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Molecular Genetics and Metabolism 77 (2002) 260–266 www.academicpress.com Brief Communication Two novel genetic lesions and a common BH4-responsive mutation of the PAH gene in Italian patients with hyperphenylalaninemia T. Bardelli,a M.A. Donati,a S. Gasperini,a F. Ciani,a F. Belli,a N. Blau,b A. Morrone,a,* and E. Zammarchia b a Metabolic and Neuromuscular Unit, Department of Pediatrics, University of Florence, Florence, Italy Division of Clinical Chemistry and Biochemistry, Department of Paediatrics, University ChildrenÕs Hospital, Zurich, Switzerland Received 30 May 2002; received in revised form 30 July 2002; accepted 31 July 2002 Abstract Hyperphenylalaninemia (HPA), due to a deficiency of phenylalanine hydroxylase (PAH) enzyme, is caused by mutations in the PAH gene. Molecular analysis in 23 Italian patients with PAH deficiency identified two novel (P281R, L287V) and 20 previously described genetic lesions in the PAH gene. The detection of the A403V amino acid substitution in combination with null mutations in patients with BH4 -responsive PAH deficiency leads us to correlate it with BH4 responsiveness. Ó 2002 Elsevier Science (USA). All rights reserved. 1. Introduction Phenylalanine hydroxylase (PAH, EC 1.14.16.1) deficiency (MIM#261600) is an autosomal recessive disorder of amino acid metabolism. The disease, characterized by hyperphenylalaninemia (HPA), has been classified into three forms: classic phenylketonuria (PKU), variant PKU, and mild HPA. The main clinical manifestations of untreated PKU and variant PKU are related to impaired brain development and include mental retardation, epilepsy, and behaviour problems. Mild HPA, generally considered a benign disorder, may also present neurological involvement [1,2]. Dietary treatment can prevent the clinical manifestations of PAH deficiency [3]. Up to now more than 400 different genetic lesions have been identified in the PAH gene (http//data.mch.mcgill.ca/pahdb_new/). On the basis of the phenotype of homozygous or ‘‘functionally hemizygous’’ patients the majority of PAH mutations has been correlated to a consistent metabolic phenotype [4,5]. Mutations in the PAH gene have also been reported in patients showing HPA normalization upon oral loading with the cofactor of PAH, tetrahydrobiopterin (BH4 ) [1,2,6–9]. This led to the treatment of BH4 -responsive PAH deficiency with BH4 [1,2,9]. Mutations of the PAH gene are heterogeneous in Southern European populations with marked differences between regions. Genetic heterogeneity has also been described in Italian patients with PAH deficiency from North and South Italy [10]. Up to now no molecular studies have been performed in patients from Tuscany, Central Italy. In this study, we characterized the genotype of 23 patients with PAH deficiency segregating into 21 unrelated families from Tuscany. Genetic analysis of the PAH gene, performed by direct sequencing of the patientsÕ genomic DNA, isolated from lymphocytes, identified two novel transversions and a common BH4 -responsive mutation. 2. Materials and methods * Corresponding author. Fax: +055/570380. E-mail addresses: malmetab@unifi.it, [email protected] (A. Morrone). We studied 23 Italian patients affected by PAH deficiency. 