CTLA4 is differently associated with autoimmune diseases in the Dutch population.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) is an important negative regulator of T-cell response and its genetic association with type 1 diabetes (T1D) has recently been demonstrated. The frequent co-association of autoimmune diseases (AID) and the implication from multiple genome scans that the CTLA4 gene region is a general autoimmune region, led us to study the role of CTLA4 in independent cohorts of T1D, coeliac disease (CD) and rheumatoid arthritis (RA) patients. We present independent data that confirm the association of CTLA4 in Dutch patients with juvenile onset T1D and show differential association of CTLA4 with CD and RA. The CTLA4 gene polymorphisms were tested for association in 350 T1D, 310 CD, 520 RA patients and 900 controls. In addition, 218 families were tested by the transmission disequilibrium test (TDT). T1D patients showed the highest association with the MH30*G: -1147*C: +49*G: CT60*G: JO37_3*G (haplotype 2) in both a case/control cohort (P=0.002, OR=1.42) and by TDT (P=0.02, OR=1.43). In contrast, this haplotype showed no association in the RA and CD cohorts. However, we observed an increased frequency of the MH30*G: -1147*T: +49*A: CT60*G: JO37_3*A (haplotype 3) in the CD patients diagnosed at a young age (OR=1.6, P=0.026, P (c)=0.052). Furthermore, when T1D and CD patients were stratified based on the HLA risk, the T1D susceptible CTLA4 haplotype 2 was over-represented in the high HLA-risk T1D and CD groups. In conclusion, we confirmed association between CTLA4 haplotype 2 and T1D in the Dutch population. Association with another CTLA4 haplotype (haplotype 3) was confirmed for CD, but only in those patients who had an early age of expression. No effect was found between RA and CTLA4. The association of the CTLA4 haplotype 2 with the high-risk HLA genotype in T1D and CD, which share DQ2 as the one of high-risk alleles, might provide a clue to understanding the common genetic background of AID.

Objective: To investigate the distribution of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) (+49 A/G) gene variants and the association of these variants with the clinical and laboratory findings in Egyptian children with Type-1 Diabetes (T1D). Methods: A case control study was done for 104 Egyptian children with T1D and 78 age and sex matched healthy control. CTLA-4 (+49 A/G) gene polymorphism typing was done by PCR amplification followed by restriction fragment length polymorphism (RFLP) method. Results: CTLA-4 G allele and GG homozygous genotype were significantly increased in T1D patients than in control group (P = 0.047, P = 0.048 respectively). There is no statistical difference between patient with optimal diabetic control (HbA1c < 8.5) and poor control (HbA1c ≥ 8.5) as regarding the CTLA-4 gene variant. The CTLA-4 GG genotype was statistically associated with younger age of patients (P = 0.027) and younger age of presentation (P = 0.036). Insignificant association was found between CTLA-4 alleles / genotypes and diabetic complications. Conclusion: The CTLA-4 +49 GG homozygous genotype is associated with T1D in Egyptian children especially with younger age of onset and in younger patients, and not associated with grades of diabetic control or diabetic complication.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is important for downregulation of T-cell activation, and CTLA-4 gene polymorphisms have been implicated as risk factors for rheumatoid arthritis (RA). Previous studies of the association between the +49 polymorphism of the CTLA-4 gene in RA have provided conflicting results. In order to determine association of the CTLA-4 gene with RA in Chinese Han population, we used denaturing gradient gel electrophoresis (DGGE) to genotype polymorphisms of four SNPs (MH30, +49, CT60 and JO31) of the CTLA-4 gene in 326 RA patients and 250 healthy controls. Furthermore, meta-analysis of all available studies relating +49 polymorphism to the risk of RA was performed to confirm the disease association. Among the SNPs examined, the genotype frequencies of CTLA-4 +49 and CT60 in RA patients differed significantly from controls (P=0.028 and 0.007). In addition, the distribution of four haplotypes constructed by these two SNPs was significantly different between patients and controls (chi(2)=10.58, d.f. =3, P=0.014). The meta-analysis also revealed that in both European and Asian populations, the CLTA-4 +49 G allele was associated with the risk of RA. These results suggested that the CTLA-4 gene might be involved in the susceptibility to RA in the Chinese Han population and both +49 and CT60 of CTLA-4 gene might be the causal variants in RA disease.

