Genome-wide analysis of mitochondrial DNA copy number reveals loci implicated in nucleotide metabolism, platelet activation, and megakaryocyte proliferation.

Mitochondrial DNA copy number (mtDNA-CN) measured from blood specimens is a minimally invasive marker of mitochondrial function that exhibits both inter-individual and intercellular variation. To identify genes involved in regulating mitochondrial function, we performed a genome-wide association study (GWAS) in 465,809 White individuals from the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) consortium and the UK Biobank (UKB). We identified 133 SNPs with statistically significant, independent effects associated with mtDNA-CN across 100 loci. A combination of fine-mapping, variant annotation, and co-localization analyses was used to prioritize genes within each of the 133 independent sites. Putative causal genes were enriched for known mitochondrial DNA depletion syndromes (p = 3.09 × 10-15) and the gene ontology (GO) terms for mtDNA metabolism (p = 1.43 × 10-8) and mtDNA replication (p = 1.2 × 10-7). A clustering approach leveraged pleiotropy between mtDNA-CN associated SNPs and 41 mtDNA-CN associated phenotypes to identify functional domains, revealing three distinct groups, including platelet activation, megakaryocyte proliferation, and mtDNA metabolism. Finally, using mitochondrial SNPs, we establish causal relationships between mitochondrial function and a variety of blood cell-related traits, kidney function, liver function and overall (p = 0.044) and non-cancer mortality (p = 6.56 × 10-4).

Genome-wide meta-analysis of variant-by-diuretic interactions as modulators of lipid traits in persons of European and African ancestry.

Hypertension (HTN) is a significant risk factor for cardiovascular morbidity and mortality. Metabolic abnormalities, including adverse cholesterol and triglycerides (TG) profiles, are frequent comorbid findings with HTN and contribute to cardiovascular disease. Diuretics, which are used to treat HTN and heart failure, have been associated with worsening of fasting lipid concentrations. Genome-wide meta-analyses with 39,710 European-ancestry (EA) individuals and 9925 African-ancestry (AA) individuals were performed to identify genetic variants that modify the effect of loop or thiazide diuretic use on blood lipid concentrations. Both longitudinal and cross sectional data were used to compute cohort-specific interaction results, which were then combined through meta-analysis in each ancestry. These ancestry-specific results were further combined through trans-ancestry meta-analysis. Analysis of EA data identified two genome-wide significant (p < 5 × 10-8) loci with single nucleotide variant (SNV)-loop diuretic interaction on TG concentrations (including COL11A1). Analysis of AA data identified one genome-wide significant locus adjacent to BMP2 with SNV-loop diuretic interaction on TG concentrations. Trans-ancestry analysis strengthened evidence of association for SNV-loop diuretic interaction at two loci (KIAA1217 and BAALC). There were few significant SNV-thiazide diuretic interaction associations on TG concentrations and for either diuretic on cholesterol concentrations. Several promising loci were identified that may implicate biologic pathways that contribute to adverse metabolic side effects from diuretic therapy.

Bidirectional Mendelian randomization to explore the causal relationships between body mass index and polycystic ovary syndrome.

