Hum Genet (1992) 89:341-342

�9 Springer-Verlag 1992

Mutation analysis of phenylketonuria in Spain: prevalence of two Mediterranean mutations

Bel~n P~rez, Lourdes R. Desviat, Mar Die, and Magdalena Ugarte

Centro de Biolog/a Molecular, Universidad Aut6noma de Madrid, E-28049 Cantoblanco (Madrid), Spain

Received September 18, 1991 / Revised December 30, 1991

Summary. We have screened seven Spanish phenylke- tonuric (PKU) families for the most prevalent Mediter- ranean and Caucasian mutations, and have subsequently found mutations P281L and IVS10. We have analyzed these two mutations in 23 of our patients. The frequen- cies found correspond to those of Mediterranean coun- tries, such as Italy. This is the first report of a mutation analysis in the Spanish PKU population.

Introduction

Classical phenylketonuria (PKU) is an autosomal reces- sive disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene. Several types of mutations have to date been established.

Molecular studies have identified a genetic basis for clinical and biochemical heterogeneity. Different restric- tion fragment length polymorphism (RFLP) haplotypes have been described associated with the PAH gene and in linkage disequilibrium with specific mutations. In North- ern Europe, the two most common mutations are R408W and IVS12, associated with haplotypes 2 and 3, respec- tively (Dilella et al. 1986, 1987). A different haplotype distribution and linked mutations have been reported in the Mediterranean countries. Thus, two prevalent muta- tions have been identified in Southern Europe: a substitu- tion of Leu for Pro (P281L) at amino acid 281 (Okano et al. 1991), and a G-to-A transition in intron 10 creating a cryptic splice acceptor site (IVS10) (Dasovich et al. 1991; Kalaydjieva et al. 1991).

In Spain, neonatal mass-screening has shown an inci- dence of PKU of approximately 1 : 10000 (Ugarte 1982). In our laboratory in Madrid, we have diagnosed 96 cases of PKU and have determined the phenotype in 60 of these cases. These data reveal a prevalence of mild PKU (57%) versus classical PKU (16%). The rest of our pa- tients correspond to non-PKU hyperphenylalaninemia (P6rez et al. 1991).

Offprint requests" to: M. Ugarte

In order to investigate the genetic heterogeneity of P A H deficiencies in Spain, an initial screening of 10 PKU mutations was performed in seven families. The two mutations found, P281L and IVS10, were further screened in a group of 23 Spanish PKU patients.

Patients and methods

The study included 23 PKU patients, most of them detected in the neonatal screening program. The patients come from different regions of Spain and were classified using the criteria described by Giittler (1980) as having classical PKU (5 cases), mild type PKU (14 cases) or non-PKU hyperphenylalaninemia (4 cases).

Mutation-containing regions of the PAH gene were amplified by the polymerase chain reaction (PCR) from the genomic DNA of each PKU patient, with the purpose of detecting PKU muta- tions (Dilella et al. 1988). Allele-specific oligonucleotide (ASO) screening was performed to identify the previously described mutations: R252W (Abadie et al. 1989), R261Q (Abadie et al. 1989), G272X (Svensson et al. 1990), $273F (Melle et al. 1992), E280K (Lyonnet et al. 1989), P281L (Okano et al. 1991), IVS10 (Dasovich et al. 1991; Kalaydjieva et al. 1991), R408W (Dilella et al. 1987), IVS12 (Dilella et al. 1986) and Y414C (Okano et al. 1991).

Direct sequencing of the purified PCR products was performed according to the method described by Reichardt and Woo (1991) and Svensson et al. (1990). In some cases, amplified exons 7 and 10 were cloned in pUC9 and sequenced.

Results and discussion

We have initiated a PKU mutation analysis in seven fam- ilies and have screened those mutations described as oc- curring most frequently in Mediterranean countries (R252W, R261Q, G272X, $273F, E280K, P281L and IVS10). Two prevalent mutations found among Cau- casians (R408W and IVS12), and one that exhibits a mild phenotype (Y414C) were also analyzed. In these seven families, two mutations were found: P281L and IVS10.

Consequently, we screened these two mutations in a total of 23 patients. Using ASO hybridization analysis, we have screened for the IVS10 mutation and have

342

Table 1. Frequencies found for the P281L and IVS10 mutations. The frequencies in Italy are according to Okano et al. (1991) and Dasovich et al. (1991)

P281L IVS10

Spain 3/46 (6%) 5/46 (11%) Italy 4/42 (9%) 10/66 (15%)

financial support of the Fundaci6n Ram6n Areces to the Centro de Biologfa Molecular is gratefully acknowledged. This work was supported by a grant from the Consejeria de Educaci6n de la Comunidad de Madrid.

References

found 5 mutant alleles. Fur the rmore , we have analyzed the P281L muta t ion and have found 3 mutan t alleles. Kindred analysis was pe r fo rmed on families bearing the P281L and the IVS10 mutat ions to determine the carrier of the mutat ion. The mutat ions in some families have been conf i rmed by sequencing the cor responding exon.

