Analysis of the PAH gene was performed by direct sequencing of the patients' genomic DNA, MLPA analysis and real-time PCR.
Among 22 independent alleles thirteen previously described mutations were detected (detection rate 100 % ), all in compound heterozygosity: p.Arg395Gly (18.2 % ), c.168 + 5 G > C (13.6 % ), p.EX3del (9 % ), c.1066-11 G > A (9 % ), p.Ala403Val (9 % ), p.Glu178Gly (9 % ), p.Ser70Pro (4.5 % ), p.Arg241His (4.5 % ), p.Phe55fs (4.5 % ), p.Arg158Gln (4.5 % ), p.Asp222Gly (4.5 % ), p.Ala300Ser (4.5 % ), p.Pro225Thr (4.5 % ). Of the ten different genotypes, three have been previously reported to be associated with a mild clinical phenotype and to respond to tetrahydrobiopterin (BH4) administration.
Marked genetic heterogeneity was found in Cypriot patients with hyperphenylalaninemia with two mutations accounting for 32 % of the alleles. Most of the mutations detected have been found in other European and Mediterranean populations.