STUDY QUESTION: What are the causal relationships between polycystic ovary syndrome (PCOS) and body mass index (BMI)? SUMMARY ANSWER: Bidirectional Mendelian randomization analyses suggest that increased BMI is causal for PCOS while the reverse is not the case. WHAT IS KNOWN ALREADY: The contribution of obesity to the pathogenesis of PCOS is controversial. To date, published genetic studies addressing this question have generated conflicting results and have not utilized the full extent of known single nucleotide polymorphisms associated with body mass index (BMI). STUDY DESIGN, SIZE, DURATION: This cross-sectional Mendelian randomization (MR) and genetic association study was conducted in 750 individuals of European origin and with PCOS and 1567 BMI-matched controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: Cases and controls were matched for BMI as well as for distribution of weight categories (normal weight, overweight, obese). Two-sample MR using inverse variance weighting (IVW) was conducted using a 92-SNP instrument variable for BMI with PCOS as the outcome, followed by two-sample MR with a 16-SNP instrument variable for PCOS with BMI as the outcome. Sensitivity analyses included MR-Egger and maximum likelihood methods. Secondary analyses assessed associations of genetic risk scores and individual SNPs with PCOS, BMI and quantitative androgen-related and glucose homeostasis-related traits. MAIN RESULTS AND THE ROLE OF CHANCE: Each standard deviation genetically higher BMI was associated with a 4.89 (95% CI 1.46-16.32) higher odds of PCOS. Conversely, genetic risk of PCOS did not influence BMI. Sensitivity analyses yielded directionally consistent results. The genetic risk score of 92 BMI SNPs was associated with the diagnosis of PCOS (OR 1.043, 95% CI 1.009-1.078, P = 0.012). Of the 92 BMI risk variants evaluated, none were associated individually with PCOS after considering multiple testing. The association of FTO SNP rs1421085 with BMI was stronger in women with PCOS (ß = 0.071, P = 0.0006) than in controls (ß = 0.046, P = 0.065). LIMITATIONS, REASONS FOR CAUTION: The current sample size, while providing good power for MR and genetic risk score analyses, had limited power to demonstrate association of individual SNPs with PCOS. Cases and controls were not matched for age; however, this was mitigated by adjusting analyses for age. Dietary and lifestyle data, which could have been used to explore the greater association of the FTO SNP with BMI in women with PCOS, was not available. WIDER IMPLICATIONS OF THE FINDINGS: Increasing BMI appears to be causal for PCOS but having PCOS does not appear to affect BMI. This study used the most comprehensive set of SNPs for BMI currently available. Prior studies using fewer SNPs had yielded conflicting results and may have been confounded because cases and controls were not matched for weight categories. The current results highlight the potential utility of weight management in the prevention and treatment of PCOS. STUDY FUNDING/COMPETING INTEREST(S): National Institutes of Health Grants R01-HD29364 and K24-HD01346 (to R.A.), Grant R01-DK79888 (to M.O.G.), Grant U54-HD034449 (to R.S.L.), Grant U19-HL069757 (to R.M.K.). The funders had no influence on the data collection, analyses or conclusions of the study. No conflict of interests to declare. TRIAL REGISTRATION NUMBER: N/A.

Generalization and fine mapping of European ancestry-based central adiposity variants in African ancestry populations.

BACKGROUND/OBJECTIVES: Central adiposity measures such as waist circumference (WC) and waist-to-hip ratio (WHR) are associated with cardiometabolic disorders independently of body mass index (BMI) and are gaining clinically utility. Several studies report genetic variants associated with central adiposity, but most utilize only European ancestry populations. Understanding whether the genetic associations discovered among mainly European descendants are shared with African ancestry populations will help elucidate the biological underpinnings of abdominal fat deposition. SUBJECTS/METHODS: To identify the underlying functional genetic determinants of body fat distribution, we conducted an array-wide association meta-analysis among persons of African ancestry across seven studies/consortia participating in the Population Architecture using Genomics and Epidemiology (PAGE) consortium. We used the Metabochip array, designed for fine-mapping cardiovascular-associated loci, to explore novel array-wide associations with WC and WHR among 15 945 African descendants using all and sex-stratified groups. We further interrogated 17 known WHR regions for African ancestry-specific variants. RESULTS: Of the 17 WHR loci, eight single-nucleotide polymorphisms (SNPs) located in four loci were replicated in the sex-combined or sex-stratified meta-analyses. Two of these eight independently associated with WHR after conditioning on the known variant in European descendants (rs12096179 in TBX15-WARS2 and rs2059092 in ADAMTS9). In the fine-mapping assessment, the putative functional region was reduced across all four loci but to varying degrees (average 40% drop in number of putative SNPs and 20% drop in genomic region). Similar to previous studies, the significant SNPs in the female-stratified analysis were stronger than the significant SNPs from the sex-combined analysis. No novel associations were detected in the array-wide analyses. CONCLUSIONS: Of 17 previously identified loci, four loci replicated in the African ancestry populations of this study. Utilizing different linkage disequilibrium patterns observed between European and African ancestries, we narrowed the suggestive region containing causative variants for all four loci.

Statin-induced expression change of INSIG1 in lymphoblastoid cell lines correlates with plasma triglyceride statin response in a sex-specific manner.

Statins are widely prescribed to lower plasma low-density lipoprotein (LDL) cholesterol levels. They also modestly reduce plasma triglyceride (TG), an independent cardiovascular disease risk factor, in most people. The mechanism and inter-individual variability of TG statin response is poorly understood. We measured statin-induced gene expression changes in lymphoblastoid cell lines derived from 150 participants of a simvastatin clinical trial and identified 23 genes (false discovery rate, FDR=15%) with expression changes correlated with plasma TG response. The correlation of insulin-induced gene 1 (INSIG1) expression changes with TG response (rho=0.32, q=0.11) was driven by men (interaction P=0.0055). rs73161338 was associated with INSIG1 expression changes (P=5.4 × 10-5) and TG response in two statin clinical trials (P=0.0048), predominantly in men. A combined model including INSIG1 expression level and splicing changes accounted for 29.5% of plasma TG statin response variance in men (P=5.6 × 10-6). Our results suggest that INSIG1 variation may contribute to statin-induced changes in plasma TG in a sex-specific manner.

Drug-gene interactions and the search for missing heritability: a cross-sectional pharmacogenomics study of the QT interval.

Variability in response to drug use is common and heritable, suggesting that genome-wide pharmacogenomics studies may help explain the 'missing heritability' of complex traits. Here, we describe four independent analyses in 33 781 participants of European ancestry from 10 cohorts that were designed to identify genetic variants modifying the effects of drugs on QT interval duration (QT). Each analysis cross-sectionally examined four therapeutic classes: thiazide diuretics (prevalence of use=13.0%), tri/tetracyclic antidepressants (2.6%), sulfonylurea hypoglycemic agents (2.9%) and QT-prolonging drugs as classified by the University of Arizona Center for Education and Research on Therapeutics (4.4%). Drug-gene interactions were estimated using covariable-adjusted linear regression and results were combined with fixed-effects meta-analysis. Although drug-single-nucleotide polymorphism (SNP) interactions were biologically plausible and variables were well-measured, findings from the four cross-sectional meta-analyses were null (Pinteraction>5.0 × 10(-8)). Simulations suggested that additional efforts, including longitudinal modeling to increase statistical power, are likely needed to identify potentially important pharmacogenomic effects.

Systematic evaluation of validated type 2 diabetes and glycaemic trait loci for association with insulin clearance.

AIMS/HYPOTHESIS: Insulin clearance is a highly heritable trait, for which few quantitative trait loci have been discovered. We sought to determine whether validated type 2 diabetes and/or glycaemic trait loci are associated with insulin clearance. METHODS: Hyperinsulinaemic-euglycaemic clamps were performed in two Hispanic-American family cohorts totalling 1329 participants in 329 families. The Metabochip was used to fine-map about 50 previously identified loci for type 2 diabetes, fasting glucose, fasting insulin, 2 h glucose or HbA1c. This resulted in 17,930 variants, which were tested for association with clamp-derived insulin clearance via meta-analysis of the two cohorts. RESULTS: In the meta-analysis, 38 variants located within seven loci demonstrated association with insulin clearance (p < 0.001). The top signals for each locus were rs10241087 (DGKB/TMEM195 [TMEM195 also known as AGMO]) (p = 4.4 × 10(-5)); chr1:217605433 (LYPLAL1) (p = 3.25 × 10(-4)); rs2380949 (GLIS3) (p = 3.4 × 10(-4)); rs55903902 (FADS1) (p = 5.6 × 10(-4)); rs849334 (JAZF1) (p = 6.4 × 10(-4)); rs35749 (IGF1) (p = 6.7 × 10(-4)); and rs9460557 (CDKAL1) (p = 6.8 × 10(-4)). CONCLUSIONS/INTERPRETATION: While the majority of validated loci for type 2 diabetes and related traits do not appear to influence insulin clearance in Hispanics, several of these loci do show evidence of association with this trait. It is therefore possible that these loci could have pleiotropic effects on insulin secretion, insulin sensitivity and insulin clearance.

Lipoprotein receptor-related protein 1 variants and dietary fatty acids: meta-analysis of European origin and African American studies.

OBJECTIVE: Low-density lipoprotein-related receptor protein 1 (LRP1) is a multi-functional endocytic receptor and signaling molecule that is expressed in adipose and the hypothalamus. Evidence for a role of LRP1 in adiposity is accumulating from animal and in vitro models, but data from human studies are limited. The study objectives were to evaluate (i) relationships between LRP1 genotype and anthropometric traits, and (ii) whether these relationships were modified by dietary fatty acids. DESIGN AND METHODS: We conducted race/ethnic-specific meta-analyses using data from 14 studies of US and European whites and 4 of African Americans to evaluate associations of dietary fatty acids and LRP1 genotypes with body mass index (BMI), waist circumference and hip circumference, as well as interactions between dietary fatty acids and LRP1 genotypes. Seven single-nucleotide polymorphisms (SNPs) of LRP1 were evaluated in whites (N up to 42 000) and twelve SNPs in African Americans (N up to 5800). RESULTS: After adjustment for age, sex and population substructure if relevant, for each one unit greater intake of percentage of energy from saturated fat (SFA), BMI was 0.104 kg m(-2) greater, waist was 0.305 cm larger and hip was 0.168 cm larger (all P<0.0001). Other fatty acids were not associated with outcomes. The association of SFA with outcomes varied by genotype at rs2306692 (genotyped in four studies of whites), where the magnitude of the association of SFA intake with each outcome was greater per additional copy of the T allele: 0.107 kg m(-2) greater for BMI (interaction P=0.0001), 0.267 cm for waist (interaction P=0.001) and 0.21 cm for hip (interaction P=0.001). No other significant interactions were observed. CONCLUSION: Dietary SFA and LRP1 genotype may interactively influence anthropometric traits. Further exploration of this, and other diet x genotype interactions, may improve understanding of interindividual variability in the relationships of dietary factors with anthropometric traits.

The M694V mutation in Armenian-Americans: a 10-year retrospective study of MEFV mutation testing for familial Mediterranean fever at UCLA.

Familial Mediterranean fever (FMF), inherited in an autosomal recessive manner, is a systemic auto-inflammatory disorder characterized by recurrent attacks of fever with peritonitis, pleuritis, synovitis and erysipeloid rash. The marenostrin-encoding fever (MEFV) gene, located on chromosome 16p13.3, is the only gene in which mutations are currently known to cause FMF. To correlate specific genotypes with adverse phenotypes of affected populations residing in the Western United States, a retrospective case series review was conducted of all MEFV gene mutation testing completed at UCLA Clinical Molecular Diagnostic Laboratory between February 2002 and February 2012, followed by clinical chart review of all subjects who either have a single or double mutation. All 12 common mutations in the MEFV gene were analyzed and the M694V variant was found to be associated with an adverse FMF clinical outcome in the Armenian-American population, manifested by earlier onset of disease, increased severity of disease, and renal amyloidosis.

Mitochondrial ribosomal RNA mutation associated with both antibiotic-induced and non-syndromic deafness.

Maternally transmitted non-syndromic deafness was described recently both in pedigrees with susceptibility to aminoglycoside ototoxicity and in a large Arab-Israeli pedigree. Because of the known action of aminoglycosides on bacterial ribosomes, we analysed the sequence of the mitochondrial rRNA genes of three unrelated patients with familial aminoglycoside-induced deafness. We also sequenced the complete mitochondrial genome of the Arab-Israeli pedigree. All four families shared a nucleotide 1555 A to G substitution in the 12S rRNA gene, a site implicated in aminoglycoside activity. Our study offers the first description of a mitochondrial rRNA mutation leading to disease, the first cases of non-syndromic deafness caused by a mitochondrial DNA mutation and the first molecular genetic study of antibiotic-induced ototoxicity.

Insulin clearance: confirmation as a highly heritable trait, and genome-wide linkage analysis.

AIMS/HYPOTHESIS: We have previously documented a high heritability of insulin clearance in a Hispanic cohort. Here, our goal was to confirm the high heritability in a second cohort and search for genetic loci contributing to insulin clearance. METHODS: Hyperinsulinaemic-euglycaemic clamps were performed in 513 participants from 140 Hispanic families. Heritability was estimated for clamp-derived insulin clearance and a two-phase genome-wide linkage scan was conducted using a variance components approach. Linkage peaks were further investigated by candidate gene association analysis in two cohorts. RESULTS: The covariate-adjusted heritability of insulin clearance was 73%, indicating that the majority of the phenotypic variance is due to genetic factors. In the Phase 1 linkage scan, no signals with a logarithm of odds (LOD) score >2 were detected. In the Phase 2 scan, two linkage peaks with an LOD >2 for insulin clearance were identified on chromosomes 15 (LOD 3.62) and 20 (LOD 2.43). These loci harbour several promising candidate genes for insulin clearance, with 12 single nucleotide polymorphisms (SNPs) on chromosome 15 and six SNPs on chromosome 20 being associated with insulin clearance in both Hispanic cohorts. CONCLUSIONS/INTERPRETATION: In a second Hispanic cohort, we confirmed that insulin clearance is a highly heritable trait and identified chromosomal loci that harbour genes regulating insulin clearance. The identification of such genes may improve our understanding of how the body clears insulin, thus leading to improved risk assessment, diagnosis, prevention and therapy of diabetes, as well as of other hyperinsulinaemic disorders, such as the metabolic syndrome and polycystic ovary syndrome.

Common genetic variants differentially influence the transition from clinically defined states of fasting glucose metabolism.

AIMS/HYPOTHESIS: Common genetic variants have been associated with type 2 diabetes. We hypothesised that a subset of these variants may have different effects on the transition from normal fasting glucose (NFG) to impaired fasting glucose (IFG) than on that from IFG to diabetes. METHODS: We identified 16 type 2 diabetes risk variants from the Illumina Broad Candidate-gene Association Resource (CARe) array genotyped in 26,576 CARe participants. Participants were categorised at baseline as NFG, IFG or type 2 diabetic (n = 16,465, 8,017 or 2,291, respectively). Using Cox proportional hazards and likelihood ratio tests (LRTs), we compared rates of progression by genotype for 4,909 (NFG to IFG) and 1,518 (IFG to type 2 diabetes) individuals, respectively. We then performed multinomial regression analyses at baseline, comparing the risk of assignment to the NFG, IFG or diabetes groups by genotype. RESULTS: The rate of progression from NFG to IFG was significantly greater in participants carrying the risk allele at MTNR1B (p = 1 × 10(-4)), nominally greater at GCK and SLC30A8 (p < 0.05) and nominally smaller at IGF2BP2 (p = 0.01) than the rate of progression from IFG to diabetes by the LRT. Results of the baseline, multinomial regression model were consistent with these findings. CONCLUSIONS/INTERPRETATION: Common genetic risk variants at GCK, SLC30A8, IGF2BP2 and MTNR1B influence to different extents the development of IFG and the transition from IFG to type 2 diabetes. Our findings may have implications for understanding the genetic contribution of these variants to the development of IFG and type 2 diabetes.

Transferability and fine-mapping of glucose and insulin quantitative trait loci across populations: CARe, the Candidate Gene Association Resource.

AIMS/HYPOTHESIS: Hyperglycaemia disproportionately affects African-Americans (AfAs). We tested the transferability of 18 single-nucleotide polymorphisms (SNPs) associated with glycaemic traits identified in European ancestry (EuA) populations in 5,984 non-diabetic AfAs. METHODS: We meta-analysed SNP associations with fasting glucose (FG) or insulin (FI) in AfAs from five cohorts in the Candidate Gene Association Resource. We: (1) calculated allele frequency differences, variations in linkage disequilibrium (LD), fixation indices (F(st)s) and integrated haplotype scores (iHSs); (2) tested EuA SNPs in AfAs; and (3) interrogated within ± 250 kb around each EuA SNP in AfAs. RESULTS: Allele frequency differences ranged from 0.6% to 54%. F(st) exceeded 0.15 at 6/16 loci, indicating modest population differentiation. All iHSs were <2, suggesting no recent positive selection. For 18 SNPs, all directions of effect were the same and 95% CIs of association overlapped when comparing EuA with AfA. For 17 of 18 loci, at least one SNP was nominally associated with FG in AfAs. Four loci were significantly associated with FG (GCK, p = 5.8 × 10(-8); MTNR1B, p = 8.5 × 10(-9); and FADS1, p = 2.2 × 10(-4)) or FI (GCKR, p = 5.9 × 10(-4)). At GCK and MTNR1B the EuA and AfA SNPs represented the same signal, while at FADS1, and GCKR, the EuA and best AfA SNPs were weakly correlated (r(2) <0.2), suggesting allelic heterogeneity for association with FG at these loci. CONCLUSIONS/INTERPRETATION: Few glycaemic SNPs showed strict evidence of transferability from EuA to AfAs. Four loci were significantly associated in both AfAs and those with EuA after accounting for varying LD across ancestral groups, with new signals emerging to aid fine-mapping.

Candidate loci for insulin sensitivity and disposition index from a genome-wide association analysis of Hispanic participants in the Insulin Resistance Atherosclerosis (IRAS) Family Study.

AIMS/HYPOTHESIS: The majority of type 2 diabetes genome-wide association studies (GWAS) to date have been performed in European-derived populations and have identified few variants that mediate their effect through insulin resistance. The aim of this study was to evaluate two quantitative, directly assessed measures of insulin resistance, namely insulin sensitivity index (S(I)) and insulin disposition index (DI), in Hispanic-American participants using an agnostic, high-density single nucleotide polymorphism (SNP) scan, and to validate these findings in additional samples. METHODS: A two-stage GWAS was performed in Hispanic-American samples from the Insulin Resistance Atherosclerosis Family Study. In Stage 1, 317,000 SNPs were assessed using 229 DNA samples. SNPs with evidence of association with glucose homeostasis and adiposity traits were then genotyped on the entire set of Hispanic-American samples (n = 1,190). This report focuses on the glucose homeostasis traits: S(I) and DI. RESULTS: Although evidence of association did not reach genome-wide significance (p = 5 x 10(-7)), in the combined analysis SNPs had admixture-adjusted p values of p (ADD) = 0.00010-0.0020 with 8 to 41% differences in genotypic means for S(I) and DI. CONCLUSIONS/INTERPRETATION: Several candidate loci were identified that are nominally associated with S(I) and/or DI in Hispanic-American participants. Replication of these findings in independent cohorts and additional focused analysis of these loci is warranted.

Linkage of Crohn's disease-related serological phenotypes: NFKB1 haplotypes are associated with anti-CBir1 and ASCA, and show reduced NF-kappaB activation.

BACKGROUND AND AIMS: Genetics studies of the serum expression of antibodies to microbial antigens may yield important clues to the pathogenesis of Crohn's disease. Our aim was to conduct a linkage study using expression of anti-CBir1, anti-I2, anti-OmpC and ASCA as quantitative traits. METHODS: Expression of antibodies to microbial antigens was measured by enzyme-linked immunosorbant assay (ELISA) and a standard approximately 10 cM whole genome microsatellite study was conducted. Single nucleotide polymorphism genotyping was performed using either Illumina or TaqMan MGB technology. Nuclear factor Kappa B (NF-kappaB) activation in cells from Epstein-Barr virus (EBV)-transformed cell lines was assessed using an electrophoretic mobility shift assay and protein was measured using ELISA and western blotting. RESULTS: Evidence for linkage to anti-CBir1 expression was detected on human chromosome 4 (logarithm of odds (LOD) 1.82 at 91 cM). We therefore directly proceeded to test the association of haplotypes in NFKB1, a candidate gene. One haplotype, H1, was associated with anti-CBir1 (p = 0.003) and another, H3, was associated with ASCA (p = 0.023). Using cell lines from Crohn's disease patients with either H1 or H3, NF-kappaB activation and NF-kappaB p105 and p50 production were significantly lower for patients with H1 compared to patients with H3. CONCLUSIONS: These results suggest that NFKB1 haplotypes induce dysregulation of innate immune responses by altering NF-kappaB expression. The results also show the use of EBV-transformed lymphoblastoid cell lines to conduct phenotypic studies of genetic variation.

A genome-wide association scan for acute insulin response to glucose in Hispanic-Americans: the Insulin Resistance Atherosclerosis Family Study (IRAS FS).

AIMS/HYPOTHESIS: This study sought to identify genes and regions in the human genome that are associated with the acute insulin response to glucose (AIRg), an important predictor of type 2 diabetes, in Hispanic-American participants from the Insulin Resistance Atherosclerosis Family Study (IRAS FS). METHODS: A two-stage genome-wide association scan (GWAS) was performed in IRAS FS Hispanic-American samples. In the first stage, 317K single nucleotide polymorphisms (SNPs) were assessed in 229 Hispanic-American DNA samples from 34 families from San Antonio, TX, USA. SNPs with the most significant associations with AIRg were genotyped in the entire set of IRAS FS Hispanic-American samples (n = 1,190). In chromosomal regions with evidence of association, additional SNPs were genotyped to capture variation in genes. RESULTS: No individual SNP achieved genome-wide levels of significance (p < 5 x 10(-7)); however, two regions (chromosomes 6p21 and 20p11) had multiple highly ranked SNPs that were associated with AIRg. Additional genotyping in these regions supported the initial evidence of variants contributing to variation in AIRg. One region resides in a gene desert between PXT1 and KCTD20 on 6p21, while the region on 20p11 has several viable candidate genes (ENTPD6, PYGB, GINS1 and RP4-691N24.1). CONCLUSIONS/INTERPRETATION: A GWAS in Hispanic-American samples identified several candidate genes and loci that may be associated with AIRg. These associations explain a small component of variation in AIRg. The genes identified are involved in phosphorylation and ion transport, and provide preliminary evidence that these processes are important in beta cell response.

An SNP linkage scan identifies significant Crohn's disease loci on chromosomes 13q13.3 and, in Jewish families, on 1p35.2 and 3q29.

Inflammatory bowel disease (IBD) is a complex genetic disorder of two major phenotypes, Crohn's disease (CD) and ulcerative colitis (UC), with increased risk in Ashkenazi Jews. Twelve genome-wide linkage screens have identified multiple loci, but these screens have been of modest size and have used low-density microsatellite markers. We, therefore, performed a high-density single-nucleotide polymorphism (SNP) genome-wide linkage study of 993 IBD multiply affected pedigrees (25% Jewish ancestry) that contained 1709 IBD-affected relative pairs, including 919 CD-CD pairs and 312 UC-UC pairs. We identified a significant novel CD locus on chromosome 13p13.3 (peak logarithm of the odds (LOD) score=3.98) in all pedigrees, significant linkage evidence on chromosomes 1p35.1 (peak LOD score=3.5) and 3q29 (peak LOD score=3.19) in Jewish CD pedigrees, and suggestive loci for Jewish IBD on chromosome 10q22 (peak LOD score=2.57) and Jewish UC on chromosome 2q24 (peak LOD score=2.69). Nominal or greater linkage evidence was present for most previously designated IBD loci (IBD1-9), notably, IBD1 for CD families at chromosome 16q12.1 (peak LOD score=4.86) and IBD6 in non-Jewish UC families at chromosome 19p12 (peak LOD score=2.67). This study demonstrates the ability of high information content adequately powered SNP genome-wide linkage studies to identify loci not observed in multiple microsatellite-based studies in smaller cohorts.

