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Characterization of Phenylketonuria Alleles in the Italian Population

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Characterization of Phenylketonuria Alleles in the Italian Population Original Paper Eur J Hum Genet 1995;3:294-302 1608180 Irma Dianzani* Characterization of Sergio Giannattasioc Luisa de Sanctis* Phenylketonuria Alleles in the Carla Alliaudi* Paolo Lattanzioc Italian Population Carlo Dionisi Viciâ Alberto Buriina6 Massimo Burronif Gianfranco Sebastio* Key Words Franco Carnevaleh Phenylketonuria • Phenylalanine hydroxylase • Vito Gazzetta * Genotype/phenotype correlation Ersilia Marra6 Clara Camaschellaa’b Abstract Alberto Ponzonea In order to identify the molecular basis of phenylketonuria a Clinica Pediatrica e Clinica Medica, (PKU) in Italy, we screened the entire coding sequence of the University of Torino, and phenylalanine hydroxylase gene in 20 Italian PKU patients, b CNR-CIOS, Torino, and whose origins are scattered throughout Italy. The frequency of 0 CNR-Centro di Studio sui Mitocondri e Metabolismo Energetico, Bari and each identified mutation and of 5 other European mutations Trani, and was determined within a panel of 92 Italian PKU patients. This d Ospedale Bambin Gesù, Rome, and approach allowed us to identify 20 different PKU mutations e Dipartimento di Pediatria, University of Padua, and and characterize 64% of the Italian PKU chromosomes. Eleven f Servizio Autonomo di Neuropsichiatria mutations (IYS10nt546, L48S, R158Q, R261Q, P281L, Infantile, Fano, and * Cattedra di Pediatria, II Facoltà di R261X, R252W, AT55, IYS7ntl, IVS12ntl, Y414C) represent Medicina, University of Naples, and 55.4% of the Italian PKU alleles, the most common mutations h Divisione Malattie Metaboliche, being IVS10nt546 (12.4%) and L48S (9%). All the other muta­ Ospedale Pediatrico Giovanni XXIII, Bari, Italy tions are very rare. These data confirm the great heterogeneity expected from previous RFLP haplotype studies. Genotype/ phenotype correlation allowed for assessment of the clinical impact of the 20 identified mutations. Introduction date, more than 160 candidate mutations have been identified [3], many of which have Cloning of the gene encoding phenylal­ proven to be causal by analysis of the expres­ anine hydroxylase (PAH) in 1985 [1] has sion of the mutated gene in vitro [4], prompted the definition of the molecular ba­ A novel approach to diagnosis, treatment, sis of phenylketonuria (PKU), the disease prognosis and also prevention of the disease caused by loss of function of this gene [2], To has been implemented by these studies. Pre- Received: December 30,1994 Dr. Irma Dianzani © 1995 Revision received: July 10,1995 Istituto di Clinica Pediatrica S. Karger AG, Basel Accepted. July 19,1995 Piazza Polonia 94 1018-4813/95/ 1-10126 Torino (Italy) 0035-0294S8.00/0 natal diagnosis and carrier detection are easily Padua), two in central Italy (Rome and Fano), and two performed by tracing the inheritance of caus­ in southern Italy (Naples and Bari). The panel of patients is representative of the Italian population, al mutations in characterized families. On since their geographic origin is uniformly spread the other hand, several polymorphic markers throughout Italy. The origin of the patients was as­ within the gene (RFLPs, VNTR, STR) [5, 6] sessed as far back as the four grandparents of the pro­ can be used to identify the mutated uncharac­ bands: 67 chromosomes were original to the northern terized alleles by comparison with an affected and central Italian regions, 110 to the southern regions. The division of Italy into a northem/central and a sibling [7], The elucidation of genotype/phe­ southern region was obtained by tracing a line below notype correlation may allow for early, opti­ Lazio and Marche. mal treatment based on the mutation [8]. PKU diagnosis was assessed after exclusion of de­ However, the possibility of using molecu­ fects in tetrahydrobiopterin [11]. PKU classification lar information in clinics is dependent on was obtained by analysing the following parameters in each patient: (a) pretreatment serum phenylalanine, both precise characterization of mutated al­ (b) time (days) necessary to normalize serum phenylal­ leles as well as mutation frequency in the anine values after introduction of a phenylalanine-free examined population. Great heterogeneity in diet, and (c) phenylalanine tolerance (maximal daily PKU mutations was shown by the earliest phenylalanine intake as required to maintain serum studies focussed on the distribution of RFLP phenylalanine below 360 gmol/1). Using the criteria mentioned above, 50 patients haplotypes at the PAH locus. However, some were assigned to classic PKU, because they showed populations were shown to be relatively ho­ pretreatment plasma phenylalanine higher than 1,500 mogeneous for PKU mutations, with a low gmol/1, dietary tolerance lower than 300 mg/day and number of the represented haplotypes, where­ required more than 4 days for normalization of plasma as others showed a large number of different phenylalanine. Twenty-two patients, assigned to the haplotypes and were expected to carry multi­ mild phenotype, showed pretreatment plasma phenyl­ alanine