The response to tetrahydrobiopterin (BH4) in patients with phenylalanine hydroxylase (PAH, EC 1.14.16.1) deficiency (OMIM 261600) has been widely reported. Here we report results of the BH4 loading test (20 mg/kg per day) in a group of 36 patients with PAH deficiency and phenotype of mild hyperphenylalaninaemia (HPA), mild phenylketonuria (PKU) or classic PKU. The patients ranged from neonates aged 7-15 days, detected in the Newborn Screening Programme for PKU in the population of Galicia (NW Spain), to adults aged up to 32 years who had been receiving a low-phenylalanine (Phe) diet for a period of years. Ten of the 36 patients showed a reduction of more than 30% in plasma Phe levels within 24 h of BH4 loading (ranging from 33.7% to 90.2%, mean 59.2%, SD 19.8%). All the patients with mild HPA (100%) showed a positive response; 57% of patients with mild PKU (4 of 7) showed a positive response. Of particular interest were positive responses in two patients with classic PKU, and in one patient with mutations of the phenylalanine hydroxylase (PAH) gene that have not to date been reported to be BH4-responsive (p.S303A and p.G46S). BH4 treatment (5-8 mg/kg per day) was commenced in 9 of the 10 BH4-responsive patients. The observed responses to treatment argue for application of the BH4 loading test in all patients with HPA or PKU, independently of genotype, phenotype or age.

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Communicated by Jürgen Horston a different RFLP and STR haplotype background than in Spanish non-Gypsies. PATIENTS AND METHODSIn this study we have examined eight Gypsy PKU families from different communities in Spain. Two of the families are related. Consanguinity among parents has not been proved. Overall, 11 affected individuals, among them three pairs of siblings, were studied. One of the patients, now 3 months old, had 13 mg Phe/dl at diagnosis; the rest of the patients had Phe values of >30 mg/dl. In most cases, patients do not follow treatment adequately, and the clinical outcome varies. In two treated patients, Phe tolerance was 350-370 mg/day.A total of 23 non-Gypsy unrelated PKU patients from different regions of Spain were also included in the study of the STR alleles. All carry mutation IVS10nt546: 8 in homozygous fashion and 15 in heterozygous fashion. The phenotypic classification and the haplotype analysis of the patients were previously described (Martínez-Pardo et al., 1994;.PCR amplification was performed using dried blood spots as the source of DNA, as described previously . The IVSl0nt546 mutation was detected by digestion of amplified exon 11 with DdeI. The R252W mutation was detected by digestion of amplified exon 7 with AvaI.RFLP in the PAH gene were determined by Southern blotting and hybridization with a PAH cDNA probe (for the EcoRI and EcoRV polymorphisms), PCR and restriction enzyme digestion (for BglII, PvuII(a), PvuII(b), MspI, and XmnI polymorphisms), and PCR and electrophoretic examination of the VNTR alleles in the 3´ end of the PAH gene (Goltsov