CTLA-4-/- mice display T cell-apoptosis resistance resembling that ascribed to autoimmune-prone non-obese diabetic (NOD) mice

The genes conferring susceptibility to autoimmune (insulin-dependent) diabetes mellitus (IDDM) are, in most cases, not defined. Among the loci so far identified as associated with murine IDDM (Idd1-19), only the nature of Idd1 has been assessed. Here we show that thymocytes and peripheral lymphocytes of the non-obese diabetic (NOD) mouse are relatively resistant to apoptosis induced by gamma-irradiation. By linkage analysis of F2 progeny mice, we map this trait to a locus on chromosome 1 containing the Idd5 diabetes susceptibility region. By the use of congenic mice, we confirm the linkage data and map this locus to a 6 cM region on proximal chromosome 1. Ctla4, being localized in this chromosomal region and mediating crucial functions in T cell biology, is a logical candidate gene in the Idd5 susceptibility region. In line with this, we demonstrate that T cells from Ctla4(-/-)deficient mice show a similar resistance to gamma-irradiation-induced apoptosis as observed in the NOD mice. This reinforces the notion that CTLA-4 contributes to the pathogenesis of autoimmune diabetes.     

Association between type 1 diabetes and polymorphism of the CTLA-4 gene in a Tunisian population]

Susceptibility to type 1 diabetes mellitus is strongly associated with particular HLA class II alleles. However, non HLA genetic factors are likely to be required for the development of disease. The candidate genes include the cytotoxic T lymphocyte associated 4 (CTLA-4) located on chromosome 2q33 and designated (IDDM12), which encodes a cell surface negative signal T molecule providing for activation. We investigated CTLA-4 exon 1 dimorphism in 74 type 1 patients and a control group of 48 healthy subjects from Tunisia using two methods PCR (polymerase chain reaction) allele specific and polymerase chain reaction restriction fragment length polymorphism (PCR RFLP). The CTLA-4/G allele was found on 68.9% in type 1 patients as compared to 51.02% in controls (p = 0.002), mostly in homozygous from 43.24% versus 22.45% (p = 0.0058). This results indicate that CTLA-4/G allele was significantly associated with predisposition to type 1 diabetes in our group from Tunisian population.     

No association of type 1 diabetes with a microsatellite marker for CTLA-4 in a Japanese population.

Susceptibility to insulin-dependent (type 1) diabetes mellitus is determined by both environmental and genetic factors. The primary gene associated with predisposition to type 1 diabetes is the human leukocyte antigen (HLA) class II gene (IDDM1). Recent studies have described linkage and association of type 1 diabetes to the cytotoxic T lymphocyte antigen-4 (CTLA-4) gene (IDDM12)in Caucasians. CTLA-4 is a candidate gene for T-cell-mediated autoimmune diseases because it is a negative regulator of T-cell proliferation. We investigated distribution of a CTLA-4 (AT)n microsatellite marker in 118 Japanese patients with type 1 diabetes and 195 control subjects. We also investigated association between this CTLA-4 gene polymorphism and GAD65 antibody positivity in 103 of the patients. CTLA-4 microsatellite marker loci were determined by polymerase chain reaction amplification of genomic DNA and resolution of the products on sequencing gels. GAD65 antibody was detected by radioligand binding assay. There was no significant difference in the distribution of CTLA-4 alleles between patients and controls, and no difference was observed in the prevalence of CTLA-4 alleles when GAD65 antibody-positive and -negative individuals with the type 1 diabetes were compared. The present study did not support an association between the CTLA-4 microsatellite marker and type 1 diabetes in our Japanese study population.     

