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H.L. Winton, L.J. Keen, J.L. Bidwell, W.J. Armitage; A Protective Tumour Necrosis Factor-Alpha Extended Haplotype in Corneal Transplantation . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4669.
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Purpose: Our study aims to investigate the role of the pro-inflammatory cytokine, tumour necrosis factor-alpha (TNF-α) gene promoter and microsatellite polymorphisms in corneal transplantation. TNF-α plays a critical role in promoting immunoinflammatory responses in the eye and previous studies have indicated direct associations between polymorphisms in gene promoter sequences and levels of mRNA or protein production. Methods: Genomic DNA extracted from peripheral blood of 315 HLA matched corneal transplant recipients, was used and divided into 2 groups. Corneal transplant recipients that had not suffered any rejection episodes were classified as "non-rejectors", whilst patients that had suffered rejection episodes were termed "rejectors". Previously identified cytokine single nucleotide polymorphisms (SNPs) associated with regulation of gene transcription were investigated: TNF-α -238, -308, -857, -863 and -1031. The upstream polymorphism +489 was also screened. PCR-induced heteroduplex generator (PCR-IHG) analysis was used to detect SNPs. Microsatellites, TNFa and TNFd, were examined by one-step PCR. Subsequent visualisation of mobility shifts was carried out using non-denaturing polyacrylamide gel electrophoresis (PAGE). Results were analysed by Arlequin software for population genetics using maximum-likelihood haplotype frequencies and Yates corrected Chi square analysis. Results: The total number of possible haplotypes was 34 for the non-rejector group and 26 for the rejectors. The extended haplotype –1031/-863/-857/-308/-238/+489 TCTGGA was found to be more prevalent in the group of non-rejectors (Chi square 7.69, p=0.006). Other frequent haplotypes CACGGG, TCCAGG, CCCGGG and TCCGGG were not found to be significantly different. Conclusion: This preliminary data suggests that the multi-locus haplotype TCTGGA may enhance graft survival. Genotyping corneal transplant recipients with respect to their TNF-α polymorphisms may help determine the risk of corneal allograft rejection and provide a guide for individualised immunosuppression for future transplant patients.
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