Rapid detection of the UGT1A1 single nucleotide polymorphism G211A using real‐time PCR with Taqman minor groove binder probes

Abstract

The UGT1A1 Taqman MGB probe single nucleotide polymorphism (SNP) genotyping assay was developed to detect nucleotide 211 of the UDP-glucoronocyltransferase 1A1 (UGT1A1) gene. Defects in this enzyme interfere with process of conjugation of bilirubin and cause unconjugated hyperbilirubinemia. Variation at nucleotide 211 in the coding region of the UGT1A1 gene has been shown to be prevalent in Japanese and Chinese. Using an ABI sequence detection system (SDS) 7000, an allele-specific real-time PCR-based genotyping method was established to detect nucleotide G211A. Cord blood from 125 infants without hyperbilirubinemia (controls) were compared with cord blood from 74 infants (cases) with severe hyperbilirubinemia (total serum bilirubin > 300 micromol/L). Homozygous variation of the UGT1A1 gene at nucleotide 211(A/A) is significantly more common in cases (14.9%) than in controls (0.8%) (P<0.001). Direct sequencing from 20 randomly selected samples showed eight samples with homozygous wild type, seven with homozygous variant, and five samples were heterozygous. The result from this assay was in complete concordance with the DNA sequencing result and clearly discriminate wild-type (G/G), homozygous variant (A/A), and heterozygous (G/A). This assay is rapid and robust for screening of SNP G211A to determine if this polymorphism plays a role in causing severe neonatal jaundice in the local context.

Neonatal hyperbilirubinemia and Gly71Arg mutation of UGT1A1 gene: a Chinese case–control study followed by systematic review of existing evidence

“…Eighteen case–control studies from six countries, including our study, with 1214 hyperbilirubinemic case subjects and 2131 control subjects were included in the meta‐analysis on the UGT1A1 211G>A mutation and neonatal hyperbilirubinemia (4,6–11,13–22) (Table 2).…”

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

“…Participants and DNA isolation 15 Chinese patients with hyperbilirubinemia were recruited at the Second Affiliated Hospital of Southeast University, Nanjing, P. R. China. All patients had total bilirubin levels (TBIL) of ≥ 220.6 umol/L, and no signs or symptoms indicative of other hepatobiliary diseases.…”

“…Fluorescent labeled probes, acrylamide-modified primers and the use of Luxscan-10K/A (CapitalBio Company, China) are expensive. Other method, such as direct sequencing method, TaqMan MGB SNP genotyping assay, DNA melting curve analysis, Restriction Fragment Length Polymorphism (RFLP) method have been used to detect mutations of UGT1A1 gene for Gilbert's Syndrome diagnosis [15][16][17]. In this study, we applied dot blot hybridization assay with molecular probes for the first time to detect the T-3279G, A(TA)6/7TAA and G211A mutation of UGT1A1 gene.…”

Restriction fragment length polymorphism effectively identifies exon 1 mutation of UGT1A1 gene in patients with Gilbert's Syndrome

“…Direct sequencing method and TaqMan MGB SNP genotyping assay have been used to identify nucleotide 211G>A mutation in the coding exon 1 of UGT1A1 gene (23)(24)(25)(26)(27)(28). Recently, melting curve analysis for the detection of nucleotide 211G>A mutation has also been described (29).…”