Lack of association between CTLA-4 gene polymorphism and IDDM in Japanese subjects

Susceptibility to insulin-dependent diabetes mellitus (IDDM) is determined by both environmental and genetic factors. The main gene associated with predisposition to IDDM is HLA. Recent studies have described linkage and association of IDDM to the CTLA-4 gene (IDDM12) in Caucasians. CTLA-4 is a candidate gene for T-cell-mediated autoimmune diseases because it is a negative regulator of T-cell proliferation. We investigated the distribution of a CTLA-4 gene polymorphism in 110 Japanese patients with IDDM and 200 control subjects. In 84 patients, we also investigated associations between this CTLA-4 gene polymorphism and GAD65 antibody positivity. An A/G transition at position 49 of exon 1 was analyzed by the polymerase chain reaction-restriction fragment length polymorphism method. GAD65 antibody was detected using a radioligand binding assay. There was no significant difference in the distribution of CTLA-4 alleles in patients and controls and no difference was observed in prevalence of CTLA-4 alleles when GAD65 antibody-positive and -negative individuals in the IDDM groups were compared. The present study did not support an association between the CTLA-4 gene and IDDM in the Japanese population.     

Genetics of type 1 diabetes mellitus

At least 20 different chromosomal regions have been linked to type 1 diabetes (T1D) susceptibility in humans, using genome screening, candidate gene testing, and studies of human homologues of mouse susceptibility genes. The largest contribution from a single locus (IDDM1) comes from several genes located in the MHC complex on chromosome 6p21.3, accounting for at least 40% of the familial aggregation of this disease. Approximately 30% of T1D patients are heterozygous for HLA-DQA1*0501-DQB1*0201/DQA1*0301-DQB1*0302 alleles (formerly referred to as HLA-DR3/4 and for simplification usually shortened to HLA-DQ2/DQ8), and a particular HLA-DQ6 molecule (HLA-DQA1*0102-DQB1*0602) is associated with dominant protection from the disease. There is evidence that certain residues important for structure and function of both HLA-DQ and DR peptide-binding pockets determine disease susceptibility and resistance. Independent confirmation of the IDDM2 locus on chromosome 11p15.5 has been achieved in both case-control and family-based studies, whereas associations with the other potential IDDM loci have not always been replicated. Several possibilities to explain these variable results from different studies are discussed, and a key factor affecting both linkage and association studies is that the genetic basis of T1D susceptibility may differ between ethnic groups. Some future strategies to address these problems are proposed. These include increasing the sample size in homogenous ethnic groups, high throughput genotyping and genomewide linkage disequilibrium (LD) mapping to establish disease associated ancestral haplotypes. Elucidation of the function of particular genes ('functional genomics') in the pathogenesis of T1D will be a most important element in future studies in this field, in addition to more sophisticated methods of statistical analyses.     

CTLA-4 gene polymorphism confers susceptibility to insulin-dependent diabetes mellitus (IDDM) independently from age and from other genetic or immune disease markers

Apart from genes in the HLA complex (IDDM1) and the variable number of tandem repeats in the 5′ region of the insulin gene (INS VNTR, IDDM2), several other loci have been proposed to contribute to IDDM susceptibility. Recently, linkage and association have been shown between the cytotoxic T lymphocyte-associated protein 4 (CTLA-4) gene on chromosome 2q and IDDM. In a registry-based group of 525 recent-onset IDDM patients < 40 years old we investigated the possible interactions of a CTLA-4 gene A-to-G transition polymorphism with age at clinical disease onset and with the presence or absence of established genetic (HLA-DQ, INS VNTR) and immune disease markers (autoantibodies against islet cell cytoplasm (ICA); insulin (IAA); glutamate decarboxylase (GAD65-Ab); IA-2 protein tyrosine phosphatase (IA-2-Ab)) determined within the first week of insulin treatment. In new-onset IDDM patients, G-allele-containing CTLA-4 genotypes (relative risk (RR) = 1.5; 95% confidence interval (CI) = 1.2–2.0; P< 0.005) were not preferentially associated with age at clinical presentation or with the presence of other genetic (HLA-DR3 or DR4 alleles; HLA-DQA1*0301-DQB1*0302 and/or DQA1*0501-DQB1*0201 risk haplotypes; INS VNTR I/I risk genotype) or immune (ICA, IAA, IA-2-Ab, GAD65-Ab) markers of diabetes. For 151 patients, thyrogastric autoantibodies (anti-thyroid peroxidase, anti-thyroid-stimulating hormone (TSH) receptor, anti-parietal cell, anti-intrinsic factor) were determined, but association between CTLA-4 risk genotypes and markers of polyendocrine autoimmunity could not be demonstrated before or after stratification for HLA- or INS-linked risk. In conclusion, the presence of a G-containing CTLA-4 genotype confers a moderate but significant RR for IDDM that is independent of age and genetic or immune disease markers.     