Background and ObjectivesMyasthenia gravis (MG) is an autoimmune disease associated with comorbid thymoma in 10%–15% of cases. Cytotoxic T lymphocyte–associated antigen 4 (CTLA4) expressed by T cells downregulates T-cell–mediated immune response. Polymorphisms in the CTLA4 gene have been associated with the development of MG. In this context, we aimed to determine whether CTLA4 expression in the thymoma differs between patients with and without MG and whether CTLA4 gene polymorphisms are associated with these differences.MethodsThis is a retrospective study of all patients, with and without MG, surgically treated at our institution for thymoma between January 2010 and December 2020. Ten samples were obtained from normal thymuses as controls. The number of CTLA4-positive cells in paraffin-embedded thymoma samples was determined by immunohistochemistry. The presence of follicular-center and regulatory T-cell lymphocytes was determined by immunohistochemistry (B-cell lymphoma [BCL]-6 expression) and double immunofluorescence–based staining of CD4-FOXP3, respectively. We evaluated the association between thymic expression of CTLA4 and the development of MG. We also determined the association between CTLA4 expression and various clinical and prognostic characteristics of MG. We sequenced the CTLA4 gene and evaluated possible associations between CTLA4 polymorphisms and thymic CTLA4 expression. Finally, we assessed the potential association between these polymorphisms and the risk of MG.ResultsForty-one patients with thymoma were included. Of them, 23 had comorbid MG (56.1%). On average, patients with MG had fewer CTLA4-positive cells in the thymoma than non-MG patients: 69.3 cells/mm2 (95% CIs: 39.6–99.1) vs 674.4 (276.0–1,024.0) cells/mm2; p = 0.001 and vs controls (200.74 [57.9–343.6] cells/mm2; p = 0.02). No between-group differences (MG vs non-MG) were observed in the number of cells positive for BCL6 or CD4-FOXP3. CTLA4 expression was not associated with differences in MG outcome or treatment refractoriness. Two polymorphisms were detected in the CTLA4 gene, rs231770 (n = 30 patients) and rs231775 (n = 17). MG was present in a similar proportion of patients for all genotypes. However, a nonsignificant trend toward a lower CTLA4-positive cell count was observed among carriers of the rs231775 polymorphism vs noncarriers: 77.9 cells/mm2 (95% CI: −51.5 to 207.5) vs 343.3 cells/mm2 (95% CI: 126.2–560.4).DiscussionReduced CTLA4 expression in thymoma may predispose to a higher risk of developing MG.

Myasthenia gravis (MG) is an autoimmune disease of striated muscle tissue mediated by autoantibodies. MG is often treated with thymectomy. Protein tyrosine phosphatase non-receptor 22 (PTPN22) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) have recently been found to be the genes that predispose to autoimmune diseases. The mechanisms of PTPN22 and CTLA-4 single nucleotide polymorphisms in resected thymomas and thymuses in MG remain unclear. In the present study, 90 patients with thymomas, including 44 patients with MG, 46 patients without MG, and 35 MG patients without thymoma were studied, with 50 healthy people as the controls. The +1858C>T polymorphism of the PTPN22 gene and the 49A>G polymorphism of the CTLA-4 gene were investigated using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The PTPN22 -1123G>C polymorphism at the promoter site was genotyped using single allele-specific primer polymerase chain reaction (SASP-PCR). The PTPN22 +1858C>T polymorphism was not significantly different between the patients and the controls. Statistically significant differences in the allelic and genotypic frequencies of PTPN 22 -1123G>C and CTLA-4 49A>G were observed between the MG(+)-thymoma group and the controls (P = 0.000, 0.003), but not between the MG(-)-thymoma and MG-thymoma(-) groups and the controls (P = 0.192/P = 0.214 and P = 0.067/P = 0.254). Statistically significant differences in allelic and genotypic frequencies of the 49A>G for CTLA-4 were observed between the MG(+)-thymoma group and the controls (P = 0.000, P = 0.003), but not between the MG(-)-thymoma and MG-thymoma(-) groups and the controls (P = 0.077/P = 0.261 and P = 0.058/P = 0.058). Individuals with the PTPN22 CC genotype and the CTLA-4 G alleles had an increased risk of developing paraneoplastic MG (odds ration [OR]= 4.722, 95% confidence interval [CI]: 1.460-15.277) compared with those with the PTPN22 G allele and the CTLA-4 AA genotype. The results show an association between the PTPN22 1123G>C genotype and thymoma-associated MG, with significant synergy with the CTLA-4 G alleles.

