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In clinical practice, it is found that autoimmune thyroid disease often additionally occurs with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). In addition, several studies showed that eye-specific autoimmune diseases may have a strong relationship with systemic autoimmune diseases. We focused on Graves' disease (GD) with ocular conditions, also known as Graves' ophthalmopathy (GO), trying to find out the potential genetic background related to GO, RA, and SLE. There were 40 GO cases and 40 healthy controls enrolled in this study. The association between single-nucleotide polymorphisms (SNPs) of the co-stimulatory molecule genes and GO was analyzed using a chi-square test. It showed that rs11571315, rs733618, rs4553808, rs11571316, rs16840252, and rs11571319 of CTLA4, rs3181098 of CD28, rs36084323 and rs10204525 of PDCD1, and rs11889352 and rs4675379 of ICOS were significantly associated with GO based on genotype analysis and/or allele analysis (p < 0.05). After summarizing the GO data and the previously published SLE and RA data, it was found that rs11571315, rs733618, rs4553808, rs16840252, rs11571319, and rs36084323 were shared in these three diseases. Furthermore, the bio-function was confirmed by dual-luciferase reporter assay. It was shown that rs733618 T > C and rs4553808 A > G significantly decreased the transcriptional activity (both p < 0.001). This study is the first to confirm that these three diseases share genetically predisposing factors, and our results support the proposal that rs733618 T > C and rs4553808 A > G have bio-functional effects on the transcriptional activity of the CTLA4 gene. [ABSTRACT FROM AUTHOR]

In a prior study, we discovered that hematopoietic stem cell transplantation (HSCT) and/or autoimmune diseases, such as systemic lupus erythematosus, were associated with the rs1234314 C/G and rs45454293 C/T polymorphisms of TNFSF4, the rs5839828 C > del and rs36084323 C > T polymorphisms of PDCD1, and the rs28541784C/T, rs200353921A/T, rs3181096C/T, and rs3181098 G/A polymorphisms of CD28. However, the association does not imply causation. These single nucleotide polymorphisms (SNPs) are all located in the promoter region of these genes, so we used the dual-luminescence reporter assay to explore the effect of single nucleotide polymorphisms (SNPs) on transcriptional activity. For each promoter–reporter with a single SNP mutation, more than 10 independent experiments were carried out, and the difference in transcription activity was compared using one-way ANOVA and Tukey's honestly significant difference test. The results showed that the G-allele of rs1234314 had 0.32 ± 0.09 times the average amount of relative light units (RLU) compared to the C-allele (p = 0.003), the T-allele of rs45454293 had 4.63 ± 0.92 times the average amount of RLU compared to the C-allele (p < 0.001), the del-allele of rs5839828 had 1.37 ± 0.24 times the average amount of RLU compared to the G-allele (p < 0.001), and the T-allele of rs36084323 had 0.68 ± 0.07 times the average amount of RLU compared to the C-allele (p < 0.001). The CD28 SNPs studied here did not affect transcriptional activity. In conclusion, the findings of this study could only confirm that the SNP had a bio-functional effect on gene expression levels. According to the findings, several SNPs in the same gene have bio-functions that affect transcriptional activity. However, some increase transcriptional activity while others decrease it. Consequently, we inferred that the final protein level should be the integration result of the co-regulation of all the SNPs with the effect on transcriptional activity. [ABSTRACT FROM AUTHOR]

Thrombocytopenia is a condition where the platelet count is under 100 × 109/L, which is caused by various disorders. However, the mechanism of thrombocytopenia is still unclear. Hence, we tried to investigate the correlation between immune thrombocytopenia (ITP) and single nucleotide polymorphisms (SNPs) of genes related to T cell activation. There were 32 ITP patients and 30 healthy controls enrolled in this study. PCR and sequencing were used to find out the significant SNPs, which we focused on the promoter region of CTLA4 and CD28. In this study, the ITP cases were divided into primary ITP group, secondary ITP group, and the combination of the two to the follow-up analysis. Moreover, dual-luciferase reporter assay was used to evaluate the transcription activity of the significant SNP. We found the − 1765_rs11571315 of CTLA4 gene was associated with primary ITP (p = 0.006), secondary ITP (p = 0.008), and the combination of the two (p = 0.003). Moreover, the −318_rs5742909 also had statistical significance in secondary ITP group that was only caused by autoimmune disease (p = 0.019). In functional study, the rs5742909 would decrease 19% of the transcription activity when it carried a T-allele at this position (p = 0.040). It was noted that CTLA4 gene polymorphism was related to ITP but not CD28. According to our results, we surmised that CTLA4 is involved in the pathogenesis of ITP, and the secondary ITP result from the lower CTLA4 expression that leads to T cell over-activation. [ABSTRACT FROM AUTHOR]

