MOLECULAR ANALYSIS OF PHENYLALANINE HYDROXYLASE GENE IN SOUTH ITALY PATIENTS AFFECTED BY PHENYLKETONURIA

Abstract

Hyperphenylalaninemias (HPAs), the most common inborn error of metabolism (1:10.000), is recessively inherited and caused by mutations in the gene encoding phenylalanine hydroxylase (PAH). PAH converts L- Phenylalanine into L-Tyrosine. PAH block results in accumulation of L-Phe and toxic metabolites. HPAs is classified according Phe serum levels as: 1.Classical PKU 2.Mild PKU 3.Mild HPA. PAH gene, located on chromosome 12, is composed of 171 Kb and 13 exons. To date more than 600 causative mutations for PKU are described in the entire PAH gene: 62% are missense mutations; 14% deletions; 11% splice site mutations; 6% nonsense mutations; 2% insertions. PAH, hepatic monooxygenase of 452 amino acids that catalyzes conversion of L-Phe to L-Tyr using 6R-l-erythro- 5,6,7,8-tetrahydrobiopterin (BH4) and molecular oxygen. PAH consists of three structural domains: 1.N-term regulatory domain 2.Large catalytic domain 3.C-term tetramerization domain. HPA is also the first metabolic disorder in which treatment (a Phe-restricted diet) was found to prevent clinical symptoms. The aim of my thesis was to perform molecular analysis of the PAH gene in 390 PKU patients and 373 carriers from the Campania Region Screening Center. The analysis of PAH mutations allowed to define the molecular epidemiology of PAH gene in Campania and compare the obtained data with those from other regions of Italy. The analysis of the PAH gene have been performed through the extraction of genomic DNA from peripheral blood, followed by amplification and direct sequencing of the entire gene. In the last phase of the study, we optimized and validated a next generation sequencing protocol for the analysis of the entire PAH gene sequence, including introns and regulatory regions to identify PKU causative mutations in patients negative. In conclusion, our study, has allowed us to define: - molecular diagnosis of HPA patients achieving a detection rate of 98.8%, - epidemiology of mutations in PAH gene of patients from Campania, - the residual risk to be carriers, and prenatal diagnosis in PKU family, - the responsiveness of BH4 in PKU patients, - good correlation between mutations and phenotypes of the disease. These data may help to reconstruct the historical and anthropological point of view, the various settlements that have occurred in our country.