We assessed the effects of non-HLA gene polymorphisms on the risk of islet autoimmunity (IA) and progression to type 1 diabetes in the Diabetes Autoimmunity Study in the Young. A total of 1,743 non-Hispanic, white children were included: 861 first-degree relatives and 882 general population children identified as having high-risk HLA-DR/DQ genotypes for type 1 diabetes. Of those, 109 developed IA and 61 progressed to diabetes. Study participants were genotyped for 20 non-HLA polymorphisms, previously confirmed as type 1 diabetes susceptibility loci. PTPN22 and UBASH3A predicted both IA and diabetes in regression models controlling for family history of type 1 diabetes and presence of HLA-DR3/4-DQB1*0302 genotype. In addition, PTPN2 predicted IA whereas INS predicted type 1 diabetes. The final multivariate regression models for both IA and type 1 diabetes included PTPN22, UBASH3A, and INS, in addition to family history of type 1 diabetes and HLA-DR3/4. In general population children, the most frequent combinations including these five significant predictors conferred hazard ratio of up to 13 for IA and >40 for type 1 diabetes. Non-HLA susceptibility alleles may help estimate risk for development of type 1 diabetes in the general population. These findings require replication in different populations.

Large-scale population sequencing studies provide a complete picture of human genetic variation within the studied populations. A key challenge is to identify, among the myriad alleles, those variants that have an effect on molecular function, phenotypes, and reproductive fitness. Most non-neutral variation consists of deleterious alleles segregating at low population frequency due to incessant mutation. To date, studies characterizing selection against deleterious alleles have been based on allele frequency (testing for a relative excess of rare alleles) or ratio of polymorphism to divergence (testing for a relative increase in the number of polymorphic alleles). Here, starting from Maruyama's theoretical prediction (Maruyama T (1974), Am J Hum Genet USA 6:669–673) that a (slightly) deleterious allele is, on average, younger than a neutral allele segregating at the same frequency, we devised an approach to characterize selection based on allelic age. Unlike existing methods, it compares sets of neutral and deleterious sequence variants at the same allele frequency. When applied to human sequence data from the Genome of the Netherlands Project, our approach distinguishes low-frequency coding non-synonymous variants from synonymous and non-coding variants at the same allele frequency and discriminates between sets of variants independently predicted to be benign or damaging for protein structure and function. The results confirm the abundance of slightly deleterious coding variation in humans.

Author Summary

A key challenge in human genetics is to identify, among the multitude of genetic differences between individuals, those that have an effect on traits. Even though new genetic variants arise through mutation in each generation, most are present only in a small proportion of individuals because they have slightly negative effects on fitness. Detecting such slightly deleterious variants is a key challenge in analyzing how genetics influence human characteristics. In this paper, we test a theoretical prediction by Takeo Maruyama from 1974 that a slightly deleterious variant is, on average, younger than a neutral (non affecting fitness) variant present at the same population frequency. Thus our method detects selection by using estimated age of variants. We applied our method to human data from the Genome of the Netherlands Project, and we show that it distinguishes low-frequency protein-modifying variants from silent variants at the same population frequency and discriminates between sets of variants predicted to be benign or damaging for protein structure and function. Our results confirm the abundance of slightly deleterious protein-coding variation in humans.

Recent advances in the identification of susceptibility genes and environmental exposures provide broad support for a post-infectious autoimmune basis for narcolepsy/hypocretin (orexin) deficiency. We genotyped loci associated with other autoimmune and inflammatory diseases in 1,886 individuals with hypocretin-deficient narcolepsy and 10,421 controls, all of European ancestry, using a custom genotyping array (ImmunoChip). Three loci located outside the Human Leukocyte Antigen (HLA) region on chromosome 6 were significantly associated with disease risk. In addition to a strong signal in the T cell receptor alpha (TRA@), variants in two additional narcolepsy loci, Cathepsin H (CTSH) and Tumor necrosis factor (ligand) superfamily member 4 (TNFSF4, also called OX40L), attained genome-wide significance. These findings underline the importance of antigen presentation by HLA Class II to T cells in the pathophysiology of this autoimmune disease.