1096-7192/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 9 6 - 7 1 9 2 ( 0 2 ) 0 0 1 6 6 - X T. Bardelli et al. / Molecular Genetics and Metabolism 77 (2002) 260–266 Sixteen patients were diagnosed by newborn screening. The other seven patients were born before newborn screening was introduced and were identified because of severe mental retardation or delayed psychomotor development. Patients were assigned to one of three categories according to pre-treatment blood Phe levels: 13 patients presented classical PKU with plasma phenylalanine (Phe) > 1200 lM, four patients showed variant PKU (plasma Phe 1200–600 lM), and six mild HPA (plasma Phe < 600 lMÞ (Tables 1 and 2). In addition, the patients have been grouped into four subsets according to their metabolic phenotype which is related to dietary Phe tolerance along the lines of Guldberg [5]: patients with classic PKU tolerate <20 mg Phe/kg body weight/day (wt/d) to keep plasma Phe value < 300 lM; patients with moderate PKU tolerate 20–25 mg Phe/kg body wt/d; patients with mild PKU tolerate 25–50 mg Phe/kg body wt/d and patients with mild HPA (MHP) tolerate >50 mg Phe/kg body wt/ d. The patientsÕ metabolic phenotypes are reported in Tables 1 and 2. In all patients a BH4 or a combined Phe/BH4 loading test was performed. The BH4 loading test was performed with oral administration of 20 mg BH4 /kg body weight and plasma phenylalanine and tyrosine were monitored at 0 h (before BH4 administration), and at 4, 8 h post BH4 administration. The combined Phe/BH4 loading test was performed with oral administration of 100 mg/kg of Phe and, after 3 h, of 20 mg BH4 /kg body weight. In the combined Phe/ BH4 loading test plasma phenylalanine and tyrosine were monitored at 0 h (before Phe administration) and at 3, 7, 11, 27 h post Phe administration. Seven patients (15.1, 16.1, 17.1, 18.1, 19.1, 20.1, and 21.1), diagnosed by newborn screening, with plasma Phe levels of 1150, 563, 240, 227, 269, 282, and 222 lM, respectively (reference range 40–130 lM) presented a decrease of plasma Phe levels in response to BH4 administration (Table 2). Patients 15.1, 16.1 (9 and 8 months old, respectively) had remained on a Phe-restricted diet since the neonatal period. Phenylalanine tolerance was 36 mg Phe/kg body wt/d in patient 15.1 and 33 mg Phe/kg body wt/d in patient 16.1. Patients 17.1, 21.1 (8 and 29 months old, respectively), who were breast fed, were started on a Phe-restricted diet from weaning because their increased plasma Phe value ð>300 lMÞ. Phenylalanine tolerance was 33 mg Phe/kg body wt/d in patient 17.1 and 40 mg Phe/kg body wt/d in patient 21.1. Patients 18.1, 19.1, 20.1 (7, 10, and 18 months old, respectively) tolerate >50 mg Phe/kg body wt/d to keep plasma Phe value <300 lM and remains on an unrestricted diet. 261 At present the seven patients with BH4 -responsive PAH deficiency show a normal psychomotor development and no neurological symptoms. The patientsÕ genomic DNA was isolated from lymphocytes. PCR amplifications of PAH DNA coding regions and intron–exon boundaries were carried out. The direct sequencing of PAH DNA fragments was performed using Applied Biosystems ABI Prism 310 Genetic Analyser (Foster City, CA, USA). The patientsÕ genetic lesions and their associated metabolic phenotype along the lines of Guldberg [5] are reported in Tables 1 and 2. The designation of the PAH gene mutations corresponds to that described by Antonarakis [11]. 