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 beenassociated with neonatal jaundice and other cases of hereditary,unconjugated hyperbilirubinemia in several Asian populations.Currently, DNA sequencing is the only method available toidentify 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 liquidchromatography (DHPLC), is increasingly used to detect variousmutations.Objective The aim of the present study was to investigate theability of DHPLC to detect the G71R mutation, in comparisonwith the gold standard of sequencing analysis.Methods Seventy-two infants were enrolled. Following genomicDNA extraction, exon 1 of the UGT1A1 gene was amplified bypolymerase chain reaction (PCR). Afterwards, the G71R mutationwas simultaneously, and blindly, determined in all subjects byDHPLC and sequence analysis. The performance of the DHPLCanalysis, compared to the sequence analysis, was assessed in termsof sensitivity and specificity.Results DHPLC detected the G71 R mutation in 31 individuals.Of these, 26 were heterozygous and 5 were homozygous for themutation. This method did not find the mutation in 41 otherindividuals. Sequence analysis produced identical results for allindividuals.Conclusion DHPLC analysis is capable of detecting the G71Rmutation in the UGT1A1 with a degree of sensitivity andspecificity (100% each) that is comparable to sequencing analysis.

Integrated Strategy for Fast and Automated Molecular Characterization of Genes Involved in Craniosynostosis

Background: Craniosynostosis, the premature fusion of 1 or more sutures of the skull, is a common congenital defect, with a prevalence of 1 in 2500 live births. Untreated progressive craniosynostosis leads to inhibition of brain growth and increased intracranial and intraorbital pressure. The heterogeneity of clinical phenotypes and the overlap of the various associated syndromes render the correct diagnosis of the different craniosynostoses particularly difficult. Methods: To identify 10 common mutations in the genes for fibroblast growth factor receptors 2 and 3 (FGFR2 and FGFR3), we developed a microelectronic microchip assay that exploited the PCR multiplexing format and coupled it with serial addressing and probe hybridization on the same pad. For the molecular characterization of patients who tested negative in the microchip screening, we also developed conditions for denaturing HPLC (DHPLC) analysis of the most mutated regions of FGFR2 and FGFR3 and the entire coding region of the TWIST1 gene. Results: In our cohort of 159 patients with various craniosynostosis syndromes, mutations were found in 100% of patients with Apert syndrome, 83.3% with Pfeiffer syndrome, 72.7% with Crouzon syndrome, 50.0% with Saethre-Chotzen syndrome, 27.7% with plagiocephaly, 31.8% with brachicephaly, 20% of complex cases, and 6.9% of mixed cases. No mutations were found in syndromic cases. Conclusions: The combined microchip-DHPLC strategy allows rapid and specific molecular diagnosis of craniosynostosis and is an effective tool for the medical and surgical management of these common congenital anomalies in a newborn or an infant with a developmental defect of the cranial vault.