The association between type 1 diabetes and the ITPR3 gene polymorphism due to linkage disequilibrium with HLA class II.

A fine mapping study of the MHC region in a Swedish case-control population sample reported a novel type 1 diabetes (T1D) association from the inositol 1-, 4-, 5-trisphosphate receptor type 3 gene (ITPR3) in a case-control study, reportedly independent of the HLA class II effect. We attempted to replicate this novel association in a family-based study of 1120 T1D families with at least one affected child, an approach immune to population stratification. We found association of the ITPR3 single nucleotide polymorphisms (SNPs) rs2296336 with T1D but in a direction opposite to that reported. Moreover, rs2296336 was in linkage disequilibrium (LD) with specific alleles of the HLA DQB1 gene. Conditional regression showed that all of the ITPR3 SNP T1D association could be accounted for by the DQB1 effect. Therefore, our findings do not support an obvious role of genetic variation of the ITPR3 gene in T1D risk.

The association between the IFIH1 locus and type 1 diabetes.

AIMS/HYPOTHESIS: We set out to validate a recently reported type 1 diabetes association from the IFIH1 gene variation in an independent cohort from a population of mixed European descent. METHODS: We genotyped five single-nucleotide polymorphisms in the IFIH1 locus, i.e. rs2111485, rs1990760, rs3747517, rs17783344 and rs984971589, in 589 type 1 diabetes nuclear family trios (1,767 individuals). RESULTS: This study independently replicated the reported genetic association using a family-based approach. CONCLUSIONS/INTERPRETATION: The reported type 1 diabetes association is from a linkage disequilibrium region including three candidate genes, i.e. FAP, IFIH1 and GCA. Further variant discovery and fine mapping could help clarify a novel type 1 diabetes mechanism.

Type 1 diabetes and the OAS gene cluster: association with splicing polymorphism or haplotype?

BACKGROUND: The 2',5'-oligoadenylate synthetase genes (OAS1, OAS2, and OAS3) map to human chromosome 12q24 and encode a family of enzymes pivotal to innate antiviral defence. Recently, the minor allele of an OAS1 single nucleotide polymorphism (SNP) that alters splicing (rs10774671) was found to be associated with increased enzymatic activity and, in a case-sibling control study, with type 1 diabetes (T1D). METHODS: We have confirmed this T1D association in 784 nuclear families (two parents and at least one affected offspring) by the transmission disequilibrium test (TDT; G:A = 386:329, p = 0.033). However, because of linkage disequilibrium within OAS1 and with the other two OAS genes, functional attribution of the association to this SNP cannot be assumed. To help answer this question, we also genotyped two non-synonymous SNPs in OAS1 exons 3 and 7. RESULTS: All three SNPs showed significant transmission distortion. Three of the eight possible haplotypes accounted for 98.4% of parental chromosomes and two of them carried the non-predisposing A allele at rs10774671. Parents heterozygous for these two haplotypes showed significant transmission distortion (p = 0.009) despite being homozygous at rs10774671. CONCLUSIONS: We confirm the T1D association with rs10774671, but we conclude that it cannot be attributed (solely) to the splicing variant rs10774671. A serine/glycine substitution in OAS1 exon 3 is more likely a functional variant.

Class III alleles of the variable number of tandem repeat insulin polymorphism associated with silencing of thymic insulin predispose to type 1 diabetes.

Type 1 diabetes results from autoimmune destruction of the insulin-producing pancreatic beta cells. The insulin gene (INS) is also expressed in human thymus, an ectopic expression site likely involved in immune tolerance. The IDDM2 diabetes susceptibility locus maps to a minisatellite composed of a variable number of tandem repeats situated 0.5 kb upstream of INS. Chromosomes carrying the protective long INS variable number of tandem repeats alleles (class III) produce higher levels of thymic INS mRNA than those with the predisposing, short class I alleles. However, complete silencing of thymic INS transcripts from the class III chromosome was found in a small proportion of heterozygous human thymus samples. We hypothesized that the specific class III alleles found on these chromosomes silence rather than enhance thymic insulin expression. To test the prediction that these alleles are predisposing, we developed a DNA fingerprinting method for detecting two putative "silencing" alleles found in two thymus samples (S1, S2). In a set of 287 diabetic children and their parents we found 13 alleles matching the fingerprint of the S1 or S2 alleles. Of 18 possible transmissions, 12 of the S1-S2 alleles were transmitted to the diabetic offspring, a frequency of 0.67, significantly higher than the 0.38 seen in the remaining 142 class III alleles; P = 0.025. This confirms our prediction and represents an additional level of correlation between thymic insulin and diabetes susceptibility, which supports a thymic enhancer effect of the INS variable number of tandem repeats as the mechanism of IDDM2 and refines the contribution of IDDM2 genotyping to diabetes risk assessment.

