Genes and mechanisms involved in common complex diseases, such as the autoimmune disorders that affect approximately 5% of the population, remain obscure. Here we identify polymorphisms of the cytotoxic T lymphocyte antigen 4 gene (CTLA4)--which encodes a vital negative regulatory molecule of the immune system--as candidates for primary determinants of risk of the common autoimmune disorders Graves' disease, autoimmune hypothyroidism and type 1 diabetes. In humans, disease susceptibility was mapped to a non-coding 6.1 kb 3' region of CTLA4, the common allelic variation of which was correlated with lower messenger RNA levels of the soluble alternative splice form of CTLA4. In the mouse model of type 1 diabetes, susceptibility was also associated with variation in CTLA-4 gene splicing with reduced production of a splice form encoding a molecule lacking the CD80/CD86 ligand-binding domain. Genetic mapping of variants conferring a small disease risk can identify pathways in complex disorders, as exemplified by our discovery of inherited, quantitative alterations of CTLA4 contributing to autoimmune tissue destruction.

Genome-wide linkage disequilibrium (LD) mapping of common disease genes could be more powerful than linkage analysis if the appropriate density of polymorphic markers were known and if the genotyping effort and cost of producing such an LD map could be reduced. Although different metrics that measure the extent of LD have been evaluated, even the most recent studies have not placed significant emphasis on the most informative and cost-effective method of LD mapping-that based on haplotypes. We have scanned 135 kb of DNA from nine genes, genotyped 122 single-nucleotide polymorphisms (SNPs; approximately 184,000 genotypes) and determined the common haplotypes in a minimum of 384 European individuals for each gene. Here we show how knowledge of the common haplotypes and the SNPs that tag them can be used to (i) explain the often complex patterns of LD between adjacent markers, (ii) reduce genotyping significantly (in this case from 122 to 34 SNPs), (iii) scan the common variation of a gene sensitively and comprehensively and (iv) provide key fine-mapping data within regions of strong LD. Our results also indicate that, at least for the genes studied here, the current version of dbSNP would have been of limited utility for LD mapping because many common haplotypes could not be defined. A directed re-sequencing effort of the approximately 10% of the genome in or near genes in the major ethnic groups would aid the systematic evaluation of the common variant model of common disease.

One very striking feature of T-cell recognition is the formation of an immunological synapse between a T cell and a cell that it is recognizing. Formation of this complex structure correlates with cytotoxicity in the case of killer (largely CD8 + ) T-cell activity, or robust cytokine release and proliferation in the case of the much longer lived synapses formed by helper (CD4 + ) T cells. Here we have used electron microscopy and 3D tomography to characterize the synapses of antigen-specific CD4 + T cells recognizing B cells and dendritic cells at different time points. We show that there are at least four distinct stages in synapse formation, proceeding over several hours, including an initial stage involving invasive T-cell pseudopodia that penetrate deeply into the antigen-presenting cell, almost to the nuclear envelope. This must involve considerable force and may serve to widen the search for potential ligands on the surface of the cell being recognized. We also show that centrioles and the Golgi complex are always located immediately beneath the synapse and that centrioles are significantly shifted toward the late contact zone with either B lymphocytes or bone marrow-derived dendritic cells such as antigenpresenting cells, and that there are dynamic, stage-dependent changes in the organization of microtubules beneath the synapse. These data reinforce and extend previous data on cytotoxic T cells that one of the principal functions of the immunological synapse is to facilitate cytokine secretion into the synaptic cleft, as well as provide important insights into the overall dynamics of this phenomenon. microtubule organizing center | centrosome A prominent feature of many T-lymphocyte interactions with other cells on which it recognizes a particular antigen is the formation of an immunological synapse (IS). The formation of this structure correlates with robust signaling, lineage commitment, and fate determination of T cells (1-6) and the directed secretion of cytokines and/or cytotoxic molecules. There is a wholesale reorganization of the T cell's cytoskeleton, surface molecules, and organelles, and the loss of polarity regulators or guidance cues that negatively affect T-cell activation (7,8). Whereas a great deal has been learned about the dynamics of cell-surface and signaling molecules within this interface (9, 10), much less is known about changes within the cell, especially in the long-lived helper T cell, namely in antigen-presenting cell (APC) interactions, which can last for 6 or more h and need continuous T-cell receptor (TCR) engagement (11). Here electron microscopic studies are particularly valuable for their very high resolution, especially with the power of 3D tomography and highpressure freezing, which preserves cell structure more faithfully. Although a number of high-resolution electron microscopy studies of T cell-APC interactions have been reported (3,(12)(13)(14)(15), these involve studies of cytotoxic T or natural killer cells and their targets for only the first few minutes of CD4 + ...