Author Summary

While there is now broad consensus that narcolepsy-hypocretin deficiency results from a highly specific autoimmune attack on hypocretin cells, little is understood regarding the initiation and progression of the underlying autoimmune process. We have taken advantage of a unique high-density genotyping platform (the ImmunoChip) designed to study variants in genes known to be important to autoimmune and inflammatory diseases. Our study of nearly 2000 narcolepsy cases compared to 10,000 controls underscored important roles for HLA DQB1*06:02 and the T cell receptor alpha genes and implicated two additional genes, Cathepsin H and TNFSF4/OX40L, in disease pathogenesis. These findings are particularly important, as these encoded proteins have key roles in antigen processing, presentation, and T cell response, and they suggest that specific interactions at the immunological synapse constitute the pathway to the disease. Further studies of these genes and encoded proteins may therefore reveal the mechanism leading to this highly selective and unique autoimmune disease.

Genetic variation in nicotinic acetylcholine receptor subunit genes (nAChRs) is associated with lung function level and chronic obstructive pulmonary disease (COPD). It is unknown whether these variants also predispose to an accelerated lung function decline. We investigated the association of nAChR susceptibility variants with lung function decline and COPD severity. The rs1051730 and rs8034191 variants were genotyped in a population-based cohort of 1,226 heavy smokers (COPACETIC) and in an independent cohort of 883 heavy smokers, of which 653 with COPD of varying severity (LEUVEN). Participants underwent pulmonary function tests at baseline. Lung function decline was assessed over a median follow-up of 3 years in COPACETIC. Current smokers homozygous for the rs1051730 A-allele or rs8034191 G-allele had significantly greater FEV1/FVC decline than homozygous carriers of wild-type alleles (3.3% and 4.3%, p = 0.026 and p = 0.009, respectively). In the LEUVEN cohort, rs1051730 AA-carriers and rs8034191 GG-carriers had a two-fold increased risk to suffer from COPD GOLD IV (OR 2.29, 95% confidence interval [CI] = 1.11–4.75; p = 0.025 and OR = 2.42, 95% [CI] = 1.18–4.95; p = 0.016, respectively). The same risk alleles conferred, respectively, a five- and four-fold increased risk to be referred for lung transplantation because of end-stage COPD (OR = 5.0, 95% [CI] = 1.68–14.89; p = 0.004 and OR = 4.06, 95% [CI] = 1.39–11.88; p = 0.010). In Europeans, variants in nAChRs associate with an accelerated lung function decline in current smokers and with clinically relevant COPD.

Recently it has become clear that only a small percentage (7%) of disease-associated single nucleotide polymorphisms (SNPs) are located in protein-coding regions, while the remaining 93% are located in gene regulatory regions or in intergenic regions. Thus, the understanding of how genetic variations control the expression of non-coding RNAs (in a tissue-dependent manner) has far-reaching implications. We tested the association of SNPs with expression levels (eQTLs) of large intergenic non-coding RNAs (lincRNAs), using genome-wide gene expression and genotype data from five different tissues. We identified 112 cis-regulated lincRNAs, of which 45% could be replicated in an independent dataset. We observed that 75% of the SNPs affecting lincRNA expression (lincRNA cis-eQTLs) were specific to lincRNA alone and did not affect the expression of neighboring protein-coding genes. We show that this specific genotype-lincRNA expression correlation is tissue-dependent and that many of these lincRNA cis-eQTL SNPs are also associated with complex traits and diseases.

Author Summary

Large intergenic non-coding RNAs (lincRNAs) are the largest class of non-coding RNA molecules in the human genome. Many genome-wide association studies (GWAS) have mapped disease-associated genetic variants (SNPs) to, or in, the vicinity of such lincRNA regions. However, it is not clear how these SNPs can affect the disease. We tested whether SNPs were also associated with the lincRNA expression levels in five different human primary tissues. We observed that there is a strong genotype-lincRNA expression correlation that is tissue-dependent. Many of the observed lincRNA cis-eQTLs are disease- or trait-associated SNPs. Our results suggest that lincRNA-eQTLs represent a novel link between non-coding SNPs and the expression of protein-coding genes, which can be exploited to understand the process of gene-regulation through lincRNAs in more detail.

Background & Aims

A limited number of genetic risk factors have been reported in primary sclerosing cholangitis (PSC). To discover further genetic susceptibility factors for PSC, we followed up on a second tier of single nucleotide polymorphisms (SNPs) from a genome-wide association study (GWAS).

Methods

We analyzed 45 SNPs in 1221 PSC cases and 3508 controls. The association results from the replication analysis and the original GWAS (715 PSC cases and 2962 controls) were combined in a meta-analysis comprising 1936 PSC cases and 6470 controls. We performed an analysis of bile microbial community composition in 39 PSC patients by 16S rRNA sequencing.