New approach in the therapy of chronic rejection? ACE- and AT1-blocker reduce the development of chronic rejection after cardiac transplantation in a rat model.

BACKGROUND: Angiotensin II is one of the most potent mitogens of smooth muscle cell proliferation and plays a central role in the development of accelerated coronary artery disease (ACAD), which remains a serious consequence after heart transplantation and limits long-term survival. We investigated the effect of an angiotensin-II blocker, Losartan (angiotensin II Type 1 [AT(1)]-blocker), and an angiotensin-converting enzyme (ACE) inhibitor, Enalapril, on experimental ACAD in a rat cardiac transplant model (Fisher to Lewis). METHODS: After grafting, recipients were treated with 10 mg/kg/day per os Losartan or 40 mg/kg/day per os Enalapril. Two groups of animals received additional pre-treatment with Losartan or Enalapril 7 days before transplantation. All study groups, including the control group, received immunosuppression with cyclosporine (3 mg/kg/day sub-cutaneously). We assessed the extent of ACAD of large and small arteries 80 days after grafting using digitizing morphometry. RESULTS: We observed significant reduction of neointimal proliferation in small arteries in Losartan pre- and post-treated and in Enalapril pre-treated recipients compared with the cyclosporine-treated group (p < 0.05). In epicardial arteries, Enalapril pre- and post-treatment as well as Losartan post-treatment significantly reduced neointimal formation compared with the control group. Reduction of neointima by Enalapril post-treatment in small arteries and Losartan pre-treatment in large arteries trended toward but failed statistical significance. CONCLUSIONS: Our results suggest the important role of the renin-angiotensin system in neointimal proliferation, which can be reduced equally with ACE inhibitors or the angiotensin-II blocker. Therefore AT(1) blockade with Losartan is a useful therapeutic strategy for inhibition of ACAD after cardiac transplantation.

Divergence between genetic determinants of IGF2 transcription levels in leukocytes and of IDDM2-encoded susceptibility to type 1 diabetes.

The IDDM2 susceptibility locus in type 1 diabetes corresponds to a variable number of tandem repeats (VNTR) upstream of the insulin (INS) and insulin-like growth factor 2 (IGF2) genes. Large VNTR alleles (class III) are dominantly protective, whereas small alleles (class I) are predisposing. IGF2 has been considered a prime candidate for mediating IDDM2-encoded susceptibility because of its proximity to the VNTR, mitogenic properties and parental effects at IDDM2 suggest the involvement of an imprinted gene. IGF2 is imprinted with exclusive expression of the paternal gene. However, there is polymorphic relaxation of IGF2 imprinting in leukocytes. VNTR allelic variation affecting either the extent of relaxation or transcription independent of parental origin might explain the IDDM2 effect. To test this, we compared IGF2 expression between chromosomes with a class III or I allele in leukocytes and stimulated lymphocytes. No significant difference was detected between the two classes. Furthermore, the (+) allele of an ApaI polymorphism in the 3'-untranslated region of IGF2 was associated with significantly higher IGF2 messenger ribonucleic acid levels than the (-) allele, but was not associated with type 1 diabetes. The absence of transcriptional effects in leukocytes on IGF2 by the VNTR, which is the disease-predisposing locus, and the presence of a strong association between IGF2 levels and ApaI, which is not associated with the disease, argue against IGF2 expression in leukocytes as the mediator of IDDM2-encoded susceptibility. Taken together, these results support studies suggesting that INS expression in the thymus is a primary target of the IDDM2 susceptibility locus.

A functional analysis of the role of IGF2 in IDDM2-encoded susceptibility to type 1 diabetes.