Co-stimulatory molecules of CD28, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), and the newly identified inducible co-stimulator (ICOS) are expressed on cell surfaces and provide regulatory signals for T-cell activation. Their genes are candidate susceptibility genes for type 1 diabetes because they co-localize to Chromosome 2q33 with the IDDM12 locus. After determining the genomic structure and screening for polymorphisms of the ICOS gene, we performed association studies between newly identified polymorphisms of the ICOS gene, together with known polymorphisms of CD28 and CTLA-4 genes, and type 1 diabetes. The 49A/G dimorphism in exon 1 and the (AT)n in the 3' untranslated region of the CTLA-4 gene were significantly associated with type 1 diabetes. Evaluation of the CTLA-4 49A-3'(AT)n 86-bp haplotype frequency in patients and controls confirmed the results from the analysis of each polymorphic site. Dimorphism in intron 3 of the CD28 gene was associated with type 1 diabetes only in the early-onset group. In contrast, there was no association with the microsatellite polymorphisms in the ICOS gene or dimorphisms in the promotor region of CTLA-4. Of the three genes encoding co-stimulatory molecules, the CTLA-4 gene appears to confer risks for the development of type 1 diabetes.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is an important molecule in the regulation of T cells, so the CTLA-4 gene has been considered as a strong candidate associated with T cell-mediated autoimmune diseases such as rheumatoid arthritis (RA). CTLA-4 has many variants and polymorphic forms, among which the +49A/G polymorphism, causing a non-synonymous substitution, has been studied most. However, previous studies of the association between the +49A/G polymorphism of the CTLA-4 gene and RA have provided conflicting results. The aim of this study was to determine the potential relationship of the CTLA-4 +49A/G polymorphism and the risk of RA in Chinese Han population. TaqMan assay was used to genotype the +49A/G polymorphism in 1,489 RA patients and 1,200 healthy controls. Furthermore, a meta-analysis of all studies relating this polymorphism in Chinese population to the risk of RA was performed. The genotype and allele frequencies of the CTLA-4 +49A/G in patients with RA differed significantly from those of controls (P = 0.03 and P = 0.007, respectively). The meta-analysis also revealed that the CTLA-4 +49G allele was associated with an increased risk of RA in Chinese population. Our results suggested that the CTLA-4 gene might contribute to the pathogenesis of RA, and the +49A/G polymorphism of this gene was a risk factor associated with increased RA susceptibility in Chinese Han population.

Type 1 diabetes (T1D) and celiac disease (CeD) are 2 distinct diseases, but there is an increased risk of developing CeD for T1D patients. Both diseases are associated with HLA-class II alleles, such as DQB1 *02:01 and DQB1 *03:02; however, their risk contribution vary between the diseases. We genotyped HLA-DRB1 and - DQB1 in 215 patients with coexisting T1D and CeD identified from a T1D cohort, and compared them to patients with T1D (N = 487) and CeD (N = 327), as well as healthy controls (N = 368). The patients with coexisting T1D and CeD had an intermediate carrier frequency (72.8%) of the DRB1 *03:01- DQB1 *02:01- DQA1 *05:01 haplotype compared to T1D (64.1%) and CeD (88.7%) patients. The DRB1 *03:01- DQB1 *02:01- DQA1 *05:01/ DRB1 *04- DQB1 *03:02- DQA1 *03 haplotype combination, encoding DQ2.5 and DQ8 molecules, was equally frequent among patients with both T1D and CeD (52.6%) and T1D patients (46.8%) but significantly lower in CeD patients (9.5%). Overall, the patients with coexisting T1D and CeD had an HLA profile more similar to T1D patients than CeD patients.

Ulcerative colitis (UC) is characterized by chronic inflammation of the colon and rectum as a result of an exaggerated T-cell response. Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is a T cell-restricted surface molecule induced with TCR or CD28 activation. There is evidence for genetic involvement of CTLA-4 in several autoimmune diseases, with the focus on the possible role of genetic variation of the CTLA-4 locus. The aim of this study was to investigate CTLA-4 gene polymorphisms in patients with UC in a Chinese population with Han nationality. The C-318T polymorphism in the promoter region and A+49G polymorphism in exon 1 of the CTLA-4 gene were studied by a polymerase chain reaction-sequence-specific primer method. We studied 82 unrelated patients with UC and 204 healthy controls in a Chinese population with Han nationality. The frequency of the haplotype 2,3 (-318C+49G/-318T+49A) was 26% in patients with UC and 41% in healthy controls (Fisher exact test P = 0.0147, odds ratio = 0.4918, 95% confidence interval: 0.2784 - 0.8688), but this significance disappeared when Bonferoni correction was applied. No other significant differences in the distribution of allele and genotype frequencies were observed between C-318T and A+49G gene polymorphisms and UC in the Chinese Han population. The C-318T and A+49G polymorphisms of the CTLA-4 gene were not associated with UC in Chinese Han patients.