Cord blood transplantation (CBT) provides a treatment scheme for hematologic diseases and leukemia in both children and adults. However, adverse reactions and transplantation-related death may still occur in patients receiving CBT even when donor and recipient have fully matched HLA in high-resolution HLA typing analysis. Single nucleotide polymorphisms (SNPs) of HLA-related and unrelated genes are known to associate with disease status of patients with unrelated stem cell transplantation. In this study, the genomic regions ranging from 500 base pairs upstream to 500 base pairs downstream of the eight SNPs that were reported as transplantation determinants by Petersdorf et al. were analyzed to evaluate whether genetic variants were associated with the survival status of patients, and the risk for severe (grades 3–4) graft-versus-host disease (GVHD) or cytomegalovirus (CMV) infection/reactivation. The analyses were performed in the mode of recipient genotype, donor genotype, and recipient-donor mismatching, respectively. By analysis of sixty-five patients and their HLA-matched unrelated donors, we found that five SNPs were associated with patient survival which included the recipient genotype with SNPs of rs107822 in the RING1 gene, and rs2070120, rs17220087 and rs17213693 in the HLA-DOB gene; and the recipient-donor mismatching with SNPs of rs9282369 in HLA-DOA gene, and rs2070120, rs17220087 and rs17213693 in the HLA-DOB gene. Five SNPs were associated with the risk for severe GVHD which included the donor genotype with SNPs of rs213210 and rs2523675; the recipient genotype with SNPs of rs9281491 in the HCP5 gene; and the recipient-donor mismatching with SNPs of rs209130 in the TRIM27 gene, and rs986522 in the COL11A2 gene. Six SNPs were related to the risk for CMV infection/reactivation which included the donor genotype with SNPs of rs435766, rs380924, and rs2523957; and the recipient-donor mismatching with SNPs of rs2070120, rs17220087, and rs17213693 in the HLA-DOB gene; and rs435766 and rs380924 in the MICD gene. This study provides the basis for larger analyses and if the results are confirmed, a way of selecting better unrelated CBT candidate donors. [ABSTRACT FROM AUTHOR]

To avoid graft rejection, the hematopoietic stem cells with matched classical human leukocyte antigen (HLA) alleles are the primary choice for clinical allogeneic transplantation. However, even if the fully HLA-matched hematopoietic stem cells are used for transplantation, some patients still have poor prognosis after hematopoietic stem cell transplantation (HSCT), suggesting that the HLA system was not the only determinant of the outcomes of HSCT. In this study, we investigated whether the single-nucleotide polymorphisms (SNPs) of the co-stimulatory genes within non-HLA regions were related to the outcomes of HSCT. The genomic DNAs of 163 patients who had acute leukemia and received HSCT and their respective donors were collected for analysis. Thirty-four SNPs located in the four co-stimulatory genes including cytotoxic T-lymphocyte associated protein 4 (CTLA4), CD28, tumor necrosis factor ligand superfamily 4 (TNFSF4), and programmed cell death protein 1 (PDCD1) were selected to explore their relationship with the adverse outcomes after transplantation, including mortality, cytomegalovirus infection, graft-versus-host disease, and relapse. Our results revealed that nine SNPs in the CTLA4 gene, five SNPs in the PDCD1 gene, two SNPs in the TNFSF4 gene, and four SNPs in the CD28 gene were significantly associated with the occurrence of adverse outcomes post-HSCT. These SNPs may play important roles in immune response to allografts post-HSCT and can be the targets for developing strategy to identify appropriate donors. [ABSTRACT FROM AUTHOR]

