Three-dimensional polyacrylamide gel-based DNA microarray method effectively identifies UDP-glucuronosyltransferase 1A1 gene polymorphisms for the correct diagnosis of Gilbert's syndrome

Abstract

Gilbert's syndrome is a mild genetic liver disorder characterized by unconjugated hyperbilirubinemia due to defects in the UDP-glucuronosyltransferase 1A1 (UGT1A1) gene. The T-3279G mutation in the phenobarbital responsive enhancer module (PBREM), the TA-insertion in the TATA box, creating the A(TA)7TAA motif instead of A(TA)6TAA and the G211A mutation in coding exon 1, particularly in Asian populations, of the human UGT1A1 gene are the three common genotypes found in patients with Gilbert's syndrome. Different approaches for detecting the T-3279G, A(TA)6/7TAA and G211A mutations of the UGT1A1 gene have been described. In this study, to the best of our knowledge, we established a three-dimensional polyacrylamide gel-based DNA microarray method for the first time, in order to study UGT1A1 gene polymorphisms. This method, based on a step-by-step three-dimensional polyacrylamide gel-based DNA microarray protocol, successfully identified all possible genotypes of T-3279G, A(TA)6/7TAA and G211A in 20 patients with hyperbilirubinemia. In addition, sequencing was performed to confirm these results. The data from the current study demonstrate that the three-dimensional polyacrylamide gel microarray method has the potential to be applied as a useful, reliable and cost-effective tool to detect the T-3279G, the A(TA)6/7TAA and the G211A mutations of the UGT1A1 gene in patients with hyperbilirubinemia and thereby aid in the diagnosis of Gilbert's syndrome.

Haplotype-Contained PCR Products Analysis by Sequencing with Selective Restriction of Primer Extension

“…Haplotype-based association studies have been proposed as a powerful approach to disease gene mapping, based on linkage disequilibrium (LD) between causal mutations and the ancestral haplotypes by which they arose. For example, a number of mutations in the regulatory and coding regions of the UGT1A1 gene have been detected to confirm the diagnosis of Gilbert's syndrome [ 11 , 22 – 24 ]. Haplotyping is usually conducted by indirect computational or direct experimental analysis.…”

“…In this study, K1637K and S1647T related to Parkinson's disease [ 10 ] and UGT1A1∗ 6 and UGT1A1∗ 28 related to Gilbert's syndrome [ 11 ] were selected as experimental sites. The former two sites are located 27 bp apart in exon 34 of the leucine-rich repeat kinase 2 (LRRK2) gene, and the latter are 268 bp apart in exon 1 of the UDP-glucuronosyltransferase 1A1 (UGT1A1) gene.…”

Macroporous Polymer Monoliths in Thin Layer Format

“…The second group is based on three-dimensional matrices and includes polyacrylamide or polysaccharide gels, nitrocellulose membranes, etc. [101][102][103]. Threedimensional supports are usually formed on a rigid inert surface, for example, glass or plastic layers [102,103].…”

Molecular probes for identification of UDP-glucuronosyltransferase 1 gene polymorphisms for Gilbert's syndrome diagnosis

“…Previously, we applied three dimensional acrylamide gel method to detect UGT1A1 gene polymorphism [14]. However, preparation of the acrylamide-modified slides is timeconsuming.…”