between 900 and 1,500 gmol/1, dietary toler­ ple mutations. Generally, greater homogene­ ance between 300 and 700 mg/day and required less ity was observed in populations from north­ than 4 days for normalization of plasma phenylala­ ern as compared to southern Europe [4]. Thir­ nine. Six patients were assigned to the benign pheno­ ty-five mutations account for 99% of mutant type, because they showed pretreatment plasma phe­ nylalanine lower than 600 gmol/1, dietary tolerance alleles in Denmark, of which only 4 account higher than 700 mg/day. Moderate PKU was assigned for 70% of the total [9]. On the other hand, the to the 14 patients who showed a pattern midway great number of different haplotypes identi­ between classic and mild phenotypes (for example: fied in the Italian population suggested great pretreatment phenylalanine values >1,500 gmol/1, but heterogeneity of PKU mutations in Italy tolerance >300 mg/day and days for normalization <4). [10]. Genomic DNA was extracted from peripheral The aim of this work was to define the blood leucocytes by standard methods. RFLP haplo­ molecular defects responsible for PKU in the type was determined for 27 patients, 12 of whom were Italian population. part of a previous analysis [10]. For the other 15 haplo- typed PKU patients, RFLPs were detected by either Southern blot analysis or multiplex PCR at the PAH locus followed by restriction digestion [12]. Materials and Methods Mutation Detection Patients Twenty patients, whose origins were spread uni­ Ninety-two Italian PKU patients, corresponding to formly throughout Italy, were selected from the panel 177 unrelated PKU chromosomes, were included in of 92 patients and their DNA was submitted to muta­ this study. Patients were followed up by six clinical tion detection techniques. Selection was made on the departments, two located in northern Italy (Turin and basis of the geographical origin of the patients’ chro- 295 mosomes: 14 chromosomes were original to the north­ evaluated by restriction digestion [16] within the panel ern and central regions (representing 20.9% of north- of the 177 unrelated alleles (table 1). If no restriction em/central chromosomes), 26 chromosomes were orig­ site was either created or abolished in the presence of inal to southern regions (representing 23.6% of south­ the mutation, mutations were introduced in the PCR ern chromosomes). Ten patients showed a classic, 5 a amplification products using mutagenic amplification moderate and 5 a mild phenotype. primers to create a new restriction site in the normal or The 13 PAH exons were amplified by PCR using the mutant allele. The experimental approach used to the appropriate primers complementary to intronic search for each PKU mutation is reported in table 1. regions. Exons 7 to 11 were analysed by chemical S359X and S231P mutations were searched by allele- cleavage method (CCM) [13,14], while exons 3,4,5,8, specific oligonucleotide (ASO) analysis [17] using 9, 10 and 11 were analysed by the combination of sin­ ASOs complementary to the mutated sequences [14, gle-strand conformation polymorphism (SSCP) and 18], heteroduplex methods. All other exons were sequenced directly (exons 1, 2, 6, 12 and 13). Since in previous reports [13,14], exon-intron boundaries of some exons had not been screened, these exons were amplified Results again using primers, allowing us to obtain products encompassing the unscreened regions, after which they Mutation Detection were subjected to direct sequencing or the combina­ Mutation detection analysis of the entire tion of SSCP and heteroduplex methods. In this way, several exons were studied with two different methods, PAH coding sequence allowed for complete but using different PCR primers. characterization of 17 out of 20 patients, with The combination of SSCP and heteroduplex meth­ only one mutation identified in the remaining ods allows a greater sensitivity in mutation detection 3 patients. The 3 uncharacterized mutations as compared to using a single method [15]. Heterodu­ might be located in unscreened regions of plexes are clearly distinguished from SSCP when a non-denatured control is included in electrophoresis: PAH, such as introns (cryptic intronic splice heteroduplexes migrate at the same level as the non- sites) or promoter regions. denatured sample in the bottom portion of the gel, We were able to characterize a total of 64% whereas single strands have a lower migration velocity. (113/177) of the Italian PKU chromosomes. The following SSCP protocol was used. To obtain Twenty mutations have been identified (ta­ labelled products, to one tenth of each PCR reaction were added 1 pi [35S]dATP (3,000 Ci/mmol) and 0.5 U ble 2). Mutations S231P, S359X and IYS7ntl of Taq polymerase, then five more PCR cycles were have been previously reported by our group. performed. 20 pi of 0.1% SDS, 10 mM EDTA pH 8, All mutations are reported in the PAH Muta­ were added to half of the labelled PCR reactions. A tion Analysis Consortium Database [3], fifth of the solution was mixed with an identical vol­ Eleven mutations (IVS10nt546, L48S, ume of stop buffer (95% formamide, 20 mM EDTA pH 8,0.05% xylene cyanol, 0.05% bromophenol blue).
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