Transfusion reactions are mainly induced by the interaction of an antigen and antibody. However, transfusion reactions still occur with the implementing of crossmatching and usage of pre-storage leukoreduced blood products. The roles of CD28 and CTLA4 gene polymorphisms in transfusion reaction have been shown, and subjects with certain single nucleotide polymorphisms (SNPs) of the CD28 or CTLA4 gene had a significantly higher risk of transfusion reactions. In total, 40 patients with transfusion reactions after receiving pre-storage leukoreduced blood products were enrolled in this study. We focused on the SNPs located in the CD28 promoter region (rs1879877, rs3181096, rs3181097, and rs3181098) to find out the significant SNP. A luciferase reporter assay was used to investigate the expression level of protein affected by promoter SNP variation. We found that the polymorphism of rs3181097 was associated with transfusion reactions (p = 0.003 in additive model and p = 0.015 in dominant model). Consequently, we investigated the biological function in the CD28 promoter polymorphisms (rs1879877 G > T, rs3181096 C > T, rs3181097 G > A, and rs3181098 G > A) by using dual-spectral luciferase reporter assay. The results showed that the ex-pression level of CD28 was decreased under the effect of rs3181097 with A-allele. This suggested that rs3181097 may regulate immune response through decreasing CD28 protein expression and then lead to development of transfusion reactions. [ABSTRACT FROM AUTHOR]

CTLA-4 and CD28 belong to co-stimulation molecules, the abnormal expression of which can regulate the T cell activation and then affect the degree of immune response. Moreover, blood transfusion reaction (TR) is a kind of immune reaction. Consequently, the hypothesis of this study was that the TR still occurred after transfusing leukoreduced blood components as a result of the sensitivity of immune system, and a small number of immune stimulations were enough to induce an immune response in patients. There were 38 cases and 36 healthy controls included in this study. The selected CD28 gene were sequenced to search single nucleotide polymorphism (SNPs), and the correlation between TR and these SNPs was analyzed. According to our data, only the rs3181097 (promoter, −1059) of CD28 gene polymorphism was associated with TR. The p value of rs3181097 under the co-dominant model was 0.031. GG was used as a reference genotype, the p value of GG vs. AG was 0.010 (OR: 0.210, 95% CI: 0.062–0.712), and GG vs. AG + AA was 0.028 (OR: 0.292, 95% CI: 0.095–0.901). In addition to CTLA-4, CD28 gene was also a crucial SNP in TR, because there was a signification for the haplotype with Grs3181097 (p = 0.015). Consequently, we suggested that the TR was related to CD28 by regulating the degree of immune response. [ABSTRACT FROM AUTHOR]

Graves' disease (GD) is an autoimmune inflammatory disease, and Graves' ophthalmopathy (GO) occurs in 25–50% of patients with GD. Several susceptible genes were identified to be associated with GO in some genetic analysis studies, including the immune regulatory gene CTLA4. We aimed to find out the correlation of CTLA4 gene polymorphism and GO. A total of 42 participants were enrolled in this study, consisting of 22 patients with GO and 20 healthy controls. Chi-square or Fisher's exact test were used to appraise the association between Graves' ophthalmopathy and CTLA4 single nucleotide polymorphisms (SNPs). All regions of CTLA4 including promoter, exon and 3'UTR were investigated. There was no nucleotide substitution in exon 2 and exon 3 of CTLA4 region, and the allele frequencies of CTLA4 polymorphisms had no significant difference between patients with GO and controls. However, the genotype frequency of "TT" genotype in rs733618 significantly differed between patients with GO and healthy controls (OR = 0.421, 95%CI: 0.290–0.611, p = 0.043), and the "CC" and "CT" genotype in rs16840252 were nearly significantly differed in genotype frequency (p = 0.052). Haplotype analysis showed that CTLA4 Crs733618Crs16840252 might increase the risk of GO (OR = 2.375, 95%CI: 1.636–3.448, p = 0.043). In conclusion, CTLA4 Crs733618Crs16840252 was found to be a potential marker for GO, and these haplotypes would be ethnicity-specific. Clinical application of CTLA4 Crs733618Crs16840252 in predicting GO in GD patients may be beneficial. [ABSTRACT FROM AUTHOR]