The frequencies found, 3/46 for the P281L muta t ion and 5/46 for the IVS10 substitution, are similar to those repor ted in Italy for these muta t ions (Okano et al. 1991; Dasovich et al. 1991) (Table 1). We found a correlat ion between haplotype 1 and the P281L mutat ion, as has been described in several Medi te r ranean countries (Okano et al. 1991). Our prel iminary study of Spanish P K U haplo- types in 10 families (unpublished results) shows a pre- valence of haplotype 1 associated both with mutant and normal chromosomes ; we have also found a major inci- dence of haplotypes 6, 9 and 27 associated with P K U al- leles. However , we have not found haplotypes 2 and 3, which are prevalent in Nor thern Eu rope (Dilella et al. 1986, 1987). These data are similar to the haplo type dis- tr ibution in Italy (Dianzani et al. 1990). In general , the Medi te r ranean countries have been found to be geneti- cally alike.

The patients bearing the P281L muta t ion (one in the homozygous and one in the he terozygous state) have a classical P K U phenotype . This is consistent with the data repor ted by O k a n o (Okano et al. 1991). The patients with the IVS10 substitution are all he terozygotes and exhibit a mild phenotype . This muta t ion has been de- scribed as being associated with classical P K U in the homozygous state (Dasovich et al. 1991). Since mild mutat ions prevail over severe mutat ions in the pheno- typic manifestation of PKU, the IVS10 substitution in our popula t ion should be combined with an as yet undef ined mild mutat ion(s) .

Ou r results suggest that the Spanish P K U popula t ion does not differ considerably f rom that of Southern Euro- pean countries, such as Italy. The two most prevalent Medi te r ranean mutat ions are those that we have de- tected to date in our patients. Nevertheless, the iden- tified mutat ions are represented in only a small number of the total P K U alleles. We are current ly sequencing the P A H exons of several P K U patients, and searching for new mutat ions that could account for the remaining Spanish P K U alleles.

Abadie V, Lyonnet S, Maurin N, Berthelon M, Caillaud C, Giraud F, Matei JF, Rey J, Rey F, Munnich A (1989) CpG dinucleo- tides are mutation hot spots in phenylketonuria. Genomics 5 : 936-939

Dasovich M, Konecki D, Lichter-Konecki U, Eisensmith RC, Gtittler F, Naughton E, Mullins C, Giovannini M, Riva E, Woo SLC (1991) Molecular characterization of a PKU allele prevalent in Southern Europe and Ireland. Somat Cell Mol Genet 17 : 303-309

Dianzani I, Devoto M, Camaschella C, Saglio G, Battista Ferrero G, Cerone R, Romano C, Romeo G, Giovannini M, Riva E, Angeneydt F, Trefz FK, Okano Y, Woo SLC (1990) Haplo- type distribution and molecular defects at the phenylalanine hydroxylase locus in Italy. Hum Genet 86 : 69-72

DiLella AG, Marvit J, Lidsky AS, Grittier F, Woo SLC (1986) Tight linkage between a splicing mutation and a specific DNA haplotype in phenylketonuria. Nature 322 : 799-803

DiLella AG, Marvit J, Brayton K, Woo SLC (1987) An amino- acid substitution involved in phenylketonuria is in linkage dis- equilibrium with DNA haplotype 2. Nature 327 : 333-336

DiLella AG, Huang WM, Woo SLC (1988) Screening for phenyl- ketonuria mutations by DNA amplification with DNA poly- merase chain reaction. Lancet I : 497-498

Giittler F (1980) Hyperphenylalaninemia: diagnosis and classifica- tion of the various types of phenylalanine hydroxylase defi- ciency in childhood. Acta Pediatr Scand 280 [Suppl] : 1-80

Kalaydjieva L, Dworniczak B, Aulehla-Scholz C, Devoto M, Romeo G, Stuhrmann M, Horst J (1991) Phenylketonuria mu- tation in Southern Europeans. Lancet 1 337 : 865

Lyonnet S, Caillaud C, Rey F, Berthelon M, Frezal J, Rey J, Mun- nich A (1989) Molecular genetics of phenylketonuria in Medi- terranean countries: a mutation associated with partial phenyl- alanine hydroxylase deficiency. Am J Hum Genet 44 : 511-5t7

Melle D, Verelst P, Rey F, Berthelon M, Francois B, Munnich A, Lyonnet S (1992) Two distinct mutations at a single BamHI site in phenylketonuria. J Med Genet (in press)

Okano Y, Wang T, Eisensmith RC, Longhi R, Riva E, Giovannini M, Cerone R, Romano C, Woo SLC (1991) Phenylketonuria missense mutations in the Mediterranean. Genomics 9 : 96 : 103

Pdrez B, Desviat LR, Garcfa MJ, Ugarte M (1991) Phenotypes and genotypes in the Spanish PKU population. Abstracts of the 29th SSIEM Annual Symposium, London 1991

Reichardt J, Woo SLC (1991) Molecular basis of galactosemia: mutations and polymorphisms in the gene encoding human galactose-l-phosphate urydiltransferase. Proc Natl Acad Sci USA 88 : 2633-2637

Svensson E, Andersson B, Hagenfeldt L (1990) Two mutations within the coding sequence of the phenylalanine hydroxylase gene. Hum Genet 85 : 300-304

Ugarte M (1982) Neonatal screening programme for aminoacid disorders and congenital hypothyroidism in Spain. In: Naruse H, Irie M (eds) Neonatal screening. International Symposium on Neonatal Screening for Inborn Errors of Metabolism. Ex- cerpta Medica, Amsterdam, pp 491-492

Acknowledgements. We thank Dr. S. L. C. Woo and his colleagues at the Baylor College of Medicine for their collaboration. The