CTLA4 gene polymorphism in Italian patients with colorectal adenoma and cancer

ABSTRACTObjectives: To investigate the association of cytotoxic T lymphocyte-associated antigen 4 (CTLA4) with immune thrombocytopenia (ITP).Methods: A case–control association analysis of 277 Chinese Han children was performed. The tagging variants rs11571315 and rs3087243 in the CTLA4 gene were detected using polymerase chain reaction-restriction fragment length polymorphism method. The expression quantitative trait loci (eQTL) analysis and quantitative real-time polymerase chain reaction were performed to determine the relationship of CTLA4 with ITP.Results: Neither SNP was significantly different between case and control groups in either the genotypic or allelic distribution. The eQTL analysis results indicated that in the spleen, the rs3087243 was significant with the expression of CTLA4. The rs11571315 has similar results. Interestingly, the transcript level of CTLA4 was found to significantly decrease in patients with ITP.Discussion: The autoimmune and gene etiology is implicated in the pathogen of ITP. The CTLA4 is important for negative regulation of T-cell activation, and CTLA-4 gene has been identified as a risk factor for some autoimmune diseases. However, association studies of ITP and CTLA4 gene have obtained conflicting results. This is the first study to systematically investigate the association of CTLA4 with ITP in Chinese Han children.Conclusions: The CTLA4 gene is suggested to correlate with ITP through its abnormal expression level instead of gene site mutation.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is a negative regulator of T cells and is, therefore, a strong candidate susceptibility gene for T cell-mediated autoimmune diseases. The association of CTLA-4 single-nucleotide polymorphisms (SNPs) with rheumatoid arthritis (RA) has been investigated previously, with inconsistent results. Recently, SNPs mapping to the gene (and not previously investigated in RA) have been associated with both type 1 diabetes mellitus and Graves' disease. The aim of this study was to investigate the association of the CTLA-4 polymorphism with RA. Primer extension methods were used to genotype 5 haplotype-tagging SNPs (htSNPs) (-1722 T/C, -1661 A/G, -658 C/T, -319 C/T, and +49 A/G), and the TaqMan 5' allelic discrimination assay was used to genotype an additional 2 SNPs (CT60 and rs1863800) mapping to the CTLA-4 gene. Association to the 5 htSNPs was investigated using the transmission disequilibrium test in RA simplex families (n = 122). Allele frequencies for the htSNPs were also investigated in affected sibling pairs (n = 96) and unrelated controls (n = 173). For the SNPs CT60 and rs1863800, unrelated patients with RA (n = 759) were compared with controls (n = 755). No evidence for association to single markers or haplotypes of the 5 htSNPs was detected in either RA simplex families or the affected sibling-control cohort. Neither of the 2 SNPs recently associated with Graves' disease showed evidence for association in the unrelated patient-control cohort. No evidence for association of CTLA-4 with RA was detected using family or case-control methods.

To investigate the densities of dendritic cells (DCs) and FOXP3(+) regulatory T cells (Tregs) and their interrelations in the small bowel mucosa in untreated celiac disease (CD) patients with and without type 1 diabetes (T1D). Seventy-four patients (45 female, 29 male, mean age 11.1 ± 6.8 years) who underwent small bowel biopsy were studied. CD without T1D was diagnosed in 18 patients, and CD with T1D was diagnosed in 15 patients. Normal small bowel mucosa was found in two T1D patients. Thirty-nine patients (mean age 12.8 ± 4.9 years) with other diagnoses (functional dyspepsia, duodenal ulcer, erosive gastritis, etc.) formed the control group. All CD patients had partial or subtotal villous atrophy according to the Marsh classification: Marsh grade IIIa in 9, grade IIIb in 21 and grade IIIc in 3 cases. Thirty-nine patients without CD and 2 with T1D had normal small bowel mucosa (Marsh grade 0). The densities of CD11c(+), IDO(+), CD103(+), Langerin (CD207(+)) DCs and FOXP3(+) Tregs were investigated by immunohistochemistry (on paraffin-embedded specimens) and immunofluorescence (on cryostat sections) methods using a combination of mono- and double-staining. Sixty-six serum samples were tested for IgA-tissue transglutaminase (tTG) using a fully automated EliA™ Celikey(®) IgA assay (Pharmacia Diagnostics, Freiburg, Germany). The density of CD11c(+) DCs was significantly increased in CD patients compared with patients with normal mucosa (21.67 ± 2.49 vs 13.58 ± 1.51, P = 0.007). The numbers of FOXP3(+) cells were significantly higher in CD patients (10.66 ± 1.50 vs 1.92 ± 0.37, P = 0.0002) and in patients with CD and coexisting T1D (8.11 ± 1.64 vs 1.92 ± 0.37, P = 0.002) compared with patients with normal mucosa. The density of FOXP3(+) cells significantly correlated with the histological grade of atrophic changes in the small bowel mucosa according to the March classification (r = 0.62; P < 0.0001) and with levels of IgA antibody (r = 0.55; P < 0.0001). The densities of IDO(+) DCs were significantly higher in CD patients (21.6 ± 2.67 vs 6.26 ± 0.84, P = 0.00003) and in patients with CD and coexisting T1D (19.08 ± 3.61 vs 6.26 ± 0.84, P = 0.004) compared with patients with normal mucosa. A significant correlation was identified between the densities of IDO(+) DCs and FOXP3(+) T cells (r = 0.76; P = 0.0001). The mean values of CD103(+) DCs were significantly higher in CD patients (10.66 ± 1.53 vs 6.34 ± 0.61, P = 0.01) and in patients with CD and associated T1D (11.13 ± 0.72 vs 6.34 ± 0.61, P = 0.00002) compared with subjects with normal small bowel mucosa. The mean value of Langerin(+) DCs was higher in CD patients compared with persons with normal mucosa (7.4 ± 0.92 vs 5.64 ± 0.46, P = 0.04). The participation of diverse DC subsets in the pathological processes of CD and the possible involvement of tolerogenic DCs in Tregs development to maintain intestinal immunological tolerance in CD patients are revealed.