3. Results and discussion Genetic analysis in 23 Italian patients with PAH deficiency led to the identification of two novel transversions ðc:842C > G; c:859C > GÞ and 20 previously described mutations in the PAH gene. The new nucleotide substitution c:842C > G, in exon 7, leads to the novel P281R amino acid change (Fig. 1A). This mutation is located in the CBR2 cofactor binding region of the catalytic domain and it disrupts the binding of the PAH enzyme with the BH4 cofactor [7,12]. Two other amino acid substitutions, P281L and P281S, were previously reported at the codon P281. The high frequency of genetic lesions at this codon could be explained by the presence of a CpG site. The new transversion c:859C > G, located in exon 8, leads to the novel L287V mutation (Fig. 1B). The amino acid Leu287 is located between the residues His285 and His290, coordinating the PAH enzyme catalytic domain to a ferric iron, and it is near the amino acid Glu286 which interacts with the BH4 cofactor [7,12]. The L287V mutation could disrupt the binding of the PAH enzyme both with the ferric iron and with the BH4 cofactor. The IVS10-11G > A mutation was detected in eight patients with classical PKU, combined with one of the following genetic lesions c.44-45delCT (L15-S16fs), c.163delT (F55fs), R158Q, R261Q, P281L, P281R, and IVS11-8G > A (2), respectively (Table 1). The other five patients with classical PKU were compound heterozygous for: R261X/R261Q (2), S67P/IVS12 + 1G > A, c.163delT (F55fs)/R158Q, and P281L/L287V (Table 1). Both the mutations R261X and R261Q, identified in two brothers, involved the same amino acid located in the CBR1 cofactor binding region: the R261Q disrupts the binding of the PAH protein with the BH4 cofactor and the R261X does not lead to a functional protein (Fig. 1C). Interestingly, the six patients with classic PKU carrying mutations located in the cofactor binding regions of the PAH enzyme (P281L, P281R, and R261Q) showed no response to BH4 loading. 262 Table 1 Phenotype and genotype in Italian patients with PAH deficiency and without BH4 responsiveness Patient Motive of diagnosis Phe at diagnosis Classification (n.v. 40–130 lM) at diagnosis Phe Metabolic tolerance phenotype (mg Phe/kg body wt/d) Genotype Allele 1 Mental retardation >1200 Classic PKU <20 Classic PKU 2.1 >1200 Classic PKU <20 Classic PKU >1200 Classic PKU <20 Classic PKU 4.1 Delayed psychomotor development Mental retardation and autistic behaviour Mental retardation >1200 Classic PKU <20 Classic PKU 5.1 Neonatal screening >1200 Classic PKU <20 Classic PKU 6.1 >1200 Classic PKU <20 Classic PKU >1200 Classic PKU <20 Classic PKU 7.2 Mental retardation and autistic behaviour Delayed psychomotor development Neonatal screening >1200 Classic PKU <20 Classic PKU 8.1 Neonatal screening >1200 Classic PKU <20 Classic PKU 8.2 Neonatal screening >1200 Classic PKU <20 Classic PKU 9.1 Neonatal screening >1200 Classic PKU <20 Classic PKU >1200 Classic PKU <20 Classic PKU 11.1 Delayed psychomotor development Neonatal screening >1200 Classic PKU <20 Classic PKU 12.1 Neonatal screening 1080 Variant PKU 13.1 Neonatal screening 780 Variant PKU 14.1 Neonatal screening 950 Variant PKU 28 (at the age of 20 months) 23 (at the age of 36 months) 28 (at the age of 36 months) 3.1 7.1 10.1 Genetic lesion Associated metabolic phenotypes Genetic lesion Associated metabolic phenotypes c44-45delCT (L15/S16fs) c163 delT (F55fs) R158Q ðc473G > AÞ R261Q ðc782G > AÞ P281L ðc842C > TÞ Classic PKU (this work) Classic PKU IVS 10-11G > A Splicing defect IVS10-11G > A Splicing defect IVS 10-11G > A Splicing defect IVS 10-11G > A Splicing defect IVS10-11G > A Splicing defect IVS10-11G > A Splicing defect IVS10-11G > A Splicing defect IVS10-11G > A Splicing defect R261Q ðc782G > AÞ R261Q ðc782G > AÞ IVS12 þ 1G > A Splicing defect R158Q ðc473G > AÞ L287V c859 C > G New mutation R158Q ðc473G > AÞ Classic PKU Classic, Moderate PKU Classic, Moderate PKU Classic PKU Classic PKU (this work) Classic PKU (this work) Classic PKU (this work) Classic PKU Classic PKU Classic PKU Classic PKU Classic PKU P281R (c842 C>G) New mutation IVS11-8G > A Splicing defect IVS11-8G > A Splicing defect R261X ðc781C > TÞ R261X ðc781C > TÞ S67P ðc199T > CÞ c163 del T (F55fs) P281L ðc842C > TÞ Classic PKU Mild PKU I65T ðc194T > CÞ Classic, Moderate, Mild PKU Moderate PKU c280–282 delATC (Del I94) ? IVS4 þ 5G > T Splicing defect ? Mild PKU D222G ðc665A > GÞ Mild PKU (this work) IVS10-11G > A Splicing defect Classic PKU Classic PKU Classic PKU (this work) Classic PKU Classic PKU Classic PKU Classic PKU Classic, Moderate PKU Classic, Moderate PKU Classic PKU Classic, Moderate PKU Classic PKU (this work) Classic, Moderate PKU T. Bardelli et al. / Molecular Genetics and Metabolism 77 (2002) 260–266 1.1 Allele2 Table 2 Phenotype, genotype and BH4 loading test in Italian patients with BH4 -responsive PAH deficiency Phe at diagnosis (n.v.40– 130 lM) Tyr at diagnosis (n.v.50– 140 lM) Classification at diagnosis Phe Tolerance (mgPhe/Kg body wt/d) Metabolic phenotype BH4 loading test Genotype Phe (n.v.40–130 lM) Tyr (n.v.50–140 lM) Allele1 T0 T4 T8 T0 T8 Genetic lesion Associated metabolic phenotypes Genetic lesion Associated metabolic phenotypes Classic, Moderate, Mild PKU, MHP Mild PKU and MHP T4 Allele2 Variant PKU 36 (at the age of 9 months) Mild PKU 1150 970 740 78 96 120 R243Q ðc728G > AÞ Classic PKU Y414C ðc1241A > GÞ 106 Mild HPA 33 (at the age of 8 months) Mild PKU 563 306 173 100 102 82 R261Q ðc782G > AÞ E390G ðc1079A > GÞ 270 118 Mild HPA Mild PKU 240 34 31 84 98 65 18.1 280 138 Mild HPA MHP 227 76 38 107 146 146 19.1 228 156 Mild HPA 33 (at the age of 7 months) >50 (at the age of 10 months) >50 (at the age of 18 months) MHP 269 78 54 216 179 174 A403V ðc1208C > TÞ A403V ðc1208C > TÞ A403V ðc1208C > TÞ Classic, ModeratePKU MHP 15.1 1026 90 16.1 508 17.1 MHP MHP IVS10-11G > A (s.d.) c1036 delG (G346fs) A300S ðc898G > TÞ Classic PKU IVS4 þ 5G > T (s.d.) IVS10-11G > A (s.d.) ? Classic PKU MHP Combined Phe/BH4 loading test Phe lM (n.v.40–130) T0 20.1 288 72 Mild HPA 21.1 276 78 Mild HPA >50 (at the age of 8 months) 40 (at the age of 29 months) Tyr lM (n.v.50–140) T3 T7 T11 T27 T0 T3 T7 T11 T27 MHP 282 864 582 96 258 66 72 114 120 72 Mild PKU 222 828 684 294 126 54 48 68 72 78 A403V ðc1208C > TÞ A403V ðc1208C > TÞ MHP MHP Classic PKU T. Bardelli et al. / Molecular Genetics and Metabolism 77 (2002) 260–266 Patient Phe, Phenylalanine; Tyr, Tyrosine; n.v., normal values; s.d., splicing defect. The BH4-responsive mutations have been underlined. T0 ¼ pre-treatment values; T4,T8 ¼ values detected at 4,8 h post BH4 administration; T3, T7, T11, T27 ¼ values detected at 3, 7, 11, 27 h post Phe administration. In the case of patient 15.1 the full effect of the BH4 administration would probably be at 24 h or later. 263 264 T. Bardelli et al. / Molecular Genetics and Metabolism 77 (2002) 260–266 Fig. 1. 1/A Nucleotide direct sequences of PAH DNA showing the wild-type sequence of exon 7/intron7 boundaries in the normal control and the new transversion c:842C > G (P281R) at heterozygous state. 1/B Nucleotide direct sequences of PAH DNA showing the wild-type sequence of exon 8 in the normal control and the new transversion c:859C > G (L287V) at heterozygous state. 