ABSTRACT
Background: Phenylalanine hydroxylase [PAH] gene is the well-known causative gene for classic Phenylketonuria [PKU] [OMIM#261600] disease, with more than 500 reported mutations. Through this study, a novel mutation in the PAH gene in an Iranian pedigree with phenylketonuria was introduced
Methods: A consanguineous family with a 10-year old affected girl was referred for genetic analysis. Mutation screening of all exons and exon-intron boundaries was performed by Sanger sequencing, and mini haplotype analysis was carried out by genotyping of Short Tandem Repeat [STR] and Variable Number Tandem Repeat [VNTR] alleles
Results: Mutation analysis revealed a novel homozygous insertion of a single adenine nucleotide at position 335 in exon 3 of the PAH gene. Based on the American College of Medical Genetics and Genomics [ACMG] guidelines, the change is interpreted as a pathogenic mutation which produces a premature termination signal [TAA] at codon 113 according to in silico assessments. The mini haplotype analysis showed that this mutation was linked to STR [15] -VNTR [3]
Conclusion: In this study, a novel mutation was reported in a patient who had PKU symptoms without any previously reported mutations in the PAH gene [NM_000277.1: p.Asp112Glufs*2] that can be responsible for the classical PKU phenotype in the Iranian population. Detection of novel mutations indicates notable allelic heterogeneity of the PAH locus among this population
ABSTRACT
Objective: The phenylalanine hydroxylase [PAH] locus has high linkage disequilibrium. Haplotypes related to this locus may thus be considered sufficiently informative for genetic diagnosis and carrier screening using multi-allelic markers. In this study, we present an efficient method for haplotype analysis of PAH locus using multiplexing dyes. In addition, we explain how to resolve the dye shift challenge in multiplex short tandem repeat [STR] genotyping
Materials and Methods: One hundred family trios were included in this descriptive study. The forward primer of a tetra-nucleotide STR and the reverse primer of a variable number tandem repeat [VNTR] were labeled with three different non-overlapping dyes 5-carboxyfluorescein [FAM], 6-carboxy-N,N,N',N'-tetramethylrhodamine [HEX] and 6-carboxy-N,N,N',N'-tetramethylrhodamine [TAMRA]. The polymerase chain reaction [PCR] products from each family trio were multiplexed for capillary electrophoresis and results were analyzed using Peak Scanner software
Results: Multiplexing trio products decreased the cost significantly. The TAMRA labeled products had a significant predictable shift [migrated at a slower electrophoretic rate] relative to the HEX and FAM labeled products. Through our methodology we achieve, the less inter-dye shift than intra-dye shift variance. Correcting the dye shift in the labeled products, according to the reference allele size, significantly decreased the inter-dye variability [P<0.001]
Conclusion: Multiplexing trio products helps to detect and resolve the dye shift accurately in each family, which otherwise would result in diagnostic error. The dye system of FAM, HEX and TAMRA is more feasible and cheaper than other dye systems