Several lines of evidence from family and twin studies have strongly suggested that genetic factors are involved in the development of diabetic microangiopathy [1--3]. Several candidate genes have been investigated to elucidate genetic factor(s) responsible for the vascular complications, but little is known about the genetic basis of these complications [4,5]. Elucidation of the genetic factors predisposing to chronic vascular complications in diabetes mellitus will permit identification of individuals genetically predisposed to the complications and, in turn, will allow us an effective intervention tailored to the specific underlying abnormalities.The renin-angiotensin system regulates the systemic circulation and local haemodynamics, and also regulates cell growth and matrix production via its action on angiotensin II production. Angiotensin I-converting enzyme (ACE; EC 3.4.15.1) is not only a key enzyme in the renin-angiotensin system but also regulates kinin metabolism [6]. The plasma ACE level is under genetic control and is strongly associated with an insertion/deletion (I/D) polymorphism of the ACE gene, defined by the presence or absence of the 287 base-pair Alu -repetitive sequence in intron 16 [7]. Accordingly, the I/D polymorphism has been investigated as a strong candidate marker for genetic predisposition to diabetic vascular complications. Association studies of the ACE genotype with diabetic complications, however, have yielded conflicting results. Diabetologia (1998) 41: 47--53 Meta-analysis of association of insertion/deletion polymorphism of angiotensin I-converting enzyme gene with diabetic nephropathy and retinopathy Summary An insertion/deletion (I/D) polymorphism in the angiotensin-converting enzyme (ACE) gene has repeatedly been shown to be associated with ischaemic heart disease, but the association of this genetic marker with diabetic microangiopathy is controversial. To assess the association of the genotypes with the development of diabetic nephropathy or retinopathy, we performed a meta-analysis of data from the literature, using Mantel-Haenszel method followed by the Breslow-Day test for assessing homogeneity among data. In a total of 4773 diabetic patients from 18 studies with (n = 2495) and without (n = 2278) renal complications, the D allele was significantly associated with diabetic nephropathy (p < 0.0001) in a dominant model (summary odds ratio 1.32, 95 % confidence interval: 1.15 to 1.51). There was no significant evidence against homogeneity of the odds ratios (c 2 = 18.9, 20 df; p = 0.53). The association was significant both in non-insulin-dependent (p < 0.005) and in insulin-dependent diabetes mellitus (p < 0.05). Likewise, in a total of 2010 diabetic patients with (n = 1008) and without (n = 1002) retinopathy, there was no association of the I/D polymorphism with diabetic retinopathy. These data suggest that the ACE I/D polymorphism affects the risk for diabetic nephropathy, but not for diabetic retinopathy. [Diabetologia (1998) 41: 47--53]

SummaryThe NSY (Nagoya-Shibata-Yasuda) mouse was established as an inbred strain of mouse with spontaneous development of diabetes mellitus, by selective breeding for glucose intolerance from outbred Jcl:ICR mice. NSY mice spontaneously develop diabetes mellitus in an age-dependent manner. The cumulative incidence of diabetes is 98 % in males and 31% in females at 48 weeks of age. Neither severe obesity nor extreme hyperinsulinaemia is observed at any age in these mice. Glucose-stimulated insulin secretion was markedly impaired in NSY mice after 24 weeks of age. In contrast, fasting plasma insulin level was higher in male NSY mice than that in male C3H/He mice (545 +73 vs 350+ 40 pmol/1, p < 0.05, at 36 weeks of age). Pancreatic insulin content was higher in male NSY mice than that in male C3H/He mice (76 + 8 vs 52 _+ 5 ng/mg wet weight, p < 0.05, at 36 weeks of age). Morphologically, no abnormal findings, such as hypertrophy or inflammatory changes in the pancreatic islets, were observed in NSY mice at any age. These data suggest that functional changes of insulin secretion in response to glucose from pancreatic beta cells may contribute to the development of non-insulin-dependent diabetes mellitus (NIDDM) in the NSY mouse. Although insulin sensitivity was not measured, fasting hyperinsulinaemia in NSY mice suggests that insulin resistance may also contribute to the pathogenesis of NIDDM. Since these findings are similar to the pathophysiologic features of human NIDDM patients, the NSY mouse is considered to be useful for investigating the pathogenesis and genetic predisposition to NIDDM. [Diabetologia (1995) 38: 503-508] Key words NSY mouse, non-insulin-dependent diabetes mellitus, animal model, insulin secretion, isolated islets.Non-insulin-dependent diabetes mellitus (NIDDM) is a heterogeneous disorder, caused by an interaction of genetic and environmental factors [1][2][3]. This heterogeneity in human NIDDM makes it difficult to clarify the genetics or pathogenesis of the disease. Animal models are invaluable for the analysis of heterogeneous disorders such as diabetes. This is eviReceived: 22 June 1994 and in revised form: 11 October 1994 Corresponding author: Dr. H. Ikegami, Department of Geriatric Medicine, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan Abbreviations: NIDDM, Non-insulin-dependent diabetes mellitus; IDDM, insulin-dependent diabetes mellitus; NSY mouse, Nagoya-Shibata-Yasuda mouse.denced by the recent progress in the understanding of the genetics and pathogenesis of insulin-dependent diabetes mellitus by use of excellent animal models, such as the nonobese diabetic (NOD) mouse and the Bio-Breeding (BB) rat [4]. Several animal models for NIDDM have been described. Although recent studies have revealed impaired insulin secretion in GK rats [5][6][7], most of the animal models for NIDDM are characterized by obesity, hyperinsulinaemia and islet hypertrophy [8].The NSY (Nagoya-Shibata-Yasuda) mouse is a spontaneous model of NIDDM with moderate obesity that was establ...