1/C Nucleotide direct sequences of PAH DNA showing the wild-type sequence of exon 7 in the normal control and the nucleotide changes c:781C > T (R261X) and c:782G > A (R261Q). One patient with moderate PKU was compound heterozygous for c.280-282delATC (delI94)/ IVS4þ 5G > T. Two patients with mild PKU metabolic phenotype were compound heterozygous for I65T/ R158Q and IVS10-11G > A=D222G, respectively (Table 1). T. Bardelli et al. / Molecular Genetics and Metabolism 77 (2002) 260–266 The IVS10-11G > A, IVS12 þ 1G > A, and P281L mutations have been previously correlated to classic PKU [5]. Combining our data with that reported in the literature the c44-45delCT, P281R, IVS11-8G > A, S67P, and L287V genetic lesions can be associated with classic PKU and the D222G mutation with the mild PKU metabolic phenotype (Table 1). We identified the R243Q/Y414C and the R261Q/ E390G genotypes in two patients with the BH4 -responsive mild PKU metabolic phenotype (Table 2). In these two patients, the BH4 load caused a significant decrease in plasma Phe levels. The Y414C mutation has been previously described at a homozygous level in one patient with BH4 -responsive PAH deficiency [13] and the E390G, detected with the null IVS10-11G > A mutation in one BH4 -responsive patient, has been previously correlated to BH4 responsiveness [9]. The other five Italian patients with BH4 -responsive PAH deficiency were compound heterozygous for the A403V mutation which was combined with IVS 10-11G > A in two patients, IVS4 þ 5G > T, A300S, and c.1036delG (G346fsdelG) (Table 2). In these patients plasma Phe concentrations were completely normalized after the BH4 or a combined Phe/BH4 loading test. The A403V amino acid substitution has been previously correlated with PAH enzyme residual activity (32%) and has been described at a heterozygous state in BH4 -responsive patients [7,12] but has not been correlated with BH4 responsiveness [8]. The detection of the A403V in combination with the severe genetic lesion IVS10-11G > A, leading to no functional protein [14], leads us to associate the A403V mutation with BH4 responsiveness. This mutation is located in a region that interacts with the secondary structural elements involved in cofactor binding and could result in a mutant enzyme with a lower binding affinity for BH4 . In this study the splicing defect IVS10-11G > A was the most common allele (11/46) among the Tuscan patients with PAH deficiency. This mutation has also been described as the most frequent PAH gene lesion in Campania, Sicily, Apulia, and Basilicata [8]. The A403V allele showed a higher frequency (5/46) than in studies involving patients from North and South Italy [8]. This genetic lesion can be easily screened for using BbvI enzymatic restriction analysis. Since in this study the A403V mutation proved to be common in Italian patients with BH4 -responsive PAH deficiency we would like to stress the importance of screening for this genetic lesion in this phenotype. Interestingly, the two patients with BH4 -responsive PAH deficiency and genotype A403V/IVS10-11G > A showed increased plasma Phe values during weaning and were started on a Phe-restricted diet. The patients with BH4 -responsive mild PKU b cdcmetabolic phenotype would probably be the best can- 265 didates for BH4 therapy as an alternative to the restrictive low-phenylalanine diet. The neurological involvement recently reported in patients with BH4 -responsive mild PAH deficiency [1,2] demonstrates the importance of a BH4 loading test and an accurate follow up even in patients with this metabolic phenotype. Acknowledgments This work was partially supported by grants from the Association AMMEC and the Azienda Ospedaliera Meyer, Florence, Italy. References [1] L. Bonafe, N. Blau, A.P. Burlina, A. Romstad, F. Guttler, A.B. Burlina, Treatable neurotransmitter deficiency in mild phenylketonuria, Neurology 57 (5) (2001) 908–911. [2] R. Koch, F. Guttler, N. Blau, Mental illness in mild PKU responds to biopterin, Mol. Genet. Metab. 