Abstract
Dihydropteridine reductase (DHPR) catalyses the conversion of quinonoid dihydrobiopterin (qBH2) to tetrahydrobiopterin (BH4), which serves as the obligatory cofactor for the aromatic amino acid hydroxylases. DHPR deficiency, caused by mutations in the QDPR gene, results in hyperphenylalaninemia and deficiency of various neurotransmitters in the central nervous system, with severe neurological symptoms as a consequence. We have studied, at the clinical and molecular levels, 17 patients belonging to 16 Turkish families with DHPR deficiency. The patients were detected at neonatal screening for hyperphenylalaninemia or upon the development of neurological symptoms. To identify the disease causing molecular defects, we developed a sensitive screening method that rapidly scans the entire open reading frame and all splice sites of the QDPR gene. This method combines PCR amplification and “GC-clamping” of each of the seven exonic regions of QDPR, resolution of mutations by denaturing gradient gel electrophoresis (DGGE), and identification of mutations by direct sequence analysis. A total of ten different mutations were identified, of which three are known (G23D, Y150C, R221X) and the remaining are novel (G17R, G18D, W35fs, Q66R, W90X, S97fs and G149R). Six of these mutations are missense variants, two are nonsense mutations, and two are frameshift mutations. All patients had homoallelic genotypes, which allowed the establishment of genotypephenotype associations. Our findings suggest that DGGE is a fast and efficient method for detection of mutations in the QDPR gene, which may be useful for confirmatory DNA-based diagnosis, genetic counselling and prenatal diagnosis in DHPR deficiency.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Abrams ES, Stanton VP (1992) Use of denaturing gradient gel electrophoresis to study conformational transitions in nucleic acids. Methods Enzymol 212: 71–104
Blau N, Dhondt J (2000) International database of tetrahydrobiopterin deficiencies http://www.bh4.org/biodefl.html (accessed June 2000)
Blau N, Heizmann CW, Sperl W, Korenke GC, Hoffmann GF, Smooker PM, Cotton RG (1992) Atypical (mild) forms of dihydropteridine reductase deficiency: neurochemical evaluation and mutation detection. Pediatr Res 32: 726–730
Blau N, Thöny B, Heizmann CW, Dhondt JL (1993) Tetrahydrobiopterin deficiency: from phenotype to genotype. Pteridines 4: 1–10
Blau N, Thöny B, Spada M, Ponzone A (1996) Tetrahydrobiopterin and inherited hyperphenylalaninemias. Turk J Pediatr 38: 19–35
Blau N, Thöny B, Dianzani I (2000) Database of mutations causing tetrahydrobiopterin deficiencies http://www.bh4.org/biomdb1. html (accessed June 2000)
Coşkun T, Özalp I, Tokatli A, Blau N, Niederwieser A (1993) Hyperphenylalaninaemia due to tetrahydrobiopterin deficiency: a report of 16 cases. J Inherit Metab Dis 16: 605–607
Dahl HM, Hutchison W, McAdam W, Wake S, Morgan FJ, Cotton RGH (1987) Human dihydropteridine reductase: characterisation of a cDNA clone and its use in analysis of patients with dihydropteridine reductase deficiency. Nucleic Acids Res 15: 1921–1932
Dianzani I, Howells DW, Ponzone A, Saleeba JA, Smooker PM, Cotton RG (1993) Two new mutations in the dihydropteridine reductase gene in patients with tetrahydrobiopterin deficiency. J Med Genet 30: 465–469
Dianzani I, de Sanctis L, Smooker PM, Gough TJ, Alliaudi C, Brusco A, Spada M, Blau N, Dobos M, Zhang H, Yang N, Ponzone A, Armarego WLF, Cotton RGH (1998) Dihydropteridine reductase deficiency: physical structure of the QDPR gene, identification of two new mutations and genotype-phenotype correlations. Hum Mutat 12: 267–273
Ferec C, Audrezet M, Mercier B, Guillermit H, Moullier P, QuereI, Verlingue C (1993) Detection of over 98% cystic fibrosis mutations in a Celtic population. Nat Genet 1: 188–191
Firgaira F, Cotton RGH, Danks DM (1981) Isolation and characterization of dihydropteridine reductase from human liver. Biochem J 197: 31–43
Firgaira FA, Cotton RGH, Danks DM, Fowler K, Lipson A, Yu JS (1983) Prenatal determination of dihydropteridine reductase in a normal fetus at risk for malignant hyperphenylalaninemia. Prenat Diagn 3: 7–11
Guldberg P, Henriksen KF, Güttier F (1993) Molecular analysis of phenylketonuria in Denmark: 99% of the mutations detected by denaturing gradient gel electrophoresis. Genomics 17: 141–146
Guldberg P, Nedergaard T, Nielsen HJ, Olsen AC, Ahrenkiel V, Zeuthen J (1997) Single-step DGGE-based mutation scanning of the p53 gene: application to genetic diagnosis of colorectal cancer. Hum Mutat 9: 348–355
Henke W, Herdel K, Jung K, Schnorr D, Loening SA (1997) Betaine improves the PCR amplification of GC-rich DNA sequences. Nucleic Acids Res 25: 3957–3958
Kuhl P, Olek K, Wardenbach P, Grzeschik KH (1979) Assignment of a gene for human quinoid-dihydropteridine reductase (QDPR, EC 1.6.5.1) to chromosome 4. Hum Genet 53: 47–49
Lerman LS, Silverstein K (1987) Computational simulation of DNA melting and its application to denaturing gradient gel electrophoresis. Methods Enzymol 155: 482–501
Lockyer J, Cook RG, Milstien S, Kaufman S, Woo SLC, Ