Genome-wide association study of ulcerative colitis identifies three new susceptibility loci, including the HNF4A region

Ulcerative colitis is a common form of inflammatory bowel disease with a complex etiology. As part of the Wellcome Trust Case Control Consortium 2, we performed a genome-wide association scan for ulcerative colitis in 2,361 cases and 5,417 controls. Loci showing evidence of association at P 1 × 10 5 were followed up by genotyping in an independent set of 2,321 cases and 4,818 controls. We find genome-wide significant evidence of association at three new loci, each containing at least one biologically relevant candidate gene, on chromosomes 20q13 (HNF4A; P = 3.2 × 10 17), 16q22 (CDH1 and CDH3; P = 2.8 × 10 8) and 7q31 (LAMB1; P = 3.0 × 10 8). Of note, CDH1 has recently been associated with susceptibility to colorectal cancer, an established complication of longstanding ulcerative colitis. The new associations suggest that changes in the integrity of the intestinal epithelial barrier may contribute to the pathogenesis of ulcerative colitis. © 2009 Nature America, Inc. All rights reserved.

Genetic analyses in a sample of individuals with high or low BMD shows association with multiple Wnt pathway genes

INTRODUCTION Although the high heritability of BMD variation has long been established, few genes have been conclusively shown to affect the variation of BMD in the general population. Extreme truncate selection has been proposed as a more powerful alternative to unselected cohort designs in quantitative trait association studies. We sought to test these theoretical predictions in studies of the bone densitometry measures BMD, BMC, and femoral neck area, by investigating their association with members of the Wnt pathway, some of which have previously been shown to be associated with BMD in much larger cohorts, in a moderate-sized extreme truncate selected cohort (absolute value BMD Z-scores = 1.5-4.0; n = 344). MATERIALS AND METHODS Ninety-six tag-single nucleotide polymorphism (SNPs) lying in 13 Wnt signaling pathway genes were selected to tag common genetic variation (minor allele frequency [MAF] > 5% with an r(2) > 0.8) within 5 kb of all exons of 13 Wnt signaling pathway genes. The genes studied included LRP1, LRP5, LRP6, Wnt3a, Wnt7b, Wnt10b, SFRP1, SFRP2, DKK1, DKK2, FZD7, WISP3, and SOST. Three hundred forty-four cases with either high or low BMD were genotyped by Illumina Goldengate microarray SNP genotyping methods. Association was tested either by Cochrane-Armitage test for dichotomous variables or by linear regression for quantitative traits. RESULTS Strong association was shown with LRP5, polymorphisms of which have previously been shown to influence total hip BMD (minimum p = 0.0006). In addition, polymorphisms of the Wnt antagonist, SFRP1, were significantly associated with BMD and BMC (minimum p = 0.00042). Previously reported associations of LRP1, LRP6, and SOST with BMD were confirmed. Two other Wnt pathway genes, Wnt3a and DKK2, also showed nominal association with BMD. CONCLUSIONS This study shows that polymorphisms of multiple members of the Wnt pathway are associated with BMD variation. Furthermore, this study shows in a practical trial that study designs involving extreme truncate selection and moderate sample sizes can robustly identify genes of relevant effect sizes involved in BMD variation in the general population. This has implications for the design of future genome-wide studies of quantitative bone phenotypes relevant to osteoporosis.

Toll-like receptor 4 and CD14 polymorphisms in ankylosing spondylitis: Evidence of a weak association in finns

Objective To investigate the association of CD14 and Toll-like receptor (TLR4) with ankylosing spondylitis (AS). Methods A promoter variant in CD14 and 2 coding polymorphisms in TLR4 were investigated in UK and Finnish families with AS and in a UK case-control study. A metaanalysis of published TLR4 and CD14 studies was performed. Results In the Finnish study the CD74-260bp T variant showed an association (p = 0.006), and the common 2-marker TLR4 haplotype showed a weak association (global p = 0.03), with AS. No associations were seen in the UK based studies or in the metaanalyses. Conclusion CD14 and TLR4 showed an association with AS in the Finns only.

Fine mapping of the MHC Class III region demonstrates association of AIF1 and rheumatoid arthritis

