Communicated by Jürgen Horst
on a different RFLP and STR haplotype background than in Spanish non-Gypsies.
PATIENTS AND METHODS
In 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; Desviat et al., 1993).
PCR amplification was performed using dried blood spots as the source of DNA, as described previously (Pérez et al., 1993). 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