Detection of the jaundice-related G71R mutation in the UGT1A1 gene by denaturing high performance liquid chromatography (DHPLC)

Background The G71R mutation in the UGT1A1 gene has been associated with neonatal jaundice and other cases of hereditary, unconjugated hyperbilirubinemia in several Asian populations. Currently, DNA sequencing is the only method available to identify the mutation, which can be time- and labor-intensive, particularly for such projects as population-based genetic studies. A relatively new method, denaturing high performance liquid chromatography (DHPLC), is increasingly used to detect various mutations. Objective The aim of the present study was to investigate the ability of DHPLC to detect the G71R mutation, in comparison with the gold standard of sequencing analysis. Methods Seventy-two infants were enrolled. Following genomic DNA extraction, exon 1 of the UGT1A1 gene was amplified by polymerase chain reaction (PCR). Afterwards, the G71R mutation was simultaneously, and blindly, determined in all subjects by DHPLC and sequence analysis. The performance of the DHPLC analysis, compared to the sequence analysis, was assessed in terms of sensitivity and specificity. Results DHPLC detected the G71 R mutation in 31 individuals. Of these, 26 were heterozygous and 5 were homozygous for the mutation. This method did not find the mutation in 41 other individuals. Sequence analysis produced identical results for all individuals. Conclusion DHPLC analysis is capable of detecting the G71R mutation in the UGT1A1 with a degree of sensitivity and specificity (100% each) that is comparable to sequencing analysis