Molecular analysis and genotype-phenotype correlation in patients affected by hyperphenylalaninemias in southern italy

Abstract

The hyperphenylalaninemia (HPA), an autosomal recessive genetic disorder, is the result of a defect of enzyme phenylalanine hydroxylase (PAH), resulting in the accumulation of phenylalanine (Phe) in the blood. In most cases (about 98%), the phenotypic expression of disease is the result of the presence of mutations in the PAH gene, coding for the enzyme PAH, with 12q24.1 locus on chromosome 12. The disease has a frequency in the Caucasian population of about 1 in 10,000 live births, corresponding to a carrier frequency of about 1 to 50; the majority of affected individuals are identified in the neonatal period by screening programs (mandatory in several countries, such as Italy) on the general population. HPA is expressed with a significant variability of phenotypic expression and with different degrees of severity. Food therapy, based on a limited intake of phenylalanine, prevents neurological damage. Phenotypes of hyperphenylalaninemia are defined based on the levels of phenylalanine in the blood at birth: classical phenylketonuria (PKU), with values of Phe in the plasma >1200 μmol/L (>20 mg/dl); mild form of PKU with phenylalaninemia levels comprised between 600 and 1200 μmol/L (10-20 mg/dl); Non-PKU hyperphenylalaninemia (HPA-non PKU or MHP) when the values of phenylalaninemia are below 600 μmol/L (<10 mg/dl). To date, through the molecular analysis of PAH gene, about 600 mutations have been identified (the majority are missense) and over 50% of patients are composite heterozygous. The considerable number of identified mutations is a substantial evidence of allelic heterogeneity of the underlying pathology that justifies the variable expressivity (even within the same family). The project aims first to increase the knowledge about the molecular and biochemical mechanisms of the disease, still not fully known, which are the basis of the etiology and variable expressivity of HPA, relying on the analysis of gene mutations responsible of the disease in patients from Southern Italy. Genotyping of patients with elevated Phe levels detected in newborn screening is often performed to complete diagnosis. From these data, we also correlated genotypes with predicted residual activities (PRA) from in vitro expression experiments tabulated in PAHdb (http://www.pahdb.mcgill.ca/), performing a genotype–phenotype correlation. The molecular bases of PKU and their implications at the metabolic level with focus on BH4 responsiveness were addressed in the final section of thesis. The quantification of PAH activity expressed in cultured cells was performed by a tandem mass spectrometry assay at the section of Dietmar-Hopp-Metabolic Center of Universitatsklinikum of Heidelberg (Germany). Mass spectrometry allows the use of stable isotopes for Phe and Tyr quantification and PAH activity measurement of PKU mutations. In summary, the data obtained in this study on the frequency and distribution of mutations in the PAH gene reinforce the idea of considerable heterogeneity of mutations in patients HPA, with particular reference to Southern Italy. This work has also led to the conclusion that the genotype is the main determinant of the biochemical phenotype in most patients with PAH deficiency and has greater value in estimation of BH4-responsiveness. In addition, calculating the residual PAH activity from the information obtained from our in vitro experiments and those available in the database may be useful for predicting and/or exclusion of potential candidates for BH4 therapy. The results presented herein provide then a clarification on PKU genotypes, on phenotypes, and response to BH4 as a reference available for clinicians, health care professionals and researchers for diagnosis and establishment of tailored treatment of patients. A significant number of PKU patients is likely to benefit from BH4 treatment which, combined with a less strict diet, or in some cases as monotherapy, may reduce nutritional deficiencies and neurological and psychological dysfunctions, contributing to a better quality of life of these patients.