Genetic studies have identified a number of loci demonstrating linkage to type 1 diabetes. One of the largest single contributors to genetic susceptibility, after the major histocompatability complex, is the IDDM2 locus, which maps to a nontranscribed variable number of tandem repeats (VNTR) minisatellite upstream of the insulin (INS) and insulin-like growth factor 2 (IGF2) genes. In a progression from population to functional studies, recent reports have shown that VNTR susceptibility alleles (class I) have different transcriptional effects on INS than protective VNTR alleles (class III) in thymus and pancreas, two tissues important in the pathogenesis of the disease. Similar VNTR transcriptional effects on IGF2 have also been proposed as a mechanism by which the IDDM2 locus confers susceptibility in addition to, or instead of, effects on INS. We evaluated this hypothesis by comparing IGF2 expression levels from chromosomes with the protective class III alleles to those with class I alleles in tissues relevant to type 1 diabetes pathogenesis. In thymus, class III alleles were associated with an IGF2 mRNA level of 4.7 +/- 0.9 (mean +/- SE, arbitrary units, n = 12) compared with 4.7 +/- 1.3 for class I alleles (n = 17). The same absence of a significant difference was found in pancreas, where class III alleles were associated with a level of 28.4 +/- 4.2 (n = 7) and class I alleles with a level of 29.5 +/- 5.2 (n = 6). There was a significant correlation between fetal age and IGF2 in both tissues, but fetal ages were not different in the genotype groups compared. We therefore did not detect any significant difference in IGF2 mRNA levels associated with the protective class of VNTR alleles as compared with the predisposing class. This is evidence against the hypotheses that have suggested IGF2 is a mediator of IDDM2-encoded susceptibility and corroborates previous studies suggesting insulin is the gene involved.

Insulin expression in human thymus is modulated by INS VNTR alleles at the IDDM2 locus.

Type 1 diabetes or insulin-dependent diabetes mellitus (IDDM) is due to autoimmune destruction of pancreatic beta-cells. Genetic susceptibility to IDDM is encoded by several loci, one of which (IDDM2) maps to a variable number of tandem repeats (VNTR) minisatellite, upstream of the insulin gene (INS). The short class I VNTR alleles (26-63 repeats) predispose to IDDM, while class III alleles (140-210 repeats) have a dominant protective effect. We have reported that, in human adult and fetal pancreas in vivo, class III alleles are associated with marginally lower INS mRNA levels than class I, suggesting transcriptional effects of the VNTR. These may be related to type 1 diabetes pathogenesis, as insulin is the only known beta-cell specific IDDM autoantigen. In search of a more plausible mechanism for the dominant effect of class III alleles, we analysed expression of insulin in human fetal thymus, a critical site for tolerance induction to self proteins. Insulin was detected in all thymus tissues examined and class III VNTR alleles were associated with 2- to 3-fold higher INS mRNA levels than class I. We therefore propose higher levels of thymic INS expression, facilitating immune tolerance induction, as a mechanism for the dominant protective effect of class III alleles.

Imprinted and genotype-specific expression of genes at the IDDM2 locus in pancreas and leucocytes.

One of the loci encoding susceptibility to insulin-dependent diabetes mellitus (IDDM) is IDDM2, mapped to a variable number of tandem repeats (VNTR) polymorphism situated 596 bp upstream of the insulin gene (INS). The shorter alleles (class I) predispose to IDDM, while the longer class III alleles are protective. Besides INS, it is possible that transcription levels of IGF2, the nearby gene encoding the insulin-like growth factor II, may be modulated by allelic forms of the VNTR. In an effort to define the pathophysiologic mechanism of the IDDM2 effect, we examined the effect, in cis, of VNTR genotype on steady-state mRNA levels of INS in samples of human fetal pancreas, and of IGF2 in leucocytes of diabetic children. Relative levels of mRNA transcripts derived from each chromosome carrying a defined VNTR allele were measured by RT-PCR, taking advantage of transcribed polymorphisms at the 3' untranslated region of each gene. In 10 samples of human fetal pancreas, INS transcripts from chromosomes carrying a class III VNTR were slightly but significantly (P = 0.015) lower than those from class I (13% lower, 95% confidence limits 3-21%). In 10 leucocyte samples, mRNA from both IGF2 alleles was seen, indicating relaxation of the parental imprinting of IGF2 in these cells. However, this relaxation was incomplete as maternal allele mRNA was systematically at a lower level than paternal. The paternal/maternal ratio varied widely among individual subjects. Two of the most extreme cases, demonstrating almost complete repression of the maternal allele, were identical twins, suggesting that this variable relaxation of imprinting is genotype-dependent. However, this genotype-dependence cannot be accounted for by the maternal VNTR, as the mean ratios of paternal/maternal IGF2 mRNA levels were not statistically different in individuals with a maternal VNTR of class I vs. class III (3.2 +/- 1.5 vs. 3.89 +/- 0.94). Thus, we present evidence that: (a) class III VNTR alleles are associated with lower INS mRNA in fetal pancreas than class I alleles. The biologic importance of this difference remains to be determined; and (b) the variable relaxation of IGF2 imprinting seen in human leucocytes is not dependent on the presence of a class I vs. a class III VNTR.