PAH gene mutations in the Sicilian population: association with minihaplotypes and expression analysis

The molecular basis of PAH deficiency in the Sicilian population is characterized by a marked heterogeneity, with 44 mutations at a single locus identified by a "gene-scanning" approach and accounting for a detection rate of 91%. The remaining 9% of PAH alleles does not bear mutations in any of the 13 exons and 24 exon/intron junctions. Three mutations IVS10nt-11 G > A, R261Q, and A300S accounted for 30.5%, whereas the remaining mutations were found at relative frequencies of less than 5% and 20 mutations were observed once only. Five mutations have been detected only in Sicilians so far. By studying the association of mutations with intragenic STR-VNTR haplotypes ("minihaplotypes"), "identity by descent" has been established for 24 mutations also detected in other populations. This finding supports the hypothesis of a multipolar origin for a large proportion of PAH mutant alleles currently detected in Sicilians. In order to improve our understanding of the clinical heterogeneity of PAH deficiency in this population, we have for the first time analyzed three missense mutations L41F, T92I, and P211T in vitro by the pCDNA3/COS-7 eukaryotic expression system and found an activity of 10, 76, and 72%, respectively, compared to normal PAH. In two HPA patients with mild PKU and mild hyperphenylalaninemia (MHP), harboring respectively L41F/R261Q and T92I/P281L genotypes, the predicted biochemical effect of these genotypes appeared to be consistent with the metabolic phenotypes. In contrast, discordant metabolic phenotypes (mild PKU and MHP) were observed in two unrelated patients bearing the same R261Q/P211T genotype, a finding which underscores the complex relationship linking genotype to phenotype in PAH deficiency. Hypotheses on the possible mechanisms responsible for the observed discordance are discussed. The spectrum of PAH gene mutations in Sicily reflects the complex demographic history of this island at the crossroad of prehistoric and historical migrations in the Mediterranean sea. The data presented in this study also add to the present knowledge on the relationship between PAH genotypes and HPA phenotype and are expected to improve PAH genotyping among individuals with hyperphenylalaninemia.