Human Leucocyte Antigen (HLA) alleles, cytokine polymorphisms and the type of factor VIII (FVIII) gene mutation are among predisposing factors for inhibitors (inh) development in children with severe haemophilia A (HA). The aim was to investigate the correlations among (i) FVIII gene intron-22 inversion, (ii) HLA alleles and haplotypes and (iii) certain cytokine polymorphisms, with the risk for FVIII inhibitors development in 52 Greek severe HA children, exclusively treated with recombinant concentrates. We performed Long-Range PCR for detection of intron-22 inversion and PCR-SSP, PCR-SSO for genotyping of HLA-A, B, C, DRB1, DQB1 alleles and also for cytokine polymorphisms of TNF-α, TGF-β1, IL-10, IL-6 and IFN-γ. Chi-squared test and Fischer's exact test were used for statistical analysis. A total of 28 children had developed inhibitors (Group I), 71.4% high responding, while 24 had not (Group II). No statistically increased intron-22 inversion prevalence was found in Group I compared with Group II (P = 0.5). Comparison of HLA allele frequencies between the two groups showed statistically significant differences in the following genotypes (i) promoting inhibitors development: DRB1*01(P = 0.014), DRB1*01:01(P = 0.011) and DQB1*05:01 (P = 0.005) and (ii) possibly protecting from inhibitors development: DRB1*11 (P = 0.011), DRB1*11:01 (P = 0.031), DQB1*03 (P = 0.004) and DQB1*03:01 (P = 0.014). Analysis of cytokines revealed a higher incidence of inhibitor detection only in homozygotes of the haplotypes ACC and ATA for IL-10 polymorphisms (P = 0.05). There is evidence that HLA alleles and cytokine polymorphisms play an important role in FVIII inh development. On the contrary, no statistically significant results were obtained for intron-22 inversion and its impact on FVIII inhibitors formation.
Various polymorphisms in cytokine genes have recently been investigated as candidate risk factors in allogeneic hematopoetic stem cell transplantation (allo-HSCT). We retrospectively analyzed specific polymorphisms in genes for interleukin (IL)-10, IL-6, tumor-necrosis factor alpha (TNF-a) and interferon gamma (IFN-c) in a pediatric cohort of 57 histocompatibility leucocyte antigen (HLA)-identical sibling myeloablative transplants. Both recipient and donor genotypes were tested for association with graft-versus-host disease (GVHD) by statistical methods including Cox regression analysis. We found a significant association between the IL-10 promoter haplotype polymorphisms at positions -1082, -819 and -592 with the occurrence of severe (grades III-IV) acute GVHD (aGVHD). Recipients with the haplotype GCC had a statistically significant decreased risk of severe aGVHD (hazard risk (HR)50.20, 95% confidence interval (CI): 0.06-0.67) in comparison with patients with other IL-10 haplotypes (P50.008). Transplant-related mortality at 1 year was significantly lower in recipients with this haplotype (HR50.17, 95% CI: 0.012-0.320) versus other IL-10 haplotypes (P50.03), whereas overall survival was not influenced by IL-10 haplotype polymorphisms. In multivariate analysis, the presence of the IL-10 GCC haplotype was found as the only variable associated with a statistically significant decreased hazard of severe aGVHD development (P50.02, HR50.21, 95% CI: 0.05-0.78). These results suggest that pediatric patients possessing the IL-10 GCC haplotype may be protected from the occurrence of severe aGVHD in the setting of matched sibling HSCT.
Synthetic modified mRNA molecules encoding pluripotency transcription factors have been used successfully in reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs). We have applied this method on bone marrow-derived mesenchymal stromal cells (BM-MSCs) obtained from a patient with β-thalassemia (β-thal) with the aim to generate trangene-free β-thal-iPSCs. Transfection of 10(4) BM-MSCs by lipofection with mRNA encoding the reprogramming factors Oct4, Klf4, Sox2, cMyc, and Lin28 resulted in formation of five iPSC colonies, from which three were picked up and expanded in β-thal-iPSC lines. After 10 serial passages in vitro, β-thal-iPSCs maintain genetic stability as shown by array comparative genomic hybridization (aCGH) and are capable of forming embryoid bodies in vitro and teratomas in vivo. Their gene expression profile compared to human embryonic stem cells (ESCs) and BM-MSCs seems to be similar to that of ESCs, whereas it differs from the profile of the parental BM-MSCs. Differentiation cultures toward a hematopoietic lineage showed the generation of CD34(+) progenitors up to 10%, but with a decreased hematopoietic colony-forming capability. In conclusion, we report herein the generation of transgene-free β-thal-iPSCs that could be widely used for disease modeling and gene therapy applications. Moreover, it was demonstrated that the mRNA-based reprogramming method, used mainly in fibroblasts, is also suitable for reprogramming of human BM-MSCs.
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