Extensive variation and low heritability of DNA methylation identified in a twin study.

Disturbance of DNA methylation leading to aberrant gene expression has been implicated in the etiology of many diseases. Whereas variation at the genetic level has been studied extensively, less is known about the extent and function of epigenetic variation. To explore variation and heritability of DNA methylation, we performed bisulfite sequencing of 1760 CpG sites in 186 regions in the human major histocompatibility complex (MHC) in CD4+ lymphocytes from 49 monozygotic (MZ) and 40 dizygotic (DZ) twin pairs. Individuals show extensive variation in DNA methylation both between and within regions. In addition, many regions also have a complex pattern of variation. Globally, there appears to be a bimodal distribution of DNA methylation in the regions, but a significant fraction of the CpG sites are also heterogeneously methylated. Classification of regions into CpG islands (intragenic and intergenic), 5' end of genes not associated with a defined CpG island, conserved noncoding regions, and random CpG sites shows region-type differences in variation and heritability. Analyses revealed slightly lower intra-pair differences among MZ than among DZ pairs, suggesting some genetic influences on DNA methylation variation, with most of the variance attributed to nongenetic factors. Overall, heritability estimates of DNA methylation were low. Our heritability estimates are, however, somewhat deflated due to the presence of batch effects that artificially inflate the estimates of shared environment.

Association analysis in type 1 diabetes of the PRSS16 gene encoding a thymus-specific serine protease.

We have previously mapped a separate type 1 diabetes (T1D) association in the extended MHC class I region, marked by D6S2223, on the DRB1*03-DQA1*0501-DQB1*0201 haplotype. The associated region encompasses a gene encoding a thymus-specific serine protease (PRSS16), presumably involved in positive selection of T cells or in T-cell regulation. Fourteen PRSS16 polymorphisms were genotyped in two steps using a total of six T1D family data sets, as well as case-control materials for both T1D and celiac disease (CD). An association with a 15 base-pair deletion in exon 12 of PRSS16 was found on the DRB1*03-DQA1*0501-DQB1*0201 haplotype for both T1D and CD, but it could not explain the more pronounced disease associations observed at marker D6S2223. We compared the performance of the 14 tested PRSS16 polymorphisms, selected after our previous comprehensive screen, against HapMap selected tag SNPs. Use of a HapMap based SNP selection strategy would result in loss of a large proportion of the genetic variation in PRSS16. Our data suggest that it is unlikely that polymorphisms within the PRSS16 gene are involved in the predisposition to T1D. However, we cannot rule out that regulatory polymorphisms located some distance away from the gene may be involved.

Polymorphisms in the cytotoxic T lymphocyte antigen-4 gene region confer susceptibility to Addison's disease.

The cytotoxic T lymphocyte antigen-4 (CTLA4) gene on chromosome 2q33 encodes a key regulator in the adaptive immune system. The CTLA4 surface molecule is expressed on activated T lymphocytes and involved in down-regulation of the immune response. Previous studies on a possible association between autoimmune Addison's disease and CTLA4 polymorphisms have shown conflicting results. A recent study identified new candidate polymorphisms in the CTLA4 region, influencing gene splicing and thereby the relative abundance of soluble CTLA4. We genotyped 134 patients with Addison's disease and 413 healthy controls from Norway and United Kingdom for these newly identified polymorphisms. Our data demonstrate that the same polymorphisms that have recently been demonstrated to confer susceptibility to autoimmune thyroid disease and type 1 diabetes also confer susceptibility to Addison's disease. This finding suggests that polymorphisms in CTLA4 confer general risk to develop autoimmunity and identifies a potential therapeutic target in the prevention of autoimmune endocrine disorders.

HLA associated type 1 diabetes risk in children of Pakistani migrants to Norway.

