Multicapillary gel electrophoresis based analysis of genetic variants in the WFS1 gene

Abstract

The WFS1 gene is one of the thoroughly investigated targets in diabetes research, variants of the gene were suggested to be the genetic components of the common forms (type 1 and type 2) of diabetes. Our project focused on the analysis of polymorphisms (rs4689388, rs148797429, rs4273545) localized in the WFS1 promoter region. Although submarine gel electrophoresis based approaches were also employed in the genetic tests, it was demonstrated that multicapillary electrophoresis offers a state of the art approach for reliable high-throughput SNP and VNTR analysis. Association studies were carried out in a case-control setup. Luciferase reporter assay was employed to test the effect of the investigated loci on the activity of gene expression in vitro. Significant association could be demonstrated between all three polymorphisms and type 2 diabetes in both allele- and genotype-wise settings even using Bonferroni correction. It is notable; however, that the three loci were in strong linkage disequilibrium, thus the observed associations cannot be considered as separate effects. Molecular analyses showed that the rs4273545 GT SNP played a role in the regulation of transcription in vitro. However, this effect took place only in the presence of the region including the rs148797429 site, although this latter locus did not have its own impact on the regulation of gene expression. The paper provides genotyping protocols readily applicable in any multiplex SNP and VNTR analyses, moreover confirms and extends previous results about the role of WFS1 polymorphisms in the genetic risk of diabetes mellitus.

High Throughput Multiplex SNP-analysis in Chronic Obstructive Pulmonary Disease and Lung Cancer

“…The aim of the present study was to clarify whether certain single nucleotide polymorphisms (SNPs) in genes encoding glycosyltransferases and glycosidases could be associated with elevated risk for chronic obstructive pulmonary disease (COPD) and lung adenocarcinoma. To this end, cohorts of COPD, lung cancer and co-morbid patients as well as healthy controls were genotyped using two different methodologies, a high-throughput, TaqMan probe based OpenArray platform approach [33] and primer extension combined with capillary electrophoresis [34].…”

Application of denaturing capillary electrophoresis for the detection of prognostic mutations in isocitrate dehydrogenase 1 and isocitrate dehydrogenase 2 genes in brain tumors

“…CGE has traditionally been an inherent part of molecular genetic testing protocols applied for DNA sequencing, DNA fragment analysis, and detection of DNA variations (mutations and polymorphisms) [16][17][18]. Over the past years, a number of publications have described a special experimental setting in which half-melted mutant homo-and heteroduplex forms are resolved by CE running at partial denaturing conditions [19][20][21].…”

Separation of single‐base sequential isomers of single‐stranded DNA by capillary electrophoresis and its application in the discrimination of single‐base DNA mutations

“…Past studies employing CE for DNA separation mainly focused on size‐based techniques using molecular‐sieving media such as gel or polymer matrixes . Recently, CE separation of sequential DNA isomers having single‐base substitutions and the same chain length has attracted the attention of researchers because it aids in the identification of gene mutations . However, DNA separation remains a highly challenging task because of a number of technical difficulties.…”