Objective. The heritability of RA has been estimated to be ∼55%, of which the MHC contributes about one-third. HLA-DRB1 alleles are strongly associated with RA, but it is likely that significant non-DRB1 MHC genetic susceptibility factors are involved. Previously, we identified two three-marker haplotypes in a 106-kb region in the MHC class III region immediately centromeric to TNF, which are strongly associated with RA on HLA-DRB1*0404 haplotypes. In the present study, we aimed to refine these associations further using a combination of genotyping and gene expression studies. Methods. Thirty-nine nucleotide polymorphisms (SNPs) were genotyped in 95 DRB1*0404 carrying unrelated RA cases, 125 DRB1*0404 - carrying healthy controls and 87 parent-case trio RA families in which the affected child carried HLA-DRB1*04. Quantitative RT-PCR was used to assess the expression of the positional candidate MHC class III genes APOM, BAT2, BAT3, BAT4, BAT5, AIF1, C6orf47, CSNK2β and LY6G5C, and the housekeeper genes, hypoxanthine-guanine phosphoribosyltransferase (HPRT) and β2-microglobulin (B2M) in 31 RA cases and 21 ethnically, age- and sex-matched healthy controls. Synovial membrane specimens from RA, PsA and OA cases were stained by an indirect immunoperoxidase technique using a mouse-anti-human AIF1 monoclonal antibody. Results. Association was observed between RA and single markers or two marker haplotypes involving AIF1, BAT3 and CSNK. AIF1 was also significantly overexpressed in RA mononuclear cells (1.5- to 1.9-fold difference, P = 0.02 vs HPRT, P = 0.002 vs B2M). AIF1 protein was clearly expressed by synovial macrophages in all the inflammatory synovial samples in contrast to the non-inflammatory OA samples. Conclusions. The results of the genotyping and expression studies presented here suggest a role for AIF1 in both the aetiology and pathogenesis of RA.

Genome-wide association studies and musculoskeletal diseases

Bone and joint diseases are major causes of morbidity and mortality worldwide, and their prevalence is increasing as the average population age increases. Most common musculoskeletal diseases show significant heritability, and few have treatments that prevent disease or can induce true treatment-free, disease-free remission. Furthermore, despite valiant efforts of hypothesis-driven research, our understanding of the etiopathogenesis of these conditions is, with few exceptions, at best moderate. Therefore, there has been a long-standing interest in genetics research in musculoskeletal disease as a hypothesis-free method for investigating disease etiopathogenesis. Important contributions have been made through the identification of monogenic causes of disease, but the holy grail of human genetics research has been the identification of the genes responsible for common diseases. The development of genome-wide association (GWA) studies has revolutionized this field, and led to an explosion in the number of genes identified that are definitely involved in musculoskeletal disease pathogenesis. However, this approach will not identify all common disease genes, and although the current progress is exciting and proves the potential of this research discipline, other approaches will be required to identify many of the types of genetic variation likely to be involved.

Association scan of 14,500 nonsynonymous SNPs in four diseases identifies autoimmunity variants

We have genotyped 14,436 nonsynonymous SNPs (nsSNPs) and 897 major histocompatibility complex (MHC) tag SNPs from 1,000 independent cases of ankylosing spondylitis (AS), autoimmune thyroid disease (AITD), multiple sclerosis (MS) and breast cancer (BC). Comparing these data against a common control dataset derived from 1,500 randomly selected healthy British individuals, we report initial association and independent replication in a North American sample of two new loci related to ankylosing spondylitis, ARTS1 and IL23R, and confirmation of the previously reported association of AITD with TSHR and FCRL3. These findings, enabled in part by increased statistical power resulting from the expansion of the control reference group to include individuals from the other disease groups, highlight notable new possibilities for autoimmune regulation and suggest that IL23R may be a common susceptibility factor for the major 'seronegative' diseases.

Association between glaucoma and at–fault motor vehicle collision involvement among older drivers

Objective To examine the association between glaucoma and motor vehicle collision (MVC) involvement among older drivers, including the role of visual field impairment that may underlie any association found. Design A retrospective population-based study Participants A sample of 2,000 licensed drivers aged 70 years and older who reside in north central Alabama. Methods At-fault MVC involvement for five years prior to enrollment was obtained from state records. Three aspects of visual function were measured: habitual binocular distance visual acuity, binocular contrast sensitivity and the binocular driving visual field constructed from combining the monocular visual fields of each eye. Poisson regression was used to calculate crude and adjusted rate ratios (RR) and 95% confidence intervals (CI). Main Outcomes Measures At-fault MVC involvement for five years prior to enrollment. Results Drivers with glaucoma (n = 206) had a 1.65 (95% confidence interval [CI] 1.20-2.28, p = 0.002) times higher MVC rate compared to those without glaucoma after adjusting for age, gender and mental status. Among those with glaucoma, drivers with severe visual field loss had higher MVC rates (RR = 2.11, 95% CI 1.09-4.09, p = 0.027), whereas no significant association was found among those with impaired visual acuity and contrast sensitivity. When the visual field was sub-divided into six regions (upper, lower, left, and right visual fields; horizontal and vertical meridians), we found that impairment in the left, upper or lower visual field was associated with higher MVC rates, and an impaired left visual field showed the highest RR (RR = 3.16, p = 0.001) compared to other regions. However, no significant association was found in deficits in the right side or along the horizontal or vertical meridian. Conclusions A population-based study suggests that older drivers with glaucoma are more likely to have a history of at-fault MVC involvement than those without glaucoma. Impairment in the driving visual field in drivers with glaucoma appears to have an independent association with at-fault MVC involvement, whereas visual acuity and contrast sensitivity impairments do not.

