetic mapping of this gene-dense region, will be critical to understand the significance of the observed association of TNF(-857C) with IBD.

The IBD6 Crohn's disease locus demonstrates complex interactions with CARD15 and IBD5 disease-associated variants.

Genetic studies in inflammatory bowel disease have identified multiple susceptibility loci, whose relevance depends critically on verification in independent cohorts. Genetic variants associated with Crohn's disease have now been identified on chromosomes 5 (IBD5/5q31 risk haplotype) and 16 (IBD1 locus, CARD15/NOD2 mutations). Stratification of genome-wide linkage analyses by disease associated variants is now possible, offering both increased power for identification of other loci and improved understanding of genetic mechanisms. We performed a genome-wide scan of 137 Crohn's disease affected relative pairs from 112 families. Multipoint non-parametric linkage analyses were performed, with further stratification of affection status by common CARD15 mutations and the IBD5 haplotype. We verified linkage of Crohn's disease to regions on chromosome 3 (P=0.0009) and X (P=0.001) in our cohort. Linkage to chromosome 16 (IBD1) was observed in Crohn's disease pairs not possessing common CARD15 mutations (P=0.0007), approximately 25 cM q telomeric of CARD15. Evidence for linkage to chromosome 19 (IBD6) was observed in Crohn's disease pairs not possessing CARD15 mutations (P=0.0001), and in pairs possessing one or two copies of the IBD5 risk haplotype (P=0.0005), with significant evidence for genetic heterogeneity and epistasis, respectively. These analyses demonstrate the complex genetic basis to Crohn's disease, and show that the discovery of disease-causing variants may be used to aid identification of further susceptibility loci in complex disease.