Phenylketonuria Diagnosis by Massive Parallel Sequencing and Genotype-Phenotype Association in Brazilian Patients.

Rafael Hencke Tresbach,Romina Soledad Heredia,Ida Vanessa Doederlein Schwartz,Monique Oliveira Poubel,Fernanda Sperb-Ludwig,Tássia Tonon,Rodrigo Ligabue-Braun,Bárbara Cátia Martins,Maria Teresa Alves Da Silva Rosa,Maria Teresinha De Oliveira Cardoso,Luiz Carlos Santana Da Silva,François Maillot

doi:10.3390/genes12010020

Abstract

Phenylketonuria (PKU) is a common inborn error of amino acid metabolism in which the enzyme phenylalanine hydroxylase, which converts phenylalanine to tyrosine, is functionally impaired due to pathogenic variants in the PAH gene. Thirty-four Brazilian patients with a biochemical diagnosis of PKU, from 33 unrelated families, were analyzed through next-generation sequencing in the Ion Torrent PGM™ platform. Phenotype–genotype correlations were made based on the BioPKU database. Three patients required additional Sanger sequencing analyses. Twenty-six different pathogenic variants were identified. The most frequent variants were c.1315+1G>A (n = 8/66), c.473G>A (n = 6/66), and c.1162G>A (n = 6/66). One novel variant, c.524C>G (p.Pro175Arg), was found in one allele and was predicted as likely pathogenic by the American College of Medical Genetics and Genomics (ACMG) criteria. The molecular modeling of p.Pro175Arg indicated that this substitution can affect monomers binding in the PAH tetramer, which could lead to a change in the stability and activity of this enzyme. Next-generation sequencing was a fast and effective method for diagnosing PKU and is useful for patient phenotype prediction and genetic counseling.

Highlights

Phenylketonuria (PKU, OMIM #261600) is an autosomal recessive inborn error of metabolism in which the conversion of phenylalanine (Phe) to tyrosine by the phenylalanine hydroxylase (EC 1.14.16.1) is defective, resulting in partial or total inactivity of the conversion due to biallelic variants in the PAH gene [1]
For the patients from RS, a BH4 deficiency was previously excluded by the measurement of 6,7-dihydropteridine reductase (DHPR) activity in the blood or dried blood spots (DBS) and of biopterins and neopterins in urine or DBS
Genotyping is a helpful way to understand how phenylalanine hydroxylase is altered in a patient, the impact of this specific alteration to the enzyme, and the enzyme’s level of residual activity with these variants

Summary

Introduction

Phenylketonuria (PKU, OMIM #261600) is an autosomal recessive inborn error of metabolism in which the conversion of phenylalanine (Phe) to tyrosine by the phenylalanine hydroxylase (EC 1.14.16.1) is defective, resulting in partial or total inactivity of the conversion due to biallelic variants in the PAH gene [1]. The treatment for PKU consists of Phe-free dietary management and supplementation with the Phe-free metabolic formula [3,4]. In Brazil, the public health system neonatal screening program performs biochemical screening for PKU by the detection of Phe in dried blood spots (DBS). If the results are abnormal, an additional blood sample is requested to confirm the diagnosis and begin treatment. The confirmatory test includes the measurement of blood Phe and tyrosine concentrations [6]

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Conclusion