An autoimmune disease-associated CTLA-4 splice variant lacking the B7 binding domain signals negatively in T cells

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) plays a critical role in downregulating T cell responses. A number of autoimmune diseases have shown genetic linkage to the CTLA-4 locus. We have cloned and expressed an alternatively spliced form of CTLA-4 that has genetic linkage with type I diabetes in the NOD mice. This splice variant of CTLA-4, named ligand-independent CTLA-4 (liCTLA-4), lacks exon2 including the MYPPPY motif essential for binding to the costimulatory ligands B7-1 and B7-2. Here we show that liCTLA-4 is expressed as a protein in primary T cells and strongly inhibits T cell responses by binding and dephosphorylating the TcRzeta chain. Expression of liCTLA-4, but not full-length CTLA-4 (flCTLA-4), was higher in memory/regulatory T cells from diabetes-resistant NOD congenic mice compared to susceptible NOD mice. These data suggest that increased expression and negative signaling delivered by the liCTLA-4 may regulate development of T cell-mediated autoimmune diseases.     

Type 1 diabetes and prediabetic state in a monozygotic triplet

Type 1 diabetes mellitus (IDDM) results from a chronic process of autoimmune destruction of beta cells of the Langerhans islets. The presence of autoantibodies (ICA, GADA, anti-IA2, IAA) in serum precedes the clinical onset of the disease. Genetic predisposition for IDDM is connected with HLA, CTLA-4 and insulin gene region. The aim of the study was the genetic and immunological analysis of a triplet. One of them developed Type 1 diabetes mellitus. We analysed HLA class II, CTLA-4 and insulin gene polymorphisms in the whole family. Besides, we investigated immunological status of three brothers. All patients present identical genotype for VNTR loci: D1S80, D17S5 and Apo B, as well as for HLA-DRB1, -DQA1, -DQB1, CTLA-4 gene and all studied insulin gene polymorphisms. That proves their monozigosity. The triplet presents strong genetic predisposition for IDDM. The two patients without overt diabetes have increased levels of ICA, GADA, IA2 and IAA.     

The cytotoxic T lymphocyte antigen-4 is a major Graves' disease locus.

Graves' disease (GD) is an autoimmune thyroid disorder that is inherited as a complex trait. We have genotyped 77 affected sib-pairs with autoimmune thyroid disease for eight polymorphic markers spanning the cytotoxic T lymphocyte antigen-4 ( CTLA-4 ) region of chromosome 2q31-q33, and for five markers spanning the major histocompatibility complex ( MHC ) region of chromosome 6p21. Non-parametric analysis showed linkage of GD to the CTLA-4 region with a peak non-parametric linkage (NPL) score of 3.43 ( P = 0.0004) at the marker D2S117. The proportion of affected full-sibs sharing zero alleles (z0) reached a minimum of 0.113 close to D2S117, giving a locus-specific lambdas for this region of 2.2. Families with brother-sister sib-pairs showed a peak NPL of 3.46 ( P = 0.0003, lambdas > 10) at D2S117, compared with 2.00 ( P = 0.02, lambdas = 1.9) in the families with only affected females, suggesting a stronger influence in families with affected males. Association between GD and the G allele of the Thr17Ala polymorphism within the CTLA-4 gene ( CTLA4A/G ) was observed using unaffected sib controls ( P = 0.005). Lesser evidence for linkage was found at the MHC locus, with a peak NPL score of 1.95 ( P = 0.026), between the markers D6S273 and TNFalpha. We demonstrate that the CTLA-4 locus (lambdas = 2.2) and the MHC locus (lambdas = 1.6) together confer approximately 50% of the inherited susceptibility to GD disease in our population.     

