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.