Phenylketonuria in Portugal: Genotype-phenotype correlations using molecular, biochemical, and haplotypic analyses.

Filipa Ferreira,Esmeralda Martins,Anabela Bandeira,Ana Marcão,Francisco Silva,Hugo Rocha,Ana Gaspar,Teresa Campos,Raquel Neiva,Célia Carmona,António Amorim,Laura Vilarinho,Esmeralda Rodrigues,Paula Garcia,Luísa Diogo,Patrícia Janeiro,Ana Cristina Ferreira,Sónia Ramos,Luísa Rodrigues,Luı́sa Azevedo ,Homero Fonseca ,Cristina Maria Miranda De Sousa ,Sílvia Sequeira

doi:10.1002/mgg3.1559

Abstract

BackgroundThe impairment of the hepatic enzyme phenylalanine hydroxylase (PAH) causes elevation of phenylalanine levels in blood and other body fluids resulting in the most common inborn error of amino acid metabolism (phenylketonuria). Persistently high levels of phenylalanine lead to irreversible damage to the nervous system. Therefore, early diagnosis of the affected individuals is important, as it can prevent clinical manifestations of the disease.MethodsIn this report, the biochemical and genetic findings performed in 223 patients diagnosed through the Portuguese Neonatal Screening Program (PNSP) are presented.ResultsOverall, the results show that a high overlap exists between different types of variants and phenylalanine levels. Molecular analyses reveal a wide mutational spectrum in our population with a total of 56 previously reported variants, most of them found in compound heterozygosity (74% of the patients). Intragenic polymorphic markers were used to assess the haplotypic structure of mutated chromosomes for the most frequent variants found in homozygosity in our population (p.Ile65Thr, p.Arg158Gln, p.Leu249Phe, p.Arg261Gln, p.Val388Met, and c.1066‐11G>A).ConclusionOur data reveal high heterogeneity at the biochemical and molecular levels and are expected to provide a better understanding of the molecular basis of this disease and to provide clues to elucidate genotype–phenotype correlations.

Highlights

Phenylketonuria (PKU; OMIM #261600) is an autosomal recessive genetic disorder caused by the deficiency in the hepatic enzyme phenylalanine hydroxylase (PAH; OMIM #612349) (Scriver et al, 1995) that converts phenylalanine into tyrosine requiring the cofactor tetrahydrobiopterin (BH4)
Whenever the enzymatic activity of PAH (EC 1.14.16.1) is impaired, the essential amino acid phenylalanine cannot be hydroxylated into tyrosine, resulting in the elevation of phenylalanine, and its metabolic derivatives in blood and other body fluids
In the late 70 s, the Portuguese Neonatal Screening Program was established by the Ministry of Health, with phenylketonuria (PKU) and later, in 1981, with congenital hypothyroidism (CH) screening (Magalhães et al, 1986; Magalhães & Osório, 1984; Osório et al, 1992; Osório & Soares, 1987)

Summary

| INTRODUCTION

Phenylketonuria (PKU; OMIM #261600) is an autosomal recessive genetic disorder caused by the deficiency in the hepatic enzyme phenylalanine hydroxylase (PAH; OMIM #612349) (Scriver et al, 1995) that converts phenylalanine into tyrosine requiring the cofactor tetrahydrobiopterin (BH4). The human PAH gene covers approximately 100 kb of genomic DNA and encodes a protein of 452 amino acids, which are assembled in a functional homotetramer This gene consists of 13 exons and 12 introns, and has been mapped on chromosome 12, band region q23.2 (Donlon et al, 2014; Scriver et al, 1995). The frequency of the PKU disease and distribution of the PAH gene variants differs between populations. Primers for the 13 exons and exonic/intronic boundaries of the PAH gene were designed employing the NCBI Primer-BLAST tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) (see Table S1). These primers were tagged with a M13 sequence for the later cycle sequencing reaction. The observed variants were referred to the NCBI reference sequence for human PAH gDNA (NG_008690.2.)

| MATERIALS AND METHODS

Findings

| DISCUSSION

| CONCLUSION