Z. M. Larsen

CTLA-4 IN AUTOIMMUNE DISEASES-A GENERAL SUSCEPTIBILITY GENE TO AUTOIMMUNITY?

For most autoimmune disorders, the pattern of inheritance is very complex. The major histocompatibility complex (MHC) gene complex has been implicated as the major genetic component in the predisposition to these diseases but other genes are likely to be involved. Based on function and experimental data, the gene encoding cytotoxic T lymphocyte-associated antigen 4 (CTLA4) has been suggested as a candidate gene for conferring susceptibility to autoimmunity. In this review, we critically evaluate the evidence for pathogenetical involvement of CTLA-4 in the different autoimmune diseases with focus on the possible role of genetic variation of the CTLA4 locus.

IDDM12 (CTLA4) on 2q33 and IDDM13 on 2q34 in Genetic Susceptibility to Type 1 Diabetes (Insulin-dependent)

Type 1 diabetes (insulin-dependent) is a multifactorial disease with polygenic susceptibility. The major genetic component (IDDM1) resides within the HLA region, but several non-HLA loci have been implicated in the genetic susceptibility. In the present study, we have analysed two such loci, IDDM12 (CTLA4) on 2q33 and IDDM13 on 2q34, in Danish (n = 254) and Spanish (n = 39) type 1 diabetic multiplex families. No significant evidence of linkage of IDDM12 was observed in any of the two studied data sets. However, when the present data were combined with previously published data, they strengthened the evidence of linkage at this locus, p = 0.00002. For the IDDM13 region, we found some positive evidence of linkage of the D2S137-D2S164-D2S1471 markers (p-values 0.007, 0.02, and 0.007, respectively) using transmission disequilibrium testing (TDT) and the Tsp version of the TDT. Importantly, random transmission of all tested alleles was observed in unaffected offspring (p > 0.3). Stratification for HLA (high risk and non-high risk genotypes) in the Danish families did not reveal heterogeneity at IDDM12 or IDDM13. In conclusion, our data on an entirely new family data set did not support the existence of IDDM12 as a type 1 diabetes susceptibility locus in the Danish population. In addition, we found support for evidence of linkage and association of the IDDM13/D2S137-D2S1471 region (approximately 3.5 cM) to type 1 diabetes, however, further studies are needed to substantiate this observation.

Characterization of new polymorphisms in the 5' UTR of the human interleukin-1 receptor type 1 (IL1R1) gene: linkage to type 1 diabetes and correlation to IL-1RI plasma level.

The human interleukin-1 type I receptor (IL-1RI) is the signal transducing receptor for IL-1, a principal proinflammatory cytokine, which is cytotoxic to pancreatic islet beta cells. The IL-1RI gene, IL1R1, maps to chromosome 2q12. We have previously examined part of the IL1R1 promoter region and in the present study we further characterized the promoter region demarcating exon 1B and 1C by sequencing and mutation scanning. New sequence was obtained 1762 bp upstream and 1609 bp downstream the known region. Within this sequence, we identified four frequent single nucleotide polymorphisms (SNPs). PCR-based RFLP assays were established and three of the polymorphisms were typed in a Danish Type 1 (insulin-dependent) diabetes mellitus (T1DM) family collection comprising 103 simplex and 150 sib-pair affected families. Linkage was evaluated by the sib-TDT (transmission disequilibrium test). One of the polymorphisms, defined by a HinfI RFLP assay, demonstrated linkage to T1DM, P(sTDT) = 0.026. Random transmission was observed to unaffected offspring from heterozygous parents, P = 0.87. No evidence for positive linkage was seen for the other tested polymorphisms, P = 0.14 and P = 0.21, respectively. To evaluate the possible functional significance of the HinfI polymorphism, we measured circulating IL-1RI plasma level in 30 T1DM patients and in 30 control subjects – 10 with each genotype in both groups. Significant differences in plasma levels in relation to genotype – independent of disease status – were found (P = 0.04). In both diabetic and non-diabetic subjects, the wt/wt genotype correlated with the highest IL-1RI plasma level, whereas the plasma levels were lowest for the mt/mt genotype.

