Multiplex Snapshot minisequencing for detection of common PAH gene mutations in Iranian patients with Phenylketonuria.

Abstract

Phenylketonuria (PKU) is one of the most common inborn faults of amino acid metabolism in the world. It is caused by an autosomal recessive insufficiency of the hepatic enzyme phenylalanine hydroxylase (PAH) that catalysis the irreversible hydroxylation of phenylalanine to tyrosine. More than 950 different disease-causing mutations in the PAH gene have been identified. The most prominent complication of PKU if not diagnosed and treated is severe mental retardation. Early diagnosis and initiation of nutritional therapy are the most important measures in preventing this mental retardation. For this reason, we developed a simple and rapid molecular test for detection of the most frequent phenylalanine hydroxylase mutations. We have developed a PCR multiplex SNaPshot reaction that targets all 10 common mutations identified in PAH gene. Multiplex assays based on the SNaPshot minisequencing approach were developed to simultaneous genotype ten mutations at the PAH gene. We have optimized the detection of this mutations in one multiplex polymerase chain reaction (PCR) followed by ten single-nucleotide extension reactions. The different genotypes were determined based on the position and the fluorescent color of the peaks in a single electropherogram. DNA sequencing assay also were used to verify genotyping results obtained by SNaPshot minisequencing, all mutations were concordant. This method can be useful as secondary test in neonatal screening for hyperphenylalaninemia in neonates with a positive screening test and also be suitable for carrier screening. The assay can be easily applied for accurate, time and cost efficient genotyping of the selected SNPs in various populations.