Blau, Nenad

Brandt, Niels Jacob

Heizmann, Claus W.

Leimbacher, Walter

Oppliger, Tanja

Scheibenreiter, Susanne

Thony, Beat

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Oppliger et a !. : Mutation analysis in patients with 6-pyruvoyl-tetrahydropterin synthase deficiency Pteridines Vol. 6, 1995, pp. 141-143 Short Communication Mutation Analysis In Patients with 6-Pyruvoyl-Tetrahydropterin Synthase Deficiency Tanja Oppliger~, Beat Thony#, Walter Leimbacher~, Susanne Scheibenreiter t, Niels Jacob Brandt t t , Claus W. Heizmann;l and Nenad Blau #§ # Division of Clinical Chemistry, Department of Pediatrics, University of Zurich, Steinwiesstrasse 75, CH-8032 Zurich, Switzerland, t Department of Pediatrics, University of Vienna, Austria, t tDivision of Clinical Genetics, University Hospital, Copenhagen, Denmark (Received June 15, 1995) Introduction 6-Pyruvoyl-tetrahydropterin synthase (PTPS) is the second enzyme involved in the biosynthesis of tetrahydrobiopterin (BH4)' , the obligatory cofactor for the aromatic amino acid hydroxylases2 as well as for all types of nitric oxide synthases3 • Defects in the PTPS are the most frequent and heterogen-eous variants of BH4 deficiency<. Three different forms of PTPS deficiency can be distinguished: (i) A central or severe type, where patients do not have sufficient PTPS activity in the brain and in the liver. Patients with this type of defect suffer from neu-rotransmitter deficiency in the central nervous sys-tem (CNS) and elevated phenylalanine concent-rations in blood and tissue. They need to be treated with BH4, L-Dopa, Carbidopa and 5-hydroxytryp-tophan to prevent irreversible brain damage. (ii) In the partial or peripheral type, patients do not have enough PTPS in the liver, but neurotransmitter biosynthesis in the brain is normal. They have to be treated with BH4 monotherapy or follow a phen-ylalanine low diet l. (iii) A transient form , where patients present clinical symptoms only in the neo-natal period, has been described in a few cases". Since the human liver cDNA was isolated?, it was § Autho r to whom correspondence should be addressed. Nenad Blau, Division of C linical Chemistry, Depart-ment of Pedia trics, U ni\"ersiIY o f Zurich Steinwiesstrasse 75 CH-8032 Zurich, Switzeri a nJ. Fax: 41-1-266-7169 Pteridines I Vol. 6 / :-; possible to investigate the cDNA in primary skin fibroblasts from PTPS deficient patients in orde r to find out the mutations causing the disease. In this work three new mutations, a missense mutation and two deletions, in two patients suffer-ing from the severe type of PTPS deficiency are described. Furthermore, a summary of all mutations found so far in the eDNA of PTPS deficient patients and of the clinical data of these patients is given. We hope that it will become possible to differentiate between variants of PTPS deficiency by finding and characterizing the mutations. Materials and Methods Pterins in urine were measured by HPLC, a fter oxidation with manganese dioxide, as described previously8. The PTPS assay is based on the measurement of BH. derived from dihydroneopterin triphosphate (substrate) (110 flM) in the presence of NADPH (1 mM), NADH (l mM), DHPR (220 mU), magnesium (10 mM), sepiapterin reductase (5 mU), and Tris-HCl buffer, pH 7.4 (0.1 M)9. Erythrocytes (50 fll) were suspended in 50 fll of 0.2 M Tris-HCl buffer, pH 7.4 and lysed by freezing and thawing. The sam-ple was saturated with carbon monoxide for 1-2 min unde r slight shaking and 50 fll of the lysate were used for the a ssay. The biopterin was measured fluorom etrically by HPLC after oxidation with 142 Oppliger et al. : Mutation analysis in patients with 6-pyruvoyl-tetrahydropterin synthase deficiency manganese dioxide at pH 1.0-1.5. Primary skin fibroblasts were cultured in Dul-becco's Modified Eagle Medium (Gibco) containing 10% fetal calf serum (Gibco) and 50 units of peni-cillin plus 50 /lg/ml streptomycin. Fibroblast lysate preparations and PTPS acivity measurements of these extracts were performed as described else-where lO• - , Total RNA was isolated from confluent