The length of the CTLA-4 microsatellite (AT)N-repeat affects the risk for type 1 diabetes. Diabetes Incidence in Sweden Study Group

CTLA-4 is important to down-regulating T cell responses and has been implicated in type 1 (insulin dependent) diabetes mellitus in both linkage and association studies. The aim of our study was to relate the polymorphic (AT)n microsatellite in the 3' untranslated sequence of the CTLA-4 gene to diabetes risk. We studied 616 consecutively diagnosed 0-34 year-old Swedish patients and 502 matched controls by PCR-based genotyping fo determine the length of the 3'-end (AT)n repeat region of the CTLA-4 gene and categorizing alleles as predominantly monomorphic short (S) or highly polymorphic (in length) long (L) alleles. The odds of type 1 diabetes of subjects with the L/L genotype was estimated to be 1.84 times that of subjects with the S/S genotype (95% CI 1.44-2.73, p=0.002). Further analysis of the long alleles, partitioned into intermediate (I) length and very long (VL) alleles, suggested that L alleles act recessively in conferring diabetes risk (p=0.0009). This study suggests that the 3'-end (AT)n repeat region of the CTLA-4 gene represents a recessive risk factor for type 1 diabetes.     

自身免疫糖尿病的分子遗传学

自身免疫糖尿病是一种T细胞介导的疾病,以β细胞免疫损伤及胰岛素缺乏为特征,与多种基因相关,除了位于人类白细胞抗原(human leukocyte antigen,HLA)基因区的IDDM1和位于人胰岛素的基因区IDDM2被公认为是1A型糖尿病的主效基因外,不断有新的潜在致病基因被发现,如PTPN22、CTLA4、IL2RA、IFIH1等.现针对这些相关性研究结果及其中可能涉及的免疫学机制作一综述.     

Analysis of the CTLA-4, CD28, and inducible costimulator (ICOS) genes in autoimmune thyroid disease

The cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) gene on 2q33 is associated with autoimmune thyroid diseases (AITDs). Our earlier study in 56 families showed linkage of 2q33 to the presence of thyroid antibodies (TAbs). The goals of this study were to confirm the linkage of the 2q33 region to TAbs, to fine map this region, and study the ICOS gene. We performed a linkage study in an expanded data set of 99 multiplex AITD-TAb families (529 individuals). The highest two-point LOD score of 2.9 was obtained for marker D2S325 on 2q33. To fine map this locus, we genotyped 238 Caucasian AITD patients and 137 controls for five additional markers in the linked locus, which contained the CTLA-4, CD28, and ICOS genes. The A/G single-nucleotide polymorphism at position 49 of CTLA-4 was associated with AITD (P=0.01, OR=1.5), while markers inside CD28 and ICOS were not. Functional studies have shown that the G allele was associated with reduced inhibition of T-cell proliferation by CTLA-4. We concluded that: (1) the AITD gene in the 2q33 locus is the CTLA-4 gene and not the CD28 or ICOS genes; and (2) the G allele is associated with decreased function of CTLA-4.     

A recently described polymorphism in the CD28 gene on chromosome 2q33 is not associated with susceptibility to type 1 diabetes

Type 1 diabetes mellitus is an autoimmune disease with a strong genetic background. The CTLA4 gene region (IDDM12) has been implicated in genetic susceptibility to type 1 diabetes by genome scanning and both family‐ and population‐based analyses. As the genes encoding the costimulatory molecules CTLA4 and CD28, which compete for the receptor B7, reside close together on chromosome 2q33 and have high sequence homology, we investigated a recently described polymorphism in intron 3 of the CD28 gene and the CLTA4 codon 17 polymorphism in 176 patients with type 1 diabetes and 220 healthy controls. Whereas CTLA4 was found to be associated with type 1 diabetes, the frequency of the CD28 polymorphism did not differ between patients and controls, either in the entire sample or after stratification for CTLA4 genotype. Thus, the CD28 intron 3 polymorphism does not appear to be associated with susceptibility to type 1 diabetes.     

A recently described polymorphism in the CD28 gene on chromosome 2q33 is not associated with susceptibility to type 1 diabetes.

