Development of a high resolution melting method for genotyping of risk HLA-DQA1 and PLA2R1 alleles and ethnic distribution of these risk alleles |
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Authors: | Guanglin Cui Lina Zhang Yujun Xu Katherine Cianflone Hu Ding Dao Wen Wang |
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Institution: | 1. Institute of Hypertension and Department of Internal Medicine, and Genetic Diagnosis Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China;2. Centre de Recherche Institut Universitaire de Cardiologie and Pneumologie de Québec, Université Laval, Québec, Canada |
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Abstract: | Recent studies have demonstrated that alleles at single nucleotide polymorphisms (SNPs) rs2187668 and rs4664308 within genes HLA-DQA1 and PLA2R1, respectively, had a significant impact on the susceptibility to idiopathic membranous nephropathy (IMN). Analysis of the two genomic loci could identify alleles for individuals at risk for IMN. Conventional methods for genotyping are labor intensive, expensive or time consuming. High resolution melting (HRM) is a new technique for genotyping and has the advantages of simplicity, speed, high sensitivity and low cost. Here, we describe genotyping of SNPs rs2187668 and rs4664308 using HRM. In this study, we identified polymorphisms of rs2187668 and rs4664308 in 480 healthy unrelated Chinese volunteers of two ethnic groups from three different geographical areas in China. The two genomic loci were genotyped by HRM using a saturating fluorescent dye SYTO® 9 on 7900 HT and RG 6000 instruments, and were further confirmed by direct DNA sequencing. Three different SNP genotypes were sufficiently distinguished by HRM with mean sensitivity of 98.8% and mean error rate of 1.9%. In addition, the allele frequencies varied greatly based on ethnic or geographic origins. In conclusion, HRM is a rapid, cost efficient, sensitive, suitable technique for genotyping, and simple enough to be readily implemented in a diagnostic laboratory. We believe this will be a valuable technique for determining the genotype of rs2187668 and rs4664308 and for assessing individual susceptibility to IMN. |
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Keywords: | MN membranous nephropathy IMN idiopathic membranous nephropathy HLA-DQA1 major histocompatibility complex class II DQ alpha 1 PLA2R1 phospholipase A2 receptor 1 SNP single nucleotide polymorphism RFLP restriction fragment length polymorphism DHLC denaturing high-performance liquid chromatography HRM high resolution melting Tm melting temperature |
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