Mutation Detection Of Phenylalanine Hydroxylase Gene In Phenylketonuria

ObjectionPhenylketonuria (PKU, OMIM 261600) is one of the routine neonatal screening programs. The majority of the patients are owing to mutations in the phenylalanine hydroxylase (PAH) gene, which caused the deficiency or loss of the activity of PAH, then phenylalanine could not be transformed into tyrosine and accumulates massively in body, finally resulting in the occurrence of the disease. In this study, we developed a new method for mutation detection of PAH gene and rapid prenatal diagnosis of PKU by using high-resolution melting (HRM), and further enrich the mutation spectrum of PAH gene in the Chinese population.Subjects and Methods1.SubjectsPeripheral blood samples were obtained from twenty-one PKU patients and their parents (patient 16 and patient 17 are monozygotic twins), amniotic fluid cell samples were collected from two families for prenatal diagnosis. Fifty normal control samples were confirmed by DNA sequencing. All the samples were collected from Guangzhou Women and Children's Medical Center.2. MethodsIn this study, mutation scanning of PAH gene was performed in twenty-one PKU patients by HRM, covering the thirteen exons and exon-intron boundaries. The HRM results were further confirmed by DNA sequencing. For family 15 and 16, prenatal diagnosis were performed by HRM and then confirmed by DNA sequencing.Results1. In this study, a total of twenty-nine different mutations were identified, including fifteen missense mutations, four nonsense mutations, threee splice mutations, two deletion mutations, three silent mutations, one single nucleotide polymorphism, and one frame-shift mutation (c.188₁96delCCCACATTG/c.197?insA). In addition, c.188₁96delCCCACATTG/c.197?insA,I94V, Q160X, H264R, G312V, and E305K are novel mutations.2. In the twenty-one patients, two pathogenic mutations were identified in each of the nineteen patients, three pathogenic mutations were identified in the remaining two patients. The fetus from family 15 was diagnosed as normal, the other fetus from family16 was diagnosed as a carrier.3. By comparing with DNA sequencing, there were neither false positive nor false negative results for HRM analysis.Therefore, in this study the sensitivity and specificity for mutation detection by HRM were both 100%.Conclusion1.This study suggested that HRM is a simple, accurate, rapid, high-throughput and cost-effective genetic analysis approach. It could be applied to mutation scanning of PAH gene, and especially useful for rapid prenatal diagnosis of classical PKU in parents with known mutations.2. This study enriched the mutation spectrum of PAH gene in the Chinese population.