The intercellular adhesion molecule-1 K469E polymorphism in type 1 diabetes.

Abstract. Type 1 (insulin-dependent) diabetes is a complex trait. The region harboring the ICAM1 gene on 19p13 links to type 1 diabetes, and a growing body of evidence indicates that intercellular adhesion molecule-1 (ICAM-1) could play a role in type 1 diabetes development. Recently, association studies of an ICAM-1 K469E polymorphism in type 1 diabetes populations have reported conflicting results. Hence, we performed a transmission disequilibrium test analysis of the ICAM-1 K469E variations in 253 Danish type 1 diabetes families. Linkage and association was not found between the ICAM-1 K469E variation and type 1 diabetes in Danish patients (Ptdt≥0.48), and our data did not indicate an interaction between ICAM1 and IDDM1 in predisposition to type 1 diabetes in Danes (P=0.78). We did not observe significant association with late-onset type 1 diabetes (Ptdt≥0.12) or differences in transmission patterns between groups of affected offspring stratified for age at onset (P≥0.19), as suggested in Japanese patients. Combined analysis of the present and previously reported transmission data comprising 728 affected offspring of Romanian, Finnish, and Danish ancestry suggested association between the ICAM-1 E469 allele and type 1 diabetes (Ptdt=0.013), but association was not found in the combined Scandinavian material. In conclusion, we found no association of the ICAM-1 K469E polymorphism with type 1 diabetes or its subsets stratified for age at onset and HLA risk in Danish patients. Analysis of ICAM-1 K469E transmissions reported in three populations suggested association to type 1 diabetes, but also demonstrated heterogeneity between populations.

Complete molecular scanning of the human Fas gene: mutational analysis and linkage studies in families with Type I diabetes mellitus

Aims/hypothesis. The human Fas gene (FAS) on chromosome 10q24.1 encoding a cell surface receptor involved in apoptosis was evaluated as a candidate susceptibility gene for Type I (insulin-dependent) diabetes mellitus. Apoptosis mediated by Fas is important in maintaining peripheral self-tolerance and in down-regulating the immune response and could have a role in immune-mediated beta-cell destruction.¶Methods. We did a molecular scan of the entire human FAS (promoter, exons 1–9 including exon-intron boundaries and the 3 ′UTR) using single strand conformational polymorphism-heteroduplex analysis.¶Results. We identified 15 mutations, of which 11 are new. Of these a g-1194A→T and a g-295Ains give rise to alterations of transcription-factor-binding consensus sequences for c-Myb, SP-1 and NF-kB, respectively. A total of 1068 people from a Danish family collection comprising 138 Type I diabetic sib-pair families (289 affected and 121 unaffected offspring) and 103 Type I diabetic parent-offspring multiplex families (103 affected and 112 unaffected offspring) were typed for the three most frequent polymorphisms with high heterozygosity indices and for a FAS microsatellite. Haplotypes were established and data analysed using the extended transmission disequilibrium test, ETDT.¶Conclusion/interpretation. We found no overall evidence for linkage of the FAS polymorphisms to Type I diabetes. We conclude that it is unlikely that the Fas gene does contribute to genetic susceptibility for Type I diabetes. [Diabetologia (2000) 43: 800–808]

Evidence for a locus (IDDM16) in the immunoglobulin heavy chain region on chromosome 14q32.3 producing susceptibility to type 1 diabetes

Type 1 diabetes results from autoimmune destruction of pancreatic islet β-cells, possibly initiated or exacerbated by viral infections. Recent studies have demonstrated that antibodies towards enterovirus and autoantibodies towards islet cell components develop in the long preclinical phase of type 1 diabetes. We therefore hypothesised that susceptibility to type 1 diabetes could be influenced by genetic factors controlling production of antiviral antibodies or autoantibodies or both. To search for evidence of linkage or association (linkage disequilibrium) between type 1 diabetes and the immunoglobulin heavy chain (IGH) region, 351 North American and British families with ⩾2 diabetic children were genotyped for IGH region microsatellites. Using affected sibpair analysis, significant evidence for linkage was obtained for three markers close to the IGH gene cluster (P values 0.004, 0.002, 0.002). No evidence was found for association using family-based methods. To attempt to confirm these findings, a smaller dataset (241 families, 138 with ⩾2 diabetic children) from Denmark, a more genetically-homogeneous population, was genotyped for one marker only. These families showed no linkage, but significant evidence for association (P = 0.019). This study suggests that a locus (assigned the symbol IDDM16) in the IGH region, possibly an IGH gene, influences susceptibility to type 1 diabetes.