75 (3) (2002) 284– 286. [3] C.R. Scriver, S. Kaufman, Hyperphenylalaninemia: phenylalanine hydroxylase deficiency, in: C.R. Scriver, A.L. Beaudet, D. Valle, W.S. Sly (Eds.), The Metabolic and Molecular Bases of Inherited Disease, 2001, pp. 1667–1724. [4] E. Kayaalp, E. Treacy, P.J. Waters, S. Byck, P. Nowacki, C.R. Scriver, Human phenylalanine hydroxylase mutations and hyperphenylalaninemia phenotypes: a metanalysis of genotype– phenotype correlations, Am. J. Hum. Genet. 61 (6) (1997) 1309– 1317. [5] P. Guldberg, F. Rey, J. Zschocke, V. Romano, B. Francois, L. Michiels, K. Ullrich, G.F. Hoffmann, P. Burgard, H. Schmidt, C. Meli, E. Riva, I. Dianzani, A. Ponzone, J. Rey, F. Guttler, A European multicenter study of phenylalanine hydroxylase deficiency: classification of 105 mutations and a general system for genotype-based prediction of metabolic phenotype, Am. J. Hum. Genet. 63 (1) (1998) 71–79. [6] S. Kure, D.C. Hou, T. Ohura, H. Iwamoto, S. Suzuki, N. Sugiyama, O. Sakamoto, K. Fujii, Y. Matsubara, K. Narisawa, Tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency, J. Pediatr. 135 (3) (1999) 375–378. [7] L.J. Spaapen, J.A. Bakker, C. Velter, W. Loots, M.E. RubioGonzalbo, P.P. Forget, L. Dorland, T.J. De Koning, B.T. PollThe, H.K. Ploos van Amstel, J. Bekhof, N. Blau, M. Duran, Tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency in Dutch neonates, J. Inherit. Metab. Dis. 24 (3) (2001) 352–358. [8] U. Lassker, J. Zschocke, N. Blau, R. Santer, Tetrahydrobiopterin responsiveness in phenylketonuria. Two new cases and a review of molecular genetic findings, J. Inherit. Metab. Dis. 25 (1) (2002) 65–70. [9] F.K. Trefz, C. Aulela-Scholz, N. Blau, Successful treatment of phenylketonuria with tetrahydrobiopterin, Eur. J. Pediatr. 160 (5) (2001) 315. [10] S. Giannattasio, I. Dianzani, P. Lattanzio, M. Spada, V. Romano, F. Cali, G. Andria, A. Ponzone, E. Marra, A. Piazza, Genetic heterogeneity in five Italian regions: analysis of PAH mutations and minihaplotypes, Hum. Hered. 52 (3) (2001) 154–159. [11] S.E. Antonarakis and the Nomenclature Working Group, Recommendations for a nomenclature system for human gene mutations, Hum. Mutat. 11 (1998) 1–3. 266 T. Bardelli et al. / Molecular Genetics and Metabolism 77 (2002) 260–266 [12] H. Erlandsen, R.C. Stevens, A structural hypothesis for BH4 responsiveness in patients with mild forms of hyperphenylalaninaemia and phenylketonuria, J. Inherit. Metab. Dis. 24 (2) (2001) 213–230. [13] R. Steinfeld, A. Kohlschutter, J. Zschocke, M. Lindner, K. Ullrich, Z. Lukacs, Tetrahydrobiopterin-responsiveness associated with common phenylalanine-hydroxylase mutations distant from the tetrahydrobiopterin binding site, J. Inherit. Metab. Dis. 24 (Suppl. 1) (2001) 29. [14] B. Dworniczak, C. Aulehla-Scholz, L. Kalaydjieva, K. Bartholome, K. Grudda, J. Horst, Aberrant splicing of phenylalanine hydroxylase mRNA: the major cause for phenylketonuria in parts of southern Europe, Genomics 11 (2) (1991) 242– 246.