Type 1 diabetes mellitus is an autoimmune disease with a strong genetic background. The CTLA4 gene region (IDDM12) has been implicated in genetic susceptibility to type 1 diabetes by genome scanning and both family- and population-based analyses. As the genes encoding the costimulatory molecules CTLA4 and CD28, which compete for the receptor B7, reside close together on chromosome 2q33 and have high sequence homology, we investigated a recently described polymorphism in intron 3 of the CD28 gene and the CLTA4 codon 17 polymorphism in 176 patients with type 1 diabetes and 220 healthy controls. Whereas CTLA4 was found to be associated with type 1 diabetes, the frequency of the CD28 polymorphism did not differ between patients and controls, either in the entire sample or after stratification for CTLA4 genotype. Thus, the CD28 intron 3 polymorphism does not appear to be associated with susceptibility to type 1 diabetes.     

CTLA-4/CD 28 region polymorphisms in children from families with autoimmune hepatitis

Susceptibility to autoimmune hepatitis is associated with particular human leucocyte antigen class II alleles. However, non-HLA genetic factors are likely to be required for development of the disease. Among the candidate genes, the cytotoxic T-lymphocyte antigen 4 (CTLA-4) and CD28 genes, located on chromosome 2q33 in humans, encode a cell surface molecule playing a dominant role in the regulation of T-cell activation. The CTLA-4 and CD28 polymorphisms were investigated in children from 32 families with autoimmune hepatitis (AIH). The transmission/disequilibrium test revealed increased transmission of the (AT)8 (dinucleotide repeat) and A (exon 1) alleles of CTLA-4 gene from heterozygous parents to affected offspring (87.5% and 83.5%) with type 1 AIH, compared with unaffected offspring (50.0% for both, p = 0.009 and 0.02, respectively). In contrast, no deviation in transmission for CTLA-4 polymorphisms was found between type 2 AIH patients and unaffected offspring. No evidence for association was found between CD28 gene polymorphism or D2S72 genetic marker and both types of AIH. This study identified the CTLA-4 gene polymorphism as a non-HLA determinant that predisposes to AIH type 1 in children. The genetic heterogeneity seen in the present study provides a new argument in favor of pathogenic differences between type 1 and type 2 AIH.     

The Length of the CTLA-4 Microsatellite (AT)N-Repeat Affects the Risk for Type 1 Diabetes: For the Swedish Childhood Diabetes Study Group

CTLA-4 is important to down-regulating T cell responses and has been implicated in type 1 (insulin dependent) diabetes mellitus in both linkage and association studies. The aim of our study was to relate the polymorphic (AT)_n microsatellite in the 3′ untranslated sequence of the CTLA-4 gene to diabetes risk. We studied 616 consecutively diagnosed 0-34 year-old Swedish patients and 502 matched controls by PCR-based genotyping to determine the length of the 3′-end (AT)_n repeat region of the CTLA-4 gene and categorizing alleles as predominantly monomorphic short (S) or highly polymorphic (in length) long (L) alleles. The odds of type 1 diabetes of subjects with the L/L genotype was estimated to be 1.84 times that of subjects with the S/S genotype (95% CI 1.44-2.73, p=0.002). Further analysis of the long alleles, partitioned into intermediate (I) length and very long (VL) alleles, suggested that L alleles act recessively in conferring diabetes risk (p=0.0009). This study suggests that the 3′-end (AT)_n repeat region of the CTLA-4 gene represents a recessive risk factor for type 1 diabetes     

Insulin gene VNTR, CTLA-4 +49A/G and HLA-DQB1 alleles distinguish latent autoimmune diabetes in adults from type 1 diabetes and from type 2 diabetes group

Recent research has underlined the need to explore pathogenic, genetic and clinical spectrum of adult onset autoimmune diabetes, also known as latent autoimmune diabetes in adults (LADA). We aimed to investigate whether genetic factors that are associated with type 1 diabetes (T1D) susceptibility, namely HLA-DQB1 alleles, cytotoxic T-lymphocyte antigen 4 gene (CTLA-4) and insulin gene (INS) polymorphisms, are also associated with an atypical subset of patients diagnosed with type 2 diabetes (T2D). The case-control study included 70 T1D, 305 T2D and 252 nondiabetic controls. The T2D group was divided into atypical T2D (LADA, n = 61) or typical T2D (n = 244) subgroups based on the presence of at least one pancreas-specific antibody. Our data suggested that HLA-DQB1 alleles of all three risk classes, INS variable number of tandem repeat (VNTR) I/I and CTLA-4 +49 GG or AG genotypes, were independent risk factors for developing LADA and could be used as a diagnostic tool to discriminate between LADA and T2D. Additionally, there was an increased association between LADA and CTLA-4 diabetes-susceptibility genotypes and decreased association with INS VNTR and high-risk HLA-DQB1 alleles, compared with T1D. Our study suggested the need for further investigation into the genetic background and functional genomics of LADA in comparison with T1D and T2D.     