No evidence for SEL1L as a candidate gene for IDDM11-conferred susceptibility

The SEL1L gene is located on human chromosome 14q24.3‐31 close to D14S67 which has been previously proposed to be a type 1 diabetes mellitus locus (IDDM11). Sel‐1 is a negative regulator of the Notch signalling pathway and SEL1L is selectively expressed in adult pancreas and islets of Langerhans. This suggests that SEL1L may be a candidate gene for IDDM11.

Identification of a Type 1 Diabetes‐Associated CD4 Promoter Haplotype with High Constitutive Activity

CD4 is a candidate gene in autoimmune diseases, including Type 1 diabetes mellitus (T1DM), because the CD4 receptor is crucial for appropriate antigen responses of CD4+ T cells. We previously found linkage between a CD4‐1188(TTTTC)5−14 promoter polymorphism and T1DM. In the present study, we screened the human CD4 promoter for mutations and identified three frequent single nucleotide polymorphisms (SNPs): CD4‐181C/G, CD4‐521C/G and CD4‐1050T/C. The SNPs are in strong linkage disequilibrium (LD) and association with the CD4‐1188(TTTTC)5−14 alleles, and we observed nine CD4 promoter haplotypes, of which four are frequent. We genotyped the SNPs in 253 Danish T1DM families (1129 individuals) and found evidence for linkage and association of a CD4 (A4‐1188T‐1050G‐521C‐181) haplotype to T1DM. In reporter studies, we show that (1) the T1DM‐associated CD4 haplotype encodes high constitutive promoter activity and (2) the CD4‐181G variant encodes higher stimulated promoter activity than the CD4‐181C variant. This difference is in part neutralized in the frequently occurring CD4 promoter haplotypes by the more upstream genetic variants. Thus, we report functional impact of a novel CD4‐181C/G SNP on stimulated CD4 promoter activity and the identification of a novel CD4 haplotype with high constitutive promoter activity that is linked and associated with T1DM.

NO LINKAGE OF P187S POLYMORPHISM IN NAD(P)H : QUINONE OXIDOREDUCTASE (NQO1/DIA4) AND TYPE 1 DIABETES IN THE DANISH POPULATION

Recent genome screening studies have identified novel regions of possible interest for susceptibility to type 1 diabetes. One of these is a 30‐35 cM region mapping to 16q22‐q24 (D16S515‐D16S520), where also the gene encoding NAD(P)H: quinone oxidoreductase (NQO1) maps. Data has suggested association of a polymorphism (P187S) in the NQO1 gene and type 1 diabetes. NQO1 is involved in protection against oxidative stress, which is likely to be involved in β‐cell destruction. By use of the transmission disequilibrium test (TDT), we analyzed the P187S polymorphism for association to type 1 diabetes in a population‐based sample of 247 Danish nuclear type 1 diabetic families. Random transmission patterns were observed to all affected offspring (ptdt = 0.82), to index cases (ptdt = 0.77), as well as to unaffected offspring (ptdt = 0.93). Hence, the NQO1 polymorphism is not likely to be an etiological mutation underlying the reported linkage of the 16q22‐q24 region. Hum Mutat 14:67–70, 1999.

Evidence for linkage on chromosome 4p16.1 in Type 1 diabetes Danish families and complete mutation scanning of the WFS1 (Wolframin) gene

Aims  To investigate whether the WFS1 gene, the gene for Wolfram syndrome, is a susceptibility gene for more common forms of diabetes in the Danish population.