Results

Seventeen SNPs representing 12 distinct genetic loci achieved nominal significance (Preplication<0.05) in the replication. The most robust novel association was detected at chromosome 1p36 (rs3748816; Pcombined=2.1×10−8) where the MMEL1 and TNFRSF14 genes represent potential disease genes. Eight additional novel loci showed suggestive evidence of association (Prepl<0.05). FUT2 at chromosome 19q13 (rs602662; Pcomb=1.9×10−6, rs281377; Pcomb = 2.1×10−6 and rs601338; Pcomb=2.7×10−6) is notable due to its implication in altered susceptibility to infectious agents. We found that FUT2 secretor status and genotype defined by rs601338 significantly influences biliary microbial community composition in PSC patients.

Conclusions

We identify multiple new PSC risk loci by extended analysis of a PSC GWAS. FUT2 genotype needs to be taken into account when assessing the influence from microbiota on biliary pathology in PSC.

Celiac disease is an inflammatory enteropathy caused by intolerance to gluten. Previous linkage studies in the Dutch, Finnish and Hungarian populations have revealed a locus on chromosome 6q21-22 conferring susceptibility to celiac disease. This locus has previously been implicated in susceptibility to other autoimmune diseases such as Crohn's disease and type 1 diabetes. We performed fine mapping on 446 independent individuals with celiac disease and 641 controls of Dutch origin, testing 872 tagging SNPs in a 22 Mb region of chromosome 6. The 12 most promising SNPs were followed up in 2071 individuals from 284 Finnish and 357 Hungarian celiac disease families to identify risk variants in this region. Multiple markers in the region were significantly associated with celiac disease in the Dutch material. Two SNPs, rs9391227 and rs4946111, were significantly associated with celiac disease in the Finnish population. The association to rs9391227 represents the strongest association signal found in the Finnish (P=0.003, OR 0.66) as well as the combined Dutch, Finnish and Hungarian populations (P=3.6 × 10−5, OR 0.76). The rs9391227 is situated downstream of the HECT domain and ankyrin repeat containing, E3 ubiquitin protein ligase 1 (HACE1) gene and is contained within a region of strong linkage disequilibrium enclosing HACE1. Two additional, independent, susceptibility variants in the 6q21-22 region were also found in a meta-analysis of the three populations. The 6q21-22 region was confirmed as a celiac disease susceptibility locus and harbors multiple independent associations, some of which may implicate ubiquitin-pathways in celiac disease susceptibility.

Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by inflammation and fibrosis of the bile ducts. Both environmental and genetic factors contribute to its pathogenesis. To further clarify its genetic background, we investigated susceptibility loci recently identified for ulcerative colitis (UC) in a large cohort of 1186PSC patients and 1748 controls. Single nucleotide polymorphisms (SNPs) tagging 13 UC susceptibility loci were initially genotyped in 854 PSC patients and 1491 controls from the Benelux (331 cases, 735 controls), Germany (265 cases, 368 controls) and Scandinavia (258 cases, 388 controls). Subsequently, a joint analysis was performed with an independent second Scandinavian cohort (332 cases, 257 controls). SNPs at chromosomes2p16 (p value 4.12×10−4), 4q27 (p value 4.10×10−5) and 9q34 (p value 8.41×10−4) were associated with PSC in the joint analysis after correcting for multiple testing. In PSC patients without inflammatory bowel disease(IBD), SNPs at 4q27and9q34 were nominally associated (p<0.05). We applied additional in silico analyses to identify likely candidate genes at PSC susceptibility loci. To identify non-random, evidence-based links we used GRAIL analysis showing interconnectivity between genes in six out of in total nine PSC-associated regions. Expression quantitative trait analysis from 1469 Dutch and UK individuals demonstrated that five out of nine SNPs had an effect on cis-gene expression. These analyses prioritized IL2, CARD9 and REL as novel candidates.

Conclusion

We have identified three UC susceptibility loci to be associated with PSC, harboring the putative candidate genes REL, IL2 and CARD9. These results add to the scarce knowledge on the genetic background of PSC and imply an important role for both innate and adaptive immunological factors.

Celiac disease is characterized by a chronic inflammatory reaction in the intestine and is triggered by gluten, a constituent derived from grains which is present in the common daily diet in the Western world. Despite decades of research, the mechanisms behind celiac disease etiology are still not fully understood, although it is clear that both genetic and environmental factors are involved. To improve the understanding of the disease, the genetic component has been extensively studied by genome-wide association studies. These have uncovered a wealth of information that still needs further investigation to clarify its importance. In this review, we summarize and discuss the results of the genetic studies in celiac disease, focusing on the “non-HLA” genes. We also present novel approaches to identifying the causal variants in complex susceptibility loci and disease mechanisms.