fibroblast cultures following a protocol using guanidinium-thiocyamate for cell lysis and subsequent centrifu-gation in cesium chloride solution II. With oligo-deoxythymidine as a primer cDNA was synthesized from this total RNAIO• cDNA was PCR-amplified and directly sequenced using the dideoxynucleotide chain termination method (USB sequencing kit, version 2.0) according to Thony et allO • Case Reports The history of the patients JRS and UT has been reported elsewhere l? LL is the first child of healthy, non related pa-rents. On the 5th day the Guthrie test was slightly elevated, and the controls on day 10 and 42 were, for plasma phenylalanine, in the high normal range. His early development was normal. At the age of 4.5 months he developed a tendency to opistotonus progressing later into frequent attacks of hyperto-nicity of the extremities. The serum phenylalanine rose to 2840 flmol/ l , and the BH4 loading test, as well as the pattern of urinary pterins confirmed the diagnosis of a defective BH" biosynthesis. The child was treated with a combination of BH., L-Dopa/ Carbidopa, and 5-hydroxytryptophan12• SS was diagnosed as hyperphenylalaninemic at the age of four days. Plasma phenylalanine ranged between 1220 and 2400 /lmol/1. At the age of 5 weeks the diagnostic test for BH4 deficiency was perfor-med; the BH. loading test was positive, neopterin was increased, and biopterin was very low. Reduced PTPS activity in the patient's erythrocytes confir-med the diagnosis. SS has been on BH. and neu-rotransmitter therapy since the age of ten weeks. At the age of 12 years her intelligence quotient was 5413. Results and Discussion Table 1 shows four PTPS deficient patients stu-died by our group (JRS, UT, LL and SS) with re-duced PTPS activity in fibroblasts and erythrocytes. Three other patients described by Shintaku l '. IS and Imamura et al. ls were all of the central type with reduced or absent activity of PTPS in the erythro-cytes. In one patient with the central type of PTPS deficiency, described by Ashida et a1. 16 , the activity of the enzyme in fibroblasts and erythrocytes was 20-fold and 5-fold lower, respectively, than that of the controls. Serum phenylalanine concentrations were elevated in all PTPS deficient patients and in Table 1. Summary of information from patients with PTPS deficiency. Patient Urinary pterins Serum Phe PTPS activity Mutation Alteration (phenotype) (mmol/mol creat) (/lmol/l) Erythrocytes Fibroblasts on on Neo Bio %Bio* (/lU/g Hb) (/lU/ mg prot.) DNA level protein level JRS 6.5 0.63 9.0 360 1.5 0.03 C ss to T Rl6C (peripheral) ~G370-G3.3 Kl20 ---+ stop lIT 9.1 0.19 2.1 1200 0 :5:0.02 G 83 to A R25Q (central) LL 34.3 0.41 1.2 IS00 1.3 :5:0.02 ~G178-GI80 ~V57 (central) ~T173 -G I 9S K54 ---+ stop SS 30.3 0.13 0.4 1220 0.5 :5:0.02 C269 to T PS7L (central) KH** 15.6 0.04 < 0.1 970 0.1 C 268 to T PS7S (central) KY** 10.4 0.D7 0.7 1550 0 C 268 to T PS7S (central) YI** 4O.S 0.12 0.3 6S0 1.0 C 268 to T P87S (central) C 295 to A D96N XY*** 5.7# 2840 0.43 A 3• 9 to G Ill4V (central) Controls 1.1-4.0 0.5-0.3 18-63 < 120 11-29 1.9-2.6 none none lINeo/Bio * %Bio= 100*Bio/ (Neo+ Bio) ** Ref. 14,15 *** Ref. 16 Pteridines / Vol. 6 / No.3  Oppliger et a!. . ... !~ ~-':: .:-. .:::-. .:. i- 515 in patients with 6-pyruvoyl-tetrahydropterin synthase deficiency 143 I1l4V I 100 bp 5' 3' j R25Q t> V57 D96N K120 -Stop Figure L Human cDNA sequence encoding PTPS, and lo-cations of mutations in patients with PTPS deficiency. all cases initially low biopterin and high neopterin levels were detected. Five patients were found to be homozygous for the mutations R25Q, P87L. 11 14V, and two patients showed the same mutation P87S (Figure 1). The other three patients harbor the compound hetero-zygote mutations R16C/K120 ~ stop, a V57 /K54 ~ stop and P87S/D96N. All mutations found in the cDNA of PTPS deficient patients are in highly conserved regions. The codon at position 87 sems to be a hot spot for mutations in the 435bp reading frame of the PTPS cDNA. Four of eight analyzed patients showed a mutation at this position. PTPS deficient