Type 1 diabetes (T1D) results from poorly defined interaction between susceptibility genes and environmental factors. The objective was to investigate Human Leukocyte Antigens (HLA) associated T1D risk among Pakistani newborns in Norway based on what published globally. DNA samples from 189 newborns, whose parents were first generation migrants from Pakistan, were analyzed. The hypothesis was tested using high resolution HLA genotyping for the -DRB1 and -DQB1 loci and high/intermediate for the -DQA1 locus. We identified 28 different DRB1, 13 DQB1 and 9 DQA1 alleles. Of the 39 different haplotypes identified, only five have been reported to confer T1D susceptibility. Among these the DR3-DQ2 (DRB1*03:01:01-DQA1*05-DQB1*02:01:01) haplotype was found in 18.5% (n=70) of the newborns, and 18.6% (n=13) of these were homozygotes. A diverse range of HLA haplotypes were identified amongst an ethnically homogenous group of newborns, with only a small proportion associated with T1D risk. The incidence of T1D among immigrants form South/East Asia is the lowest in the Norwegian Type 1 Diabetes Registry. The few included so far, are children of first generation immigrants. If incidence of T1D rises in the Norwegian Pakistani childhood population, as observed in the UK, then environmental triggers rather than genetic susceptibility will be the explanation.

Multiple loci in the HLA complex are associated with Addison's disease.

CONTEXT: A strong association between autoimmune Addison's disease (AAD) and major histocompatibility complex class II-encoded HLA-DRB1-DQA1-DQB1 haplotypes is well known. Recent evidence from other autoimmune diseases has suggested that class I-encoded HLA-A and HLA-B gene variants confer HLA-DRB1-DQA1-DQB1-independent effects on disease. OBJECTIVE: We aimed to explore AAD predisposing effects of HLA-A and -B and further investigate the role of MICA and HLA-DRB1-DQA1-DQB1 in a much larger material than has previously been studied. DESIGN: HLA-A, -B, -DRB1, and -DQB1 and a microsatellite in MICA were genotyped in 414 AAD patients and 684 controls of Norwegian origin. RESULTS: The strongest association was observed for the DRB1 locus, in which the DRB1*03:01 and DRB1*04:04 conferred increased risk of AAD, particularly in a heterozygous combination [odds ratio 22.13; 95% confidence interval (11.39-43.98); P = 6 × 10(-20)]. After conditioning on DRB1, association with AAD was still present for HLA-B and MICA, suggesting the presence of additional risk factors. CONCLUSIONS: The major histocompatibility complex harbors multiple risk loci for AAD, in which DRB1 appears to represent the main risk factor.

FOXP3 polymorphisms in type 1 diabetes and coeliac disease.

The FOXP3 gene encodes a transcription factor thought to be essential for the development and function of T regulatory cells. Two previous studies have tested common polymorphisms in FOXP3 for association with type 1 diabetes (T1D) with conflicting results. The aim of our study was to see whether there is any evidence of association between the FOXP3 polymorphisms previously reported to be associated with T1D, in a Caucasian population regarding T1D and coeliac disease (CD). We further looked for evidence of interaction between FOXP3 polymorphisms and HLA-DR3 in conferring susceptibility to T1D. Initially, we analysed two microsatellites in the FOXP3 gene in 363 T1D nuclear families. Our results indicated an association between FOXP3 and T1D (global p=0.004) and a possible interaction between FOXP3 and the HLA-DR3-DQ2 susceptibility haplotype. We then genotyped an additional independent set of 826 T1D patients and 1459 controls as well as one CD dataset consisting of 325 families. A similar tendency was revealed in the CD family material (pnc=0.055 for the associated allele). On the other hand, we were unable to reproduce our initial findings in the T1D case-control dataset (global p=0.6). Our results suggest that the tested FOXP3 markers do not have any major impact on susceptibility for these diseases.

CTLA4 polymorphisms are associated with vitiligo, in patients with concomitant autoimmune diseases.

The cytotoxic T lymphocyte antigen4 (CTLA4) gene plays a critical role in the control of T cell activation. The gene encodes a surface molecule with inhibitory effects on activated T cells. Several studies have disclosed an association between the previously known variants of the CTLA4 gene and autoimmune disorders, but no study has as yet found any definite association between vitiligo and the CTLA4 polymorphisms. A recent study identified new candidate susceptibility polymorphisms in this region, associated with differential gene splicing and thereby the relative abundance of soluble CTLA4. To assess these new polymorphisms in patients with vitiligo, we genotyped 100 vitiligo patients and 140 healthy controls from the UK, for these novel polymorphisms. No association was found in patients with isolated vitiligo, but a significant association was seen in patients with vitiligo and other autoimmune diseases. The results indicate that the polymorphisms in the CTLA4 gene region confer susceptibility to vitiligo when occurring together with other autoimmune diseases, but not in patients with isolated vitiligo. This raises the possibility that there are two distinct forms of vitiligo where only a subgroup of patients may have a disease caused by the autoimmune destruction of melanocytes.