Vesico-ureteral reflux (VUR) is the retrograde passage of urine from the bladder to the urinary tract and causes 8.5% of end-stage renal disease in children. It is a complex genetic developmental disorder, in which ectopic embryonal ureteric budding is implicated in the pathogenesis. VUR is part of the spectrum of Congenital Anomalies of the Kidney and Urinary Tract (CAKUT). We performed an extensive association study for primary VUR using a two-stage, case-control design, investigating 44 candidate genes in the ureteric budding pathway in 409 Dutch VUR patients. The 44 genes were selected from the literature and a set of 567 single nucleotide polymorphisms (SNPs) capturing their genetic variation was genotyped in 207 cases and 554 controls. The 14 SNPs with p<0.005 were included in a follow-up study in 202 cases and 892 controls. Of the total cohort, ∼50% showed a clear-cut primary VUR phenotype and ∼25% had both a duplex collecting system and VUR. We also looked for association in these two extreme phenotype groups. None of the SNPs reached a significant p-value. Common genetic variants in four genes (GREM1, EYA1, ROBO2 and UPK3A) show a trend towards association with the development of primary VUR (GREM1, EYA1, ROBO2) or duplex collecting system (EYA1 and UPK3A). SNPs in three genes (TGFB1, GNB3 and VEGFA) have been shown to be associated with VUR in other populations. Only the result of rs1800469 in TGFB1 hinted at association in our study. This is the first extensive study of common variants in the genes of the ureteric budding pathway and the genetic susceptibility to primary VUR.

Background

Genome-wide association studies in Japanese populations recently identified common variants in the KCNQ1 gene to be associated with type 2 diabetes. We examined the association of these variants within KCNQ1 with type 2 diabetes in a Dutch population, investigated their effects on insulin secretion and metabolic traits and on the risk of developing complications in type 2 diabetes patients.

Methodology

The KCNQ1 variants rs151290, rs2237892, and rs2237895 were genotyped in a total of 4620 type 2 diabetes patients and 5285 healthy controls from the Netherlands. Data on macrovascular complications, nephropathy and retinopathy were available in a subset of diabetic patients. Association between genotype and insulin secretion/action was assessed in the additional sample of 335 individuals who underwent a hyperglycaemic clamp.

Principal Findings

We found that all the genotyped KCNQ1 variants were significantly associated with type 2 diabetes in our Dutch population, and the association of rs151290 was the strongest (OR 1.20, 95% CI 1.07–1.35, p = 0.002). The risk C-allele of rs151290 was nominally associated with reduced first-phase glucose-stimulated insulin secretion, while the non-risk T-allele of rs2237892 was significantly correlated with increased second-phase glucose-stimulated insulin secretion (p = 0.025 and 0.0016, respectively). In addition, the risk C-allele of rs2237892 was associated with higher LDL and total cholesterol levels (p = 0.015 and 0.003, respectively). We found no evidence for an association of KCNQ1 with diabetic complications.

Conclusions

Common variants in the KCNQ1 gene are associated with type 2 diabetes in a Dutch population, which can be explained at least in part by an effect on insulin secretion. Furthermore, our data suggest that KCNQ1 is also associated with lipid metabolism.

Objectives

Microvillus inclusion disease (MVID) is a rare autosomal recessive enteropathy characterized by intractable diarrhea and malabsorption. Recently, various MYO5B gene mutations have been identified in MVID patients. Interestingly, several MVID patients showed only a MYO5B mutation in one allele (heterozygous) or no mutations in the MYO5B gene, illustrating the need to further functionally characterize the cell biological effects of the MYO5B mutations.

Methods

The genomic DNA of nine patients diagnosed with microvillus inclusion disease was screened for MYO5B mutations, and qPCR and immunohistochemistry on the material of two patients was performed to investigate resultant cellular consequences.

Results

We demonstrate for the first time that MYO5B mutations can be correlated with altered myosin Vb mRNA expression and with an aberrant subcellular distribution of the myosin Vb protein. Moreover, we demonstrate that the typical and myosin Vb–controlled accumulation of rab11a-and FIP5-positive recycling endosomes in the apical cytoplasm of the cells is abolished in MVID enterocytes, which is indicative for altered myosin Vb function. Also, we report 8 novel MYO5B mutations in 9 MVID patients of various etnic backgrounds, including compound heterozygous mutations.

Conclusions

Our functional analysis indicate that MYO5B mutations can be correlated with an aberrant subcellular distribution of the myosin Vb protein and apical recycling endosomes which, together with the additional compound heterozygous mutations, significantly strengthen the link between MYO5B and MVID.