Study of miRNA polymorphisms and their potential association with breast cancer risk in Australian Caucasian populations

This project established a large and well characterised prospective breast cancer DNA biobank and used this biobank to conduct genetic studies in breast cancer. The thesis presented the results of these high-throughput genotyping studies in two separate Australian Caucasian case-control populations and identified association between three novel genetic variants in microRNA genes and breast cancer risk.

Association between biomechanical structural stresses of atherosclerotic carotid plaques and subsequent ischaemic cerebrovascular events - A longitudinal in vivo magnetic resonance imaging-based finite element study

Background: High-resolution magnetic resonance (MR) imaging has been used for MR imaging-based structural stress analysis of atherosclerotic plaques. The biomechanical stress profile of stable plaques has been observed to differ from that of unstable plaques; however, the role that structural stresses play in determining plaque vulnerability remains speculative. Methods: A total of 61 patients with previous history of symptomatic carotid artery disease underwent carotid plaque MR imaging. Plaque components of the index artery such as fibrous tissue, lipid content and plaque haemorrhage (PH) were delineated and used for finite element analysis-based maximum structural stress (M-C Stress) quantification. These patients were followed up for 2 years. The clinical end point was occurrence of an ischaemic cerebrovascular event. The association of the time to the clinical end point with plaque morphology and M-C Stress was analysed. Results: During a median follow-up duration of 514 days, 20% of patients (n=12) experienced an ischaemic event in the territory of the index carotid artery. Cox regression analysis indicated that M-C Stress (hazard ratio (HR): 12.98 (95% confidence interval (CI): 1.32-26.67, pZ0.02), fibrous cap (FC) disruption (HR: 7.39 (95% CI: 1.61e33.82), p Z 0.009) and PH (HR: 5.85 (95% CI: 1.27e26.77), p Z 0.02) are associated with the development of subsequent cerebrovascular events. Plaques associated with future events had higher M-C Stress than those which had remained asymptomatic (median (interquartile range, IQR): 330 kPa (229e494) vs. 254 kPa (166-290), p Z0.04). Conclusions: High biomechanical structural stresses, in addition to FC rupture and PH, are associated with subsequent cerebrovascular events.

Association between aneurysm shoulder stress and abdominal aortic aneurysm expansion: A longitudinal follow-up study

Background Aneurysm expansion rate is an important indicator of the potential risk of abdominal aortic aneurysm (AAA) rupture. Stress within the AAA wall is also thought to be a trigger for its rupture. However, the association between aneurysm wall stresses and expansion of AAA is unclear. Methods and Results Forty-four patients with AAAs were included in this longitudinal follow-up study. They were assessed by serial abdominal ultrasonography and computed tomography scans if a critical size was reached or a rapid expansion occurred. Patient-specific 3-dimensional AAA geometries were reconstructed from the follow-up computed tomography images. Structural analysis was performed to calculate the wall stresses of the AAA models at both baseline and final visit. A nonlinear large-strain finite element method was used to compute the wall-stress distribution. The relationship between wall stresses and expansion rate was investigated. Slowly and rapidly expanding aneurysms had comparable baseline maximum diameters (median, 4.35 cm [interquartile range, 4.12 to 5.0 cm] versus 4.6 cm [interquartile range, 4.2 to 5.0 cm]; P=0.32). Rapidly expanding AAAs had significantly higher shoulder stresses than slowly expanding AAAs (median, 300 kPa [interquartile range, 280 to 320 kPa] versus 225 kPa [interquartile range, 211 to 249 kPa]; P=0.0001). A good correlation between shoulder stress at baseline and expansion rate was found (r=0.71; P=0.0001). Conclusion A higher shoulder stress was found to have an association with a rapidly expanding AAA. Therefore, it may be useful for estimating the expansion of AAAs and improve risk stratification of patients with AAAs.

