Background: Phenylketonuria is a common inborn defect of amino acid metabolism in the world. This failure is caused by an autosomal recessive insufficiency of the hepatic enzyme hyperphenylalaninemia (PAH), which catalyzes the irreversible hydroxylation of phenylalanine to tyrosine. More than 1,040 different disease-causing mutations have already been identified in the PAH gene. The most prominent complication of Phenylketonuria, if not diagnosed and treated, is severe mental retardation. Hence, early diagnosis and initiation of nutritional therapy are the most significant measures in preventing this mental disorder. Given these data, we developed a simple and rapid molecular test to detect the most frequent PAH mutations.
Methods: Multiplex assay was developed based on the SNaPshot minisequencing approach to simultaneously perform genotyping of the 10 mutations at the PAH gene. We optimized detection of these mutations in one multiplex PCR, followed by 10 single-nucleotide extension reactions. DNA sequencing assay was also used to verify genotyping results obtained by SNaPshot minisequencing.
Result: All 10 genotypes were determined based on the position and the fluorescent color of the peaks in a single electropherogram. Sequencing results of these frequent mutations showed that by using this method, a 100% detection rate could be achieved in the Iranian population.
Conclusion: SNaPshot minisequencing can be useful as a secondary test in neonatal screening for HPA in neonates with a positive screening test, and it is also suitable for carrier screening. The assay can be easily applied for accurate and time- and cost-efficient genotyping of the selected SNPs in various population.
Keywords: Hperphenylalaninemia; Multiplex polymerase chain reaction; Phenylketonurias; Polymorphism.