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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]

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]

Adverse reactions may still occur in some patients after receiving haematopoietic stem cell transplantation (HSCT), even when choosing a human leukocyte antigen (HLA)-matched donor. The adverse reactions of transplantation include disease relapse, graft-versus-host disease (GVHD), mortality and CMV infection. However, only the relapse was discussed in our previous study. Therefore, in this study, we investigated the correlation between the gene polymorphisms within the HLA region and the adverse reactions of post-HSCT in patients with acute leukaemia (n = 176), where 72 patients were diagnosed with acute lymphocytic leukaemia (ALL) and 104 were acute myeloid leukaemia (AML). The candidate single nucleotide polymorphisms were divided into three models: donor, recipient, and donor-recipient pairs and the data of ALL and AML were analysed individually. Based on the results, we found 16 SNPs associated with the survival rates, the risk of CMV infection, or the grade of GVHD in either donor, recipient, or donor-recipient matching models. In the ALL group, the rs209132 of TRIM27 in the donor group was related to CMV infection (p = 0.021), the rs213210 of RING1 in the recipient group was associated with serious GVHD (p = 0.003), and the rs2227956 of HSPA1L in the recipient group correlated with CMV infection (p = 0.001). In the AML group, the rs3130048 of BAG6 in the donor-recipient pairs group was associated with serious GVHD (p = 0.048). Moreover, these SNPs were further associated with the duration time of survival after transplantation. These results could be applied to select the best donor in HSCT. [ABSTRACT FROM AUTHOR]

Background: ABO subgroups would be considered when discrepancies in ABO grouping occur. Serological methods including adsorption-elution test, salivary ABH inhibition test, and anti-A1 (lectin) saline method could be used. However, these serological methods are laboring and obscure. Therefore, reliable and affordable method to assess the ABO subgroups is of particular interest.Methods: To solve this problem, the multiplex SNaPshot-based assays were designed to determine rare A and B subgroups. Primers used as probes for determination of rare ABO blood groups known in Taiwanese population were designed. Many ABO subtype samples were used to validate the accuracy and reproducibility of our SNaPshot panel.Results: A panel of primer probes were successfully designed in determining 8 SNP sites (261, 539, 838, 820, 745, 664, IVS6 +5, and 829 in exon 6 and 7) for A phenotype and 6 SNP sites (261, 796, IVS3 +5, 247, 523, and 502 in exon 2, 6 and 7 and intron 3) for B phenotype. SNaPshot analysis for defining blood group A alleles (A1, A2, A3, Am and Ael) and blood group B alleles (B1, B3, Bw and Bel) was therefore available.Conclusion: SNaPshot analysis could be used in reference laboratories for typing known rare subgroups of A and B without DNA cloning and traditional sequencing. Moreover, this method would help to construct databases of genotyped blood donors, and it potentially plays a role in determining fetal-maternal ABO incompatibility. [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]

Leukocytes and cytokines in blood units have been known to be involved in febrile non-hemolytic transfusion reaction (FNHTR), and these adverse reactions still occur while using pre-storage leukoreduced blood products. Blood transfusion is similar to transplantation because both implant allogeneic cells or organs into the recipient. CTLA4 gene polymorphism was found to be associated with graft-versus-host disease in hematopoietic stem cell transplantation. We performed a prospective cohort study at a major tertiary care center to investigate the correlation of CTLA4 gene polymorphism and transfusion reactions. Selected CTLA4 gene SNPs were genotyped and compared between patients with transfusion-associated adverse reactions (TAARs) and healthy controls. Nineteen patients and 20 healthy subjects were enrolled. There were 4 SNPs showing differences in allele frequency between patients and controls, and the frequency of "A" allele of rs4553808, "G" allele of rs62182595, "G" allele of rs16840252, and "C" allele of rs5742909 were significantly higher in patients than in controls. Moreover, these alleles also showed significantly higher risk of TAARs (OR = 2.357, 95%CI: 1.584–3.508, p = 0.02; OR = 2.357, 95%CI: 1.584–3.508, p = 0.02; OR = 2.462, 95%CI: 1.619–3.742, p = 0.008; OR = 2.357, 95%CI: 1.584–3.508, p = 0.02; OR = 2.357, 95%CI: 1.584–3.508, p = 0.02, respectively). The present study demonstrated the correlation of CTLA4 gene polymorphism and transfusion reaction, and alleles of 4 CTLA4 SNPs with an increased risk of TAARs were found. It is important to explore the potential immune regulatory mechanism affected by SNPs of costimulatory molecules, and it could predict transfusion reaction occurrence and guide preventive actions. [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]

Abstract: Background: An accurate analysis of chimerism kinetics permits early detection of hematopoietic stem cell transplantation (HSCT) in patients with high risks of graft-versus-host disease or those liable to relapse. Although short tandem repeats-PCR (STR-PCR) is the golden standard for quantitative chimerism analysis in most of the clinical laboratories, it has a relatively low sensitivity of 5% and the detection of low percentage in mixed chimerism is usually delayed. In this study, we developed a real-time PCR for chimerism analysis based on the informative biallelic polymorphisms (BP). Methods: The allele frequencies of 19 selective biallelic polymorphic markers were analyzed using the genomic DNA from 100 healthy Taiwanese volunteers. The informative biallelic polymorphic markers with high discrimination power in the Taiwanese population were identified. The TaqMan probe-based real-time BP-PCR for amplification of the informative loci was designed and the detection sensitivity was determined. Clinical application of real-time BP-PCR in chimerism monitoring was evaluated and was compared with the conventional STR-PCR by analyzing the DNA samples obtained at different time points post-HSCT from 4 relapsed and 10 non-relapsed patients. Results: Allele distribution analysis revealed that the loci of S01a, S03, S04a, S05b, S06, S07b, S08b, S09b, S10b and S11a had a relatively high discrimination power and were the informative BP for chimerism monitoring in the Taiwanese population. Real-time BP-PCRs for these 10 BP loci were set up with the detection sensitivity equivalent to 0.003–0.006%. Real-time BP-PCR of the 4 HSCT patients revealed the presence of recipient-specific DNA at early time point than STR-PCR for 3 of the patients, whereas real-time BP-PCR was as effective as STR-PCR in uncovering the sign of relapse for one of the patients. In addition, the baseline value for the patients with no sign of relapse was 0.127±0.193% of recipient DNA. Conclusion: We conclude that real-time BP-PCR is a sensitive and reliable method for chimerism monitoring and is superior to the STR-PCR in identifying patients who are at high risk for relapse after transplantation. [Copyright &y& Elsevier]