Association of NOS1AP variants and depression phenotypes in schizophrenia

Background The nitric oxide synthase 1 adaptor protein gene (NOS1AP) has previously been recognised as a schizophrenia susceptibility gene due to its role in glutamate neurotransmission. The gene is believed to inhibit nitric oxide (NO) production activated by the N-methyl-d-aspartate (NMDA) receptor and reduced NO levels have been observed in schizophrenia patients. However, association studies investigating NOS1AP and schizophrenia have produced inconsistent results, most likely because schizophrenia is a clinically heterogeneous disorder. This study aims to investigate the association between NOS1AP variants and defined depression phenotypes of schizophrenia. Methods Nine NOS1AP SNPs, rs1415259, rs1415263, rs1858232, rs386231, rs4531275, rs4656355, rs4657178, rs6683968 and rs6704393 were genotyped in 235 schizophrenia subjects screened for various phenotypes of depression. Result One NOS1AP SNP (rs1858232) was associated with the broad diagnosis of schizophrenia and eight SNPs were associated with depression related phenotypes within schizophrenia. The rs1415259 SNP showed strong association with sleep dysregulation phenotypes of depression. Conclusion Results suggest that NOS1AP variants are associated with various forms of depression in schizophrenia and are more prevalent in males. Limitation Schizophrenia is a clinically heterogeneous disease that can vary greatly between different ethnic and geographic populations so our observations should be viewed with caution until they are independently replicated, particularly in larger patient cohorts.

Unbiased estimating equations from working correlation models for irregularly timed repeated measures

The method of generalized estimating equation-, (GEEs) has been criticized recently for a failure to protect against misspecification of working correlation models, which in some cases leads to loss of efficiency or infeasibility of solutions. However, the feasibility and efficiency of GEE methods can be enhanced considerably by using flexible families of working correlation models. We propose two ways of constructing unbiased estimating equations from general correlation models for irregularly timed repeated measures to supplement and enhance GEE. The supplementary estimating equations are obtained by differentiation of the Cholesky decomposition of the working correlation, or as score equations for decoupled Gaussian pseudolikelihood. The estimating equations are solved with computational effort equivalent to that required for a first-order GEE. Full details and analytic expressions are developed for a generalized Markovian model that was evaluated through simulation. Large-sample ".sandwich" standard errors for working correlation parameter estimates are derived and shown to have good performance. The proposed estimating functions are further illustrated in an analysis of repeated measures of pulmonary function in children.

Association of microRNA 17–92 cluster host gene (MIR17HG) polymorphisms with breast cancer

Breast cancer incidence and mortality rates are increasing despite our current knowledge on the disease. Ninety-five percent of breast cancer cases correspond to sporadic forms of the disease and are believed to involve an interaction between environmental and genetic determinants. The microRNA 17–92 cluster host gene (MIR17HG) has been shown to regulate expression of genes involved in breast cancer development and progression. Study of single-nucleotide polymorphisms (SNPs) located in this cluster gene could help provide a further understanding of its role in breast cancer. Therefore, this study investigated six SNPs in the MIR17HG using two independent Australian Caucasian case–control populations (GRC-BC and GU-CCQ BB populations) to determine association to breast cancer susceptibility. Genotyping was undertaken using chip-based matrix assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry (MS). We found significant association between rs4824505 and breast cancer at the allelic level in both study cohorts (GRC-BC p = 0.01 and GU-CCQ BB p = 0.03). Furthermore, haplotypic analysis of results from our combined population determined a significant association between rs4824505/rs7336610 and breast cancer susceptibility (p = 5 × 10−4). Our study is the first to show that the A allele of rs4824505 and the AC haplotype of rs4824505/rs7336610 are associated with risk of breast cancer development. However, definitive validation of this finding requires larger cohorts or populations in different ethnical backgrounds. Finally, functional studies of these SNPs could provide a deeper understanding of the role that MIR17HG plays in the pathophysiology of breast cancer.

Genetic association analysis of miRNA SNPs implicates MIR145 in breast cancer susceptibility

Background MicroRNAs (miRNAs) are important small non-coding RNA molecules that regulate gene expression in cellular processes related to the pathogenesis of cancer. Genetic variation in miRNA genes could impact their synthesis and cellular effects and single nucleotide polymorphisms (SNPs) are one example of genetic variants studied in relation to breast cancer. Studies aimed at identifying miRNA SNPs (miR-SNPs) associated with breast malignancies could lead towards further understanding of the disease and to develop clinical applications for early diagnosis and treatment. Methods We genotyped a panel of 24 miR-SNPs using multiplex PCR and chip-based matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis in two Caucasian breast cancer case control populations (Primary population: 173 cases and 187 controls and secondary population: 679 cases and 301 controls). Association to breast cancer susceptibility was determined using chi-square (X 2 ) and odds ratio (OR) analysis. Results Statistical analysis showed six miR-SNPs to be non-polymorphic and twelve of our selected miR-SNPs to have no association with breast cancer risk. However, we were able to show association between rs353291 (located in MIR145) and the risk of developing breast cancer in two independent case control cohorts (p = 0.041 and p = 0.023). Conclusions Our study is the first to report an association between a miR-SNP in MIR145 and breast cancer risk in individuals of Caucasian background. This finding requires further validation through genotyping of larger cohorts or in individuals of different ethnicities to determine the potential significance of this finding as well as studies aimed to determine functional significance. Keywords: Association analysis; Breast cancer; microRNA; miR-SNPs; MIR145