Conditional linkage disequilibrium analysis of a complex disease superlocus, IDDM1 in the HLA region, reveals the presence of independent modifying gene effects influencing the type 1 diabetes risk encoded by the major HLA-DQB1, -DRB1 disease loci

Type 1 diabetes mellitus is a common disease with a complex mode of inheritance. Its aetiology is underpinned by a major locus, insulin-dependent diabetes mellitus 1 (IDDM1) in the human leukocyte antigen (HLA) region of chromosome 6p21, and an unknown number of loci of lesser individual effect. In linkage analyses IDDM1 is a single peak, but it is evident that the linkage is caused by allelic variation of three adjacent genes in a 75 kb region, namely the class II genes, HLA-DRB1, -DQA1 and -DQB1. However, even these three genes may not explain all of the HLA association. We investigated, in the founder population of Sardinia, whether non-DQ/DR polymorphic markers within a 9.452 Mb region encompassing the whole HLA complex further influence the disease risk, after taking into account linkage disequilibrium with the disease loci HLA-DQB1, -DQA1 and -DRB1. We generalized the conditional association test, the haplotype method, to detect marker associations that are independent of the main DR/DQ disease associations. Three regions were identified as risk modifiers. These associations were not only independent of the polymorphic exon 2 sequences of HLA-DQB1, -DQA1 and -DRB1, but also independent of each other. The individual contributions of these risk modifiers were relatively modest but their combined impact was highly significant. Together, alleles of single nucleotide polymorphisms at the DMB and DOB genes, and the microsatellite locus TNFc, identified approximately 40% of Sardinian DR3 haplotypes as non-predisposing. This conditional analysis approach can be applied to any chromosome region involved in the predisposition to complex traits.     

Analysis of chromosome 6q in Basque families with type 1 diabetes. GEPV-N. Basque-Navarre Endocrinology and Paediatric Group

Over the last few years several studies of linkage between non-HLA loci and type 1 diabetes mellitus have mapped several putative susceptibility genes on chromosome 6q; in fact, positive evidence of linkage and/or association of IDDM5 (6q25), IDDM8 (6q27) and IDDM15 (6q21) with type 1 diabetes has been reported. We have studied these loci in diabetic families of Basque origin, a genetically homogeneous population, to avoid artifactual association results due to admixture within the sample analysed. Statistical analyses of linkage were performed using a transmission disequilibrium test (TDT). We could not confirm linkage for IDDM5, IDDM8 and IDDM15 in our population, possibly due to population-specific differences in genetic susceptibility and/or environmental triggering factors to type 1 diabetes.     

CTLA-4 gene polymorphism and its association with Graves' disease in the Lebanese population.

Graves' disease is an organ-specific autoimmune disease that has a female predominance. It is probably the result of a complex interaction of genetic and environmental factors. This disease is characterized by immune system activation, evidenced by elevated serum thyroid-specific autoantibodies and lymphocytic infiltration of the target organ (the thyroid gland), associated with raised levels of circulating activated T lymphocytes. Several reports have demonstrated genetic linkage and association between the genetic markers of the CTLA-4 gene on chromosome 2q33 and Graves' disease. In order to confirm this association in the Lebanese population, a bi-allelic A/G polymorphism at position 49 of CTLA-4 exon 1 was studied in 34 patients with Graves' disease, and in 38 healthy individuals, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. The results showed a significant increase in allele and genotype frequencies in patients with Graves' disease compared to controls. This suggests that the CTLA-4 gene might play a role in the development of Graves' disease in the Lebanese population.