Ulcerative colitis: a genetically heterogeneous disorder defined by genetic (HLA class II) and subclinical (antineutrophil cytoplasmic antibodies) markers.

Newly described distinct associations of HLA class II genes with ulcerative colitis (UC) (DR2) and Crohn's disease (CD) (DR1/DQ5) provide strong evidence for genetic heterogeneity of susceptibility between these two forms of inflammatory bowel disease. A familial distribution of antineutrophil cytoplasmic antibodies (ANCAs, a subclinical marker of UC) in UC families has further implied the existence of heterogeneity within UC. To test the hypothesis that the heterogeneity within UC indicated by ANCAs has a genetic basis that resides within the HLA region, we studied 89 UC cases and an ethnically matched control group (n = 50). Serological and molecular typing techniques were applied to define HLA class II genes (DR, DQ). ANCAs were detected using an enzyme-linked immunosorbent assay, and positive values were confirmed by indirect immunofluorescence. We observed that ANCA-positive UC patients (n = 70) had a significantly increased frequency of DR2 compared with ANCA-negative controls (n = 46) (44% vs 22%, P = 0.01). In contrast, the frequency of DR2 in ANCA-negative UC cases (21%) was virtually identical to that in controls (22%, P = 0.9). Furthermore, the ANCA-negative UC patients had an increase in the DR4 allele compared with ANCA-positive UC (P = 0.004). Thus, with the combination of a subclinical marker (ANCAs) and molecular genetic markers, genetic heterogeneity has been demonstrated within UC: ANCA-positive UC associated with DR2, and ANCA-negative UC likely associated with DR4.

Complex genetic contribution of the Apo AI-CIII-AIV gene cluster to familial combined hyperlipidemia. Identification of different susceptibility haplotypes.

Familial combined hyperlipidemia (FCH) is a common genetic lipid disorder in Western societies. In a recent report (Dallinga-Thie, G.M., X.D. Bu, M. van Linde-Sibenius Trip, J.I. Rotter, A.J. Lusis, and T.W.A. de Bruin. J. Lipid Res., 1996, 36:136-147) we have studied three restriction enzyme polymorphisms: XmnI, and MspI sites 5' of the apo AI gene and SstI site in the 3' untranslated region of exon 4 of the apo CIII gene in 18 FCH pedigrees, including 18 probands, 178 hyperlipidemic relatives, 210 normolipidemic relatives, and 176 spouses. DNA variations in the apo AI-CIII-AIV gene cluster had a modifying effect on plasma triglycerides, LDL cholesterol, and apolipoprotein CIII levels. In this study, combinations of haplotypes were analyzed to further characterize their interactions and effect on the expression of severe hyperlipidemia in FCH subjects. A specific combination of haplotypes with one chromosome carrying the X1M1S2 (1-1-2) haplotype and the other the X2M2S1 haplotype (2-2-1) was significantly more frequent in hyperlipidemic relatives (6%) than in normolipidemic relatives (3%) and spouses (0.5%). Associated with this combination of haplotypes were significantly elevated plasma cholesterol (P < 0.0001), triglycerides (P < 0.0001), and apo CIII (P < 0.001) levels when compared to the wild type combination of haplotypes 1-1-1/1-1-1. The only spouse with this specific combination of haplotypes showed a severe hyperlipidemic phenotype, similar to FCH. Furthermore, nonparametric sibpair linkage analysis revealed significant linkage between these markers in the gene cluster and the FCH phenotype (MspI P = 0.0088, SstI P = 0.044, and XMS haplotype P = 0.037). The present findings confirm that the apo AI-CIII-IV gene cluster contributes to the FCH phenotype, but this contribution is genetically complex. An epistatic interaction between different haplotypes of the gene cluster was demonstrated. The S2 allele on one haplotype was synergistic to the X2M2 allele on the other haplotype in its hyperlipidemic effect. Therefore, two different susceptibility loci exist in the gene cluster, demonstrating the paradigm of complex genetic contribution to FCH.