patients with loower PTPS acti-vities in erythrocytes and in fibroblasts showed also higher serum phenylalanine levels, higher neopterin and lower biopterin levels. Data presented in Table I and those from previous studies l ? suggested that patients with the peripheral type of PTPS deficiency presented intermediate biochemical abnormalities. Biochemical characterization including kinetic and crosslinking experiments will reveal structural and functional differences between mutant proteins and the wild type PTPS. There was so far no correlation between the phenotype and genotype. Acknowledgments We thank L. Kierat, A. Matasovic, and S. Holm for technical help and M .Killen for editorial help. This work was supported by the Swiss National Science Foundation project no. 31-33897.92. References I. Takikawa S. Curtius HCh, Redweik U, Leimbacher W, Ghisla S. Biosynthesis of tetrahydrobiopterin. Purifica-nlion and characterization of 6-pyruvoyl-tetrahydropte-rin synthast from human liver. Eur 1 Biochem. 1986; 161: 295-302. 2. Nichol CA. Smi lh GK Ouch DS. Biosynthesis and metabolism of ltlr J h\orobiopterin and molybdopterin. Annu. Rev. Biochem. 1985; 54: 729-764. 3. Nathan C, Xie QW. Regulation of biosynthesis of nitric oxide. 1. BioI. Chern. 1994; 269: 13725-13728. 4. Blau N, Thony B, Heizmann CW, Dhondt lL. Tetra-hydrobiopterin deficiency: From phenotype to genotype. Pteridines. 1993; 4: 1-10. 5. Niederwieser A, Shintaku H, Leimbacher W, Curtius HC, Hyanek 1, Zeman 1, Endres W . "Peripheral" tetra-hydrobiopterin deficiency with hyperphenylalaninaemia due to incomplete 6-pyruvoyl tetrahydropterin synthase deficiency or heterozygosity. Eur. 1. Pediatr. 1987; 146: 228-232. 6. Takahashi T. Kodama S. Nishio H. Takumi T, Matsuo T, Hase Y. Sawada y. Transient hyperphenylalaninaemia with a high neopterin to biopterin ratio in urine. 1 Inberit Metab Dis. 1985; 8: 105-108. 7. Thony B, Leimbacher W , Biirgisser D. Heizmann CW. Human 6-pyruvoyltetrahydropterin synthase: cDNA cloning and heterologous expression of the recombinant enzyme. Biochem Biophys Res. Commun. 1992; 189: 1437-1443. 8. Niederwieser A, Staudenmann W, Wetzel E. High-per-formance liquid chromatography with column switching for the analysis of biogenic amine metabolites and pte-rins. J. Chromatogr. 1984; 290: 237-246. 9. Shintaku H, Niederwieser A, Leimbacher W, Curtius He. Tetrahydrobiopterin deficiency: assay for 6-pyru-voyl-tetrahydropterin synthase activity in erythrocytes. and detection of patients and heterozygous carriers. Eur 1. Pediatr. 1988; 147: 15-19. 10. Thony B, Leimbacher W, Blau N, Harvie A, Heizmann CWo Hyperphenylalaninemia due to defect in tetrahyd-robiopterin metabolism: molecular characterization of mutations in 6-pyruvoyl-tetrahydropterin synthase. Am. J. Hum. Genet. 1994; 54: 728-792. I L Sambrook J, Fritsch EF, Maniatis T, Eds .. Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor, 1989 12. Beck N, Brandt Nl. Christensen A, Pedersen PS. Dia-gnostic and therapeutic aspects of dihydrobiopterin de-ficiency. Acta Paediatr Scand. 1993; 72: 449-454. 13. Birnbacher R, Scheibenreiter S, Blau N, Bieglmayer C. Frisch H, Wald chauser F. Tetrahydrobiopterin deficie-ncy: a model for cerebral catecholamine and serotonin shortage. Endocrine studies in an affected girl. 1 Clin Endocr Metab. 1995; submitted. 14. Shintaku H. Early diagnosis of 6-pyruvoyl-tetrahydrop-terin synthase deficiency. Pteridines. 1994; 5: 18-27. 15. Imamura T, Okano Y, Sawada Y, Hase Y, Oura T, Isshiki G , Shintaku H. A missense mutation of 6-pyruvoyl-tet-rahydropterin synthase deficiency in Japanese. Pteridi-nes. 1994; 5: 31. 16. Ashida A. Owada M , Hatakeyama K. A missense mu-tation(A to G) of 6-pyruvoyltelrahydropterin synthase in tetrahydrobiopterin-deficient form of hyperphenylalani-nemia. Genomics. 1994; 24: 408-410. 17. Blau N, Dhondt JL. BlODEF: International database of tetrahydrobiopterin deficiencies. Ziirich & Lille. 1995. Pteridines / VoL 6 / No.3 

Mutation Analysis In Patients with 6-Pyruvoyl-Tetrahydropterin Synthase Deficiency

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