Muscle strength is important in functional activities of daily living and the prevention of common pathologies. We describe the two-staged fine mapping of a previously identified linkage peak for knee strength on chr12q12-14. First, 209 tagSNPs in/around 74 prioritized genes were genotyped in 500 Caucasian brothers from the Leuven Genes for Muscular Strength study (LGfMS). Combined linkage and family-based association analyses identified activin receptor 1B (ACVR1B) and inhibin β C (INHBC), part of the transforming growth factor β pathway regulating myostatin – a negative regulator of muscle mass – signaling, for follow-up. Second, 33 SNPs, selected in these genes based on their likelihood to functionally affect gene expression/function, were genotyped in an extended sample of 536 LGfMS siblings. Strong associations between ACVR1B genotypes and knee muscle strength (P-values up to 0.00002) were present. Of particular interest was the association with rs2854464, located in a putative miR-24-binding site, as miR-24 was implicated in the inhibition of skeletal muscle differentiation. Rs2854464 AA individuals were ∼2% stronger than G-allele carriers. The strength increasing effect of the A-allele was also observed in an independent replication sample (n=266) selected from the Baltimore Longitudinal Study of Aging and a Flemish Policy Research Centre Sport, Physical Activity and Health study. However, no genotype-related difference in ACVR1B mRNA expression in quadriceps muscle was observed. In conclusion, we applied a two-stage fine mapping approach, and are the first to identify and partially replicate genetic variants in the ACVR1B gene that account for genetic variation in human muscle strength.

It is known that genetic variants can affect gene expression, but it is not yet completely clear through what mechanisms genetic variation mediate this expression. We therefore compared the cis-effect of single nucleotide polymorphisms (SNPs) on gene expression between blood samples from 1,240 human subjects and four primary non-blood tissues (liver, subcutaneous, and visceral adipose tissue and skeletal muscle) from 85 subjects. We characterized four different mechanisms for 2,072 probes that show tissue-dependent genetic regulation between blood and non-blood tissues: on average 33.2% only showed cis-regulation in non-blood tissues; 14.5% of the eQTL probes were regulated by different, independent SNPs depending on the tissue of investigation. 47.9% showed a different effect size although they were regulated by the same SNPs. Surprisingly, we observed that 4.4% were regulated by the same SNP but with opposite allelic direction. We show here that SNPs that are located in transcriptional regulatory elements are enriched for tissue-dependent regulation, including SNPs at 3′ and 5′ untranslated regions (P = 1.84×10−5 and 4.7×10−4, respectively) and SNPs that are synonymous-coding (P = 9.9×10−4). SNPs that are associated with complex traits more often exert a tissue-dependent effect on gene expression (P = 2.6×10−10). Our study yields new insights into the genetic basis of tissue-dependent expression and suggests that complex trait associated genetic variants have even more complex regulatory effects than previously anticipated.

Author Summary

Gene expression can be affected by genetic variation, e.g. single nucleotide polymorphisms (SNPs). These are called expression-affecting SNPs or eSNPs. Gene expression levels are known to vary across different tissues in the same individual, despite the fact that genetic variation is the same in these tissues. We explored the different mechanisms by which genetic variants can mediate tissue-dependent gene expression. We observed that the genetic variants that associated with complex traits are more likely to affect gene expression in a tissue-dependent manner. Our results suggest that complex traits are even more complex than we had anticipated, and they underline the great importance of using expression data from tissues relevant to the disease being studied in order to further the understanding of the biology underlying the disease association.

Canine copper toxicosis is an autosomal recessive disorder characterized by hepatic copper accumulation resulting in liver fibrosis and eventually cirrhosis. We have identified COMMD1 as the gene underlying copper toxicosis in Bedlington terriers. Although recent studies suggest that COMMD1 regulates hepatic copper export via an interaction with the Wilson disease protein ATP7B, its importance in hepatic copper homeostasis is ill-defined. In this study, we aimed to assess the effect of Commd1 deficiency on hepatic copper metabolism in mice. Liver-specific Commd1 knockout mice (Commd1Δhep) were generated and fed either a standard or a copper-enriched diet. Copper homeostasis and liver function were determined in Commd1Δhep mice by biochemical and histological analyses, and compared to wild-type littermates. Commd1Δhep mice were viable and did not develop an overt phenotype. At six weeks, the liver copper contents was increased up to a 3-fold upon Commd1 deficiency, but declined with age to concentrations similar to those seen in controls. Interestingly, Commd1Δhep mice fed a copper-enriched diet progressively accumulated copper in the liver up to a 20-fold increase compared to controls. These copper levels did not result in significant induction of the copper-responsive genes metallothionein I and II, neither was there evidence of biochemical liver injury nor overt liver pathology. The biosynthesis of ceruloplasmin was clearly augmented with age in Commd1Δhep mice. Although COMMD1 expression is associated with changes in ATP7B protein stability, no clear correlation between Atp7b levels and copper accumulation in Commd1Δhep mice could be detected. Despite the absence of hepatocellular toxicity in Commd1Δhep mice, the changes in liver copper displayed several parallels with copper toxicosis in Bedlington terriers. Thus, these results provide the first genetic evidence for COMMD1 to play an essential role in hepatic copper homeostasis and present a valuable mouse model for further understanding of the molecular mechanisms underlying hepatic copper homeostasis.