The number and exact locations of the major histocompatibility complex (MHC)-linked diabetogenic genes

A possible pathogenic mutation in the beta 3-adrenergic-receptor gene (Trp64Arg) has been reported to be associated with an earlier age of onset of non-insulin-dependent diabetes mellitus (NIDDM) and clinical features of the insulin resistance syndrome in Pima Indian, Finnish and French subjects. Since marked heterogeneity has been reported in the association of mutations of candidate genes with NIDDM between Japanese and other ethnic groups, we investigated the association of Trp64Arg with NIDDM in Japanese subjects. The allele frequency of the mutation (Arg) was slightly, but not significantly, higher in NIDDM than in control subjects (70 out of 342 alleles [20.5%] vs 40 out of 248 [16.1%], respectively, p > 0.2). When our data were combined with those of Pima Indian and Finnish subjects, however, the Arg/Arg genotype was significantly associated with NIDDM as compared with the other two genotypes (p < 0.005, relative risk [RR] 2.13, 95% confidence interval [CI] 1.28-3.55). The Arg allele was also associated with NIDDM (p < 0.05, RR 1.27, 95% CI 1.06-1.52). Japanese subjects homozygous for the mutation had a significantly higher body mass index (mean +/- SD: 25.5 +/- 3.9 kg/m2) than heterozygotes (22.6 +/- 4.1, p < 0.05) and normal homozygotes (22.8 +/- 3.8, p < 0.05). NIDDM patients homozygous for the mutation tended to have an earlier age of onset of NIDDM than those with other genotypes. These data suggest that the Trp64Arg mutation not only contributes to weight gain and age-at-onset of NIDDM but is also associated with susceptibility to NIDDM.

Type 2 diabetes is a complex trait with both genes and environmental factors contributing to susceptibility. Except for rare subtypes with monogenic inheritance, the genetic basis of type 2 diabetes is unknown because of the complex and heterogeneous nature of the disease. By using the NSY mouse, an inbred mouse model of type 2 diabetes, we genetically dissected late-onset type 2 diabetes and demonstrated age-dependent changes in the genetic control of type 2 diabetes as well as polygenic inheritance. Three major loci (Nidd1nsy, Nidd2nsy, Nidd3nsy) were mapped on mouse chromosomes (Chr) 11, 14, and 6, respectively. The existence of a fourth locus (Nidd4nsy) with an age-dependent effect was suggested by longitudinal, but not cross-sectional, analysis of linkage data. Nidd1nsy and Nidd4nsy appear to affect insulin secretion, whereas Nidd2nsy and Nidd3nsy appear to affect insulin sensitivity. A locus on Chr 6 was significantly linked to epididymal fat weight. A candidate disease gene (Tcf2) on Chr 11, encoding hepatic nuclear factor-1beta, was shown to have a rare sequence variant in the DNA binding domain in the model. The mouse model we used will serve as a useful model for future studies on the etiology of late-onset polygenic type 2 diabetes in humans.