Susceptibility to autoimmune insulin-dependent (type 1) diabetes mellitus is determined by a combination of environmental and genetic factors, which include variation in MHC genes on chromosome 6p21 (IDDM1) and the insulin gene on chromosome 11p15 (IDDM2). However, linkage to IDDM1 and IDDM2 cannot explain the clustering of type 1 diabetes in families, and a role for other genes is inferred. In the present report we describe linkage and association of type 1 diabetes to the CTLA-4 gene (cytotoxic T lymphocyte associated-4) on chromosome 2q33 (designated IDDM12). CTLA-4 is a strong candidate gene for T cell-mediated autoimmune disease because it encodes a T cell receptor that mediates T cell apoptosis and is a vital negative regulator of T cell activation. In addition, we provide supporting evidence that CTLA-4 is associated with susceptibility to Graves' disease, another organ-specific autoimmune disease.

Antibodies against islet cell antigens are used as predictive markers of type 1 diabetes, but it is unknown whether they reflect an ongoing autoimmune process in islet tissue. We investigated whether organs from adult donors that are positive for autoantibodies (aAbs) against islet cell antigens exhibit insulitis and/or a reduced ␤-cell mass. Serum from 1,507 organ donors (age 25-60 years) was analyzed for islet cell antibodies (ICAs), glutamate decarboxylase aAbs (GADAs), insulinoma-associated protein 2 aAbs (IA2As), and insulin aAbs. T ype 1 diabetes results from a specific and major loss of insulin-producing ␤-cells presumably through a T-cell-mediated process (1-5). At clinical onset, patients present circulating autoantibodies (aAbs) against islet cell antigens, which can appear many years before hyperglycemia is established and which are therefore used for prediction of the disease. In firstdegree relatives of type 1 diabetic patients, the risk for developing the disease is higher when multiple positivity is present for aAbs against the islet cell cytoplasm (islet cell antibodies [ICA]), insulin (insulin aAbs [IAA]), the 65,000 M r isoform of glutamate decarboxylase (glutamate decarboxylase aAbs [GADA]), or the insulinoma-associated protein 2 tyrosine phosphatase (insulinoma-associated protein 2 aAbs [IA-2A]) (6 -12). Antibody positivity is therefore used for patient recruitment in prevention trials, but it is still unknown whether it corresponds to an insulitis process in the pancreas and, if so, for which combination. There are only few studies available on the histopathology of the pancreas in antibody ϩ nondiabetic individuals (13,14). In the four reported cases, no leukocytic islet infiltrate or signs of ␤-cell damage were noticed; three of them were GADA ϩ patients with polyendocrinopathy, and one was an IA-2A ϩ organ donor. In the present study we investigated the pancreas in 62 aAb ϩ organ donors. This larger series allowed us to identify two cases with insulitis, one of whom presenting signs of ␤-cell proliferation, and to correlate these histopathological findings to the small subgroup of patients with three or four aAbs and a high-risk genotype. RESEARCH DESIGN AND METHODSCollection of pancreatic tissue. Pancreas biopsies were obtained from the Beta Cell Bank, which operates for a clinical trial on islet cell transplantation in Belgium (15,16). They were taken as part of a quality control procedure that was approved by the ethics committees of the Belgian Diabetes Registry and participating hospitals. Tissue (ϳ0.5 cm 3 ) was excised from the body region of cold-preserved (UW flushed) donor organs that were provided by Eurotransplant Foundation (Leiden, The Netherlands). It was fixed in 4% (vol/vol) phosphate-buffered formaldehyde, pH 7.4, or Bouin's fixative; embedded in paraffin; and then histologically analyzed. Between 1989 and 2004, a total of 1,507 biopsies were collected from patients aged 25-60 years for whom serum or plasma was also available for islet cell antibody assays. For none of t...

XY females (n = 17) were analysed for mutations in SRY (sex-determining region Y gene), a gene that has recently been equated with the testis determining factor (TDF). SRY sequences were amplified by the polymerase chain reaction (PCR) and analysed by both the single strand conformational polymorphism assay (SSCP) and DNA sequencing. The DNA from two individuals gave altered SSCP patterns; only these two individuals showed any DNA sequence variation. In both cases, a single base change was found, one altering a tryptophan codon to a stop codon, the other causing a glycine to arginine amino acid substitution. These substitutions lie in the high mobility group (HMG)-related box of the SRY protein, a potential DNA-binding domain. The corresponding regions of DNA from the father of one individual and the paternal uncle of the other, were sequenced and found to be normal. Thus, in both cases, sex reversal is associated with de novo mutations in SRY. Combining this data with two previously published reports, a total of 40 XY females have now been analysed for mutations in SRY. The number of de novo mutations in SRY is now doubled to four, adding further strength to the argument that SRY is TDF.

Both GAD65- and IA-2-Abs exhibit higher prevalences in presence of HLA-DQ- and/or INS-genetic risk markers. Their respective associations differ with age at clinical onset, suggesting a possible usefulness in the identification of subgroups in this heterogeneous disease.

Aims/hypothesis: Prevention trials in first-degree relatives of type 1 diabetic patients are hampered by large interindividual differences in progression rate to diabetes. We investigated whether specific combinations of immune and genetic markers can identify subgroups with more homogeneous progression to clinical onset. Methods: Antibodies against islet cell cytoplasm (ICA), insulin (IAA), glutamate decarboxylase (GADA) and IA-2 protein (IA-2A) were measured in 790 non-diabetic control subjects and 4,589 first-degree relatives under age 40. Results: On first sampling, 11.1% of the siblings presented at least one antibody type (p<0.001 vs other relatives). During follow-up (median 52 months) 43 subjects developed type 1 diabetes (31 siblings, ten offspring of a diabetic father, two offspring of a diabetic mother). Using Kaplan-Meier survival analysis and Cox regression, IA-2A conferred the highest 5-year diabetes risk (>50%) irrespective of the number of antibodies present. In initially IA-2A-positive relatives (n=58) progression to hyperglycaemia depended more on HLA DQ status than on type of kinship (84% progression in the presence of DQ2/DQ8 vs 32% in its absence; p<0.003). In IA-2A-negative relatives (n=4,531) 5-year progression to diabetes increased with the number of other antibodies (ICA, GADA and/or IAA) (p<0.001) but overall did not exceed 10% even for two or more antibodies. Among relatives initially positive for one or more antibody type other than IA-2A (n=315), there was significantly more progression to diabetes (overall still <10%) in carriers of DQ2 (p<0.001 vs no DQ2), regardless of DQ8 status. Conclusions/interpretation: These observations suggest that the HLA-DQ-inferred risk of diabetes can proceed through two distinct pathways distinguished by IA-2A status. Combined positivity for DQ2/DQ8 and IA-2A defines a more homogeneous high-risk population for prevention trials than those used so far.