BACKGROUND

Fertility problems are frequently followed by early menopause, and early menopause has been associated with increased risk of type 2 diabetes (T2D). Thus far, it is unknown whether low fertility is independently associated with future T2D risk.

METHODS

We assessed the association between measures of low fertility and T2D in the Prospect-European Prospective Investigation into Cancer and Nutrition (EPIC) cohort of 17 357 Dutch women, aged 49–70 years at baseline using Cox proportional hazards models, adjusted for various confounders. To investigate whether BMI and waist circumference influence the observed associations, analyses were additionally adjusted for these variables.

RESULTS

At baseline, 332 women had T2D. During a mean follow-up of 9.1 ± 3.6 years, 535 T2D cases occurred. Out of 15 707 Prospect-EPIC women who wanted to get pregnant, 1940 consulted a physician for fertility problems and 700 remained childless. No relation was found between consulting a physician for fertility problems or nulliparity and T2D risk. Of all women who wanted to get pregnant, 3946 (25.1%) had one or more miscarriages, with an average of 1.4 (±0.9) miscarriages and a maximum of 10 miscarriages. Women who had one or more miscarriage showed the same risk for T2D as women who had no miscarriage. Also, none of the other measures of low fertility were associated with increased risk for T2D.

CONCLUSIONS

Generally, measures of low fertility were not independently associated with a risk of T2D in a cohort of 17 357 Dutch women.

The successes of genome-wide association (GWA) studies have mainly come from studies performed in populations of European descent. Since complex traits are characterized by marked genetic heterogeneity, the findings so far may provide an incomplete picture of the genetic architecture of complex traits. However, the recent GWA studies performed on East Asian populations now allow us to globally assess the heterogeneity of association signals between populations of European ancestry and East Asians, and the possible obstacles for multi-ethnic GWA studies. We focused on four different traits that represent a broad range of complex phenotypes, which have been studied in both Europeans and East Asians: type 2 diabetes, systemic lupus erythematosus, ulcerative colitis and height. For each trait, we observed that most of the risk loci identified in East Asians were shared with Europeans. However, we also observed that a significant part of the association signals at these shared loci seems to be independent between populations. This suggests that disease aetiology is common between populations, but that risk variants are often population specific. These variants could be truly population specific and result from natural selection, genetic drift and recent mutations, or they could be spurious, caused by the limitations of the method of analysis employed in the GWA studies. We therefore propose a three-stage framework for multi-ethnic GWA analyses, starting with the commonly used single-nucleotide polymorphism-based analysis, and followed by a gene-based approach and a pathway-based analysis, which will take into account the heterogeneity of association between populations at different levels.

Background

We previously showed that activation of the bile salt nuclear receptor Farnesoid X Receptor (FXR) protects against intestinal inflammation in mice. Reciprocally, these inflammatory mediators may decrease FXR activation. We investigated whether FXR activation is repressed in the ileum and colon of inflammatory bowel disease (IBD) patients in remission. Additionally, we evaluated whether genetic variation in FXR is associated with IBD.

Methods

mRNA expression of FXR and FXR target gene SHP was determined in ileal and colonic biopsies of patients with Crohn's colitis (n = 15) and ulcerative colitis (UC; n = 12), all in clinical remission, and healthy controls (n = 17). Seven common tagging SNPs and two functional SNPs in FXR were genotyped in 2355 Dutch IBD patients (1162 Crohn's disease (CD) and 1193 UC) and in 853 healthy controls.

Results

mRNA expression of SHP in the ileum is reduced in patients with Crohn's colitis but not in patients with UC compared to controls. mRNA expression of villus marker Villin was correlated with FXR and SHP in healthy controls, a correlation that was weaker in UC patients and absent in CD patients. None of the SNPs was associated with IBD, UC or CD, nor with clinical subgroups of CD.

