DNA base bulge vs unmatched end formation in probe-based diagnostic insertion/deletion genotyping: Genotyping the UGT1A1 (TA)(n) polymorphism by real-time fluorescence PCR

Abstract

Background: Gilbert syndrome is a clinically inconsequential entity of mild unconjugated hyperbilirubinemia caused by an A(TA)(n)TAA insertion polymorphism (UGTIA1*28) in the promoter region of the gene coding for the enzyme UDP-glucuronosyltransferase 1 (EC 2.4.1.17; UGT1A1). Present methods for genotyping this polymorphism are laborious. Methods: Hybridization probes were designed complementary to the wild type (TA)6 and to alleles with (TA)7 and (TA)8 repeats in the promoter region. Melting points were measured in samples representing all currently known alleles with (TA)5 to (TA)8 repeats. Probe melting points were predicted with a thermodynamic nearest-neighbor model for Watson-Crick paired probes. The dominant secondary structures resulting from probe hybridization were predicted by thermodynamic free energy calculations. Alternatively samples were genotyped based on amplicon size resolved by high-resolution polyacrylamide gel electrophoresis. Results: Only short probes (22-24 bases) could be successfully used for genotyping this locus because of the very low stability of this TA repeat. Assays based on (TA)7 or (TA)8 genotype-compatible hybridization probes effectively discriminated five to eight TA repeats. The consecutive use of two different detection probes was necessary for better discrimination of some heterozygous genotypes. All results were in concordance with the alternative genotyping method. Of 100 investigated Caucasians (50 males, 50 females), 9 (9%) were homozygous for the (TA)7 allele. Conclusions: The presented method for genotyping the (TA)(n) promoter polymorphism of the UGT1A1 gene with the LightCycler has the potential to genotype all currently known (TA)(n) repeats in a single assay and is sensitive toward possible new genotypes. Our findings also show that thermodynamic calculations are of practical value for the design of hybridization probe assays for the genotyping of insertion/deletion polymorphisms. (C) 2000 American Association for Clinical Chemistry.