Background & objectives:Celiac disease (CD) can exist in various forms in type 1 diabetes (T1D) patients and can remain undetected, leading to severe complications. This study was aimed to evaluate five commercially available anti-tissue transglutaminase (tTG) ELISA kits with distinct formats for the detection of CD and potential CD in T1D patients. Clinical and demographic profiles of the patients with different disease subsets were also studied.Methods:Fifty T1D patients with classical and non-classical symptoms of CD and 100 T1D patients without any symptoms of CD were included in this study. Anti-tTG autoantibody levels were estimated by five ELISA kits followed by histological examination of duodenal biopsy. HLA DQ2-DQ8 and DRB1-DQB1 typing was done, and serum levels for transforming growth factor (TGF)-β1 were also estimated.Results:Assay format detecting anti-tTG IgA antibodies against recombinant antigens along with neopeptides of gliadin was most efficient in the detection of CD in symptomatic patients, and assay format detecting IgA+IgG helped in the detection of potential CD in asymptomatic T1D patients. These findings were supported by histological examination and human leucocyte antigen analysis. Patients with potential CD were found to have markedly deranged glycaemic control parameters and also had significantly raised serum levels of TGF-β1, (P<0.05) compared to T1D patients.Interpretation & conclusions:Potential CD can be frequently seen in T1D patients. This can be attributed to the dietary patterns prevalent in the subcontinent and the genetic basis of the disease. Anti-tTG IgA+IgG antibodies can be useful in the detection of these potential CD cases in T1D patients. Early intervention with gluten-free diet can be considered in these patients for better disease management.

BackgroundCytotoxic T lymphocyte-associated antigen-4 (CTLA-4) plays a critical role in downregulation of antigen-activated immune response and polymorphisms at the CTLA-4 gene have been shown to be associated with several autoimmune diseases including type-1 diabetes (T1D). The etiological mutation was mapped to the CT60-A/G single nucleotide polymorphism (SNP) that is believed to control the processing and production of soluble CTLA-4 (sCTLA-4).MethodsWe therefore determined sCTLA-4 protein levels in the sera from 82 T1D patients and 19 autoantibody positive (AbP) subjects and 117 autoantibody negative (AbN) controls using ELISA. The CT-60 SNP was genotyped for these samples by using PCR and restriction enzyme digestion of a 268 bp DNA segment containing the SNP. Genotyping of CT-60 SNP was confirmed by dye terminating sequencing reaction.ResultsHigher levels of sCTLA-4 were observed in T1D (2.24 ng/ml) and AbP (mean = 2.17 ng/ml) subjects compared to AbN controls (mean = 1.69 ng/ml) with the differences between these subjects becoming significant with age (p = 0.02). However, we found no correlation between sCTLA-4 levels and the CTLA-4 CT-60 SNP genotypes.ConclusionConsistent with the higher serum sCTLA-4 levels observed in other autoimmune diseases, our results suggest that sCTLA-4 may be a risk factor for T1D. However, our results do not support the conclusion that the CT-60 SNP controls the expression of sCTLA-4.