Conclusions

FXR activation in the ileum is decreased in patients with Crohn's colitis. This may be secondary to altered enterohepatic circulation of bile salts or transrepression by inflammatory signals but does not seem to be caused by the studied SNPs in FXR. Increasing FXR activity by synthetic FXR agonists may have benefit in CD patients.

Genome-wide association (GWA) studies have identified numerous, replicable, genetic associations between common single nucleotide polymorphisms (SNPs) and risk of common autoimmune and inflammatory (immune-mediated) diseases, some of which are shared between two diseases. Along with epidemiological and clinical evidence, this suggests that some genetic risk factors may be shared across diseases—as is the case with alleles in the Major Histocompatibility Locus. In this work we evaluate the extent of this sharing for 107 immune disease-risk SNPs in seven diseases: celiac disease, Crohn's disease, multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, and type 1 diabetes. We have developed a novel statistic for Cross Phenotype Meta-Analysis (CPMA) which detects association of a SNP to multiple, but not necessarily all, phenotypes. With it, we find evidence that 47/107 (44%) immune-mediated disease risk SNPs are associated to multiple—but not all—immune-mediated diseases (SNP-wise PCPMA<0.01). We also show that distinct groups of interacting proteins are encoded near SNPs which predispose to the same subsets of diseases; we propose these as the mechanistic basis of shared disease risk. We are thus able to leverage genetic data across diseases to construct biological hypotheses about the underlying mechanism of pathogenesis.

Author Summary

Over the last five years we have found over 100 genetic variants predisposing to common diseases affecting the immune system. In this study we analyze 107 such variants across seven diseases and find that almost half are shared across diseases. We also find that the patterns of sharing across diseases cluster these variants into groups; proteins encoded near variants in the same group tend to interact. This suggests that genetic variation may influence entire pathways to create risk to multiple diseases.

For many complex traits, genetic variants have been found associated. However, it is still mostly unclear through which downstream mechanism these variants cause these phenotypes. Knowledge of these intermediate steps is crucial to understand pathogenesis, while also providing leads for potential pharmacological intervention. Here we relied upon natural human genetic variation to identify effects of these variants on trans-gene expression (expression quantitative trait locus mapping, eQTL) in whole peripheral blood from 1,469 unrelated individuals. We looked at 1,167 published trait- or disease-associated SNPs and observed trans-eQTL effects on 113 different genes, of which we replicated 46 in monocytes of 1,490 different individuals and 18 in a smaller dataset that comprised subcutaneous adipose, visceral adipose, liver tissue, and muscle tissue. HLA single-nucleotide polymorphisms (SNPs) were 10-fold enriched for trans-eQTLs: 48% of the trans-acting SNPs map within the HLA, including ulcerative colitis susceptibility variants that affect plausible candidate genes AOAH and TRBV18 in trans. We identified 18 pairs of unlinked SNPs associated with the same phenotype and affecting expression of the same trans-gene (21 times more than expected, P<10−16). This was particularly pronounced for mean platelet volume (MPV): Two independent SNPs significantly affect the well-known blood coagulation genes GP9 and F13A1 but also C19orf33, SAMD14, VCL, and GNG11. Several of these SNPs have a substantially higher effect on the downstream trans-genes than on the eventual phenotypes, supporting the concept that the effects of these SNPs on expression seems to be much less multifactorial. Therefore, these trans-eQTLs could well represent some of the intermediate genes that connect genetic variants with their eventual complex phenotypic outcomes.

Author Summary

Many genetic variants have been found associated with diseases. However, for many of these genetic variants, it remains unclear how they exert their effect on the eventual phenotype. We investigated genetic variants that are known to be associated with diseases and complex phenotypes and assessed whether these variants were also associated with gene expression levels in a set of 1,469 unrelated whole blood samples. For several diseases, such as type 1 diabetes and ulcerative colitis, we observed that genetic variants affect the expression of genes, not implicated before. For complex traits, such as mean platelet volume and mean corpuscular volume, we observed that independent genetic variants on different chromosomes influence the expression of exactly the same genes. For mean platelet volume, these genes include well-known blood coagulation genes but also genes with still unknown functions. These results indicate that, by systematically correlating genetic variation with gene expression levels, it is possible to identify downstream genes, which provide important avenues for further research.

