Rapid Analysis of Colipase Gene Variants by Multicapillary Electrophoresis.

Abstract

Despite of the fact that the Human Genome Project was completed more than a decade ago, identification of the genetic background of polygenic diseases is still challenging. Several somewhat different approaches are available to investigate inheritable factors of complex phenotypes, all require, however efficient, high-throughput techniques for SNP genotyping. In this paper we report a robust and reliable multiplex PCR-RFLP for genotype and haplotype analysis of 6 SNPs (rs41270082, rs3748051, rs142027015, rs3748048, rs73404011 and rs72925892) of the CLPS gene. A multicapillary (12 capillaries) electrophoresis unit was used for high throughput and sensitive analysis of the digestion fragments. A Microsoft Excel-based spreadsheet was designed for the flexible visualization and evaluation of the electrophoretic separations, which is readily adaptable for any kind of electrophoresis application. Haplotype analysis of the two loci localized in close proximity of each other was carried out by molecular method, extended haplotypes including all 5 SNPs in the 5' upstream region were calculated. The techniques were applied in a case-control association study of type 2 diabetes mellitus. Although, single marker analysis did not reveal any significant association, it was observed that the rare GGCCG haplotype of the five 5' upstream region SNPs was about three times more frequent among patients compared to healthy control population. Our results demonstrated the applicability of multicapillary CGE in large-scale, high-throughput SNP analysis and suggested that the CLPS gene polymorphisms might be considered as genetic risk factor for type 2 diabetes mellitus.