Menkes disease (MD) is an X-linked recessive disorder characterized by copper deficiency resulting in a diminished function of copper-dependent enzymes. Most MD patients die in early childhood, although mild forms of MD have also been described. A diversity of mutations in the gene encoding of the Golgi-resident copper-transporting P1B-type ATPase ATP7A underlies MD. To elucidate the molecular consequences of the ATP7A mutations, various mutations in ATP7A associated with distinct phenotypes of MD (L873R, C1000R, N1304S, and A1362D) were analyzed in detail. All mutants studied displayed changes in protein expression and intracellular localization parallel to a dramatic decline in their copper-transporting capacity compared to ATP7A the wild-type. We restored these observed defects in ATP7A mutant proteins by culturing the cells at 30°C, which improves the quality of protein folding, similar to that which as has recently has been demonstrated for misfolded ATP7B, a copper transporter homologous to ATP7A. Further, the effect of the canine copper toxicosis protein COMMD1 on ATP7A function was examined as COMMD1 has been shown to regulate the proteolysis of ATP7B proteins. Interestingly, in addition to adjusted growth temperature, binding of COMMD1 partially restored the expression, subcellular localization, and copper-exporting activities of the ATP7A mutants. However, no effect of pharmacological chaperones was observed. Together, the presented data might provide a new direction for developing therapies to improve the residual exporting activity of unstable ATP7A mutant proteins, and suggests a potential role for COMMD1 in this process.

Electronic supplementary material

The online version of this article (doi:10.1007/s00018-011-0743-1) contains supplementary material, which is available to authorized users.

Many genetic studies in autism have been performed, resulting in the identification of multiple linkage regions and cytogenetic aberrations, but little unequivocal evidence for the involvement of specific genes exists. By identifying novel symptoms in these patients, enhanced phenotyping of autistic individuals not only improves understanding and diagnosis but also helps to define biologically more homogeneous groups of patients, improving the potential to detect causative genes. Supported by recent copy number variation findings in autism, we hypothesized that for some susceptibility loci, autism resembles a contiguous gene syndrome, caused by aberrations within multiple (contiguous) genes, which jointly increases autism susceptibility. This would result in various different clinical manifestations that might be rather atypical, but that also co-occur with autism. To test this hypothesis, 13 susceptibility loci, identified through genetic linkage and cytogenetic analyses, were systematically analyzed. The Online Mendelian Inheritance in Man database was used to identify syndromes caused by mutations in the genes residing in each of these loci. Subsequent analysis of the symptoms expressed within these disorders allowed us to identify 33 symptoms (significantly more than expected, P=0.037) that were over-represented in previous reports mapping to these loci. Some of these symptoms, including seizures and craniofacial abnormalities, support our hypothesis as they are already known to co-occur with autism. These symptoms, together with ones that have not previously been described to co-occur with autism, might be considered for use as inclusion or exclusion criteria toward defining etiologically more homogeneous groups for molecular genetic studies of autism

Background

IgE-mediated peanut allergy is a complex trait with strong heritability, but its genetic basis is currently unknown. Loss-of-function mutations within the filaggrin gene are associated with atopic dermatitis and other atopic diseases; therefore, filaggrin is a candidate gene in the etiology of peanut allergy.

Objective

To investigate the association between filaggrin loss-of-function mutations and peanut allergy.

Methods

Case-control study of 71 English, Dutch, and Irish oral food challenge–positive patients with peanut allergy and 1000 non peanut-sensitized English population controls. Replication was tested in 390 white Canadian patients with peanut allergy (defined by food challenge, or clinical history and skin prick test wheal to peanut ≥8 mm and/or peanut-specific IgE ≥15 kUL−1) and 891 white Canadian population controls. The most prevalent filaggrin loss-of-function mutations were assayed in each population: R501X and 2282del4 in the Europeans, and R501X, 2282del4, R2447X, and S3247X in the Canadians. The Fisher exact test and logistic regression were used to test for association; covariate analysis controlled for coexistent atopic dermatitis.

Results

Filaggrin loss-of-function mutations showed a strong and significant association with peanut allergy in the food challenge–positive patients (P = 3.0 × 10−6; odds ratio, 5.3; 95% CI, 2.8-10.2), and this association was replicated in the Canadian study (P = 5.4 × 10−5; odds ratio, 1.9; 95% CI, 1.4-2.6). The association of filaggrin mutations with peanut allergy remains significant (P = .0008) after controlling for coexistent atopic dermatitis.

Conclusion

Filaggrin mutations represent a significant risk factor for IgE-mediated peanut allergy, indicating a role for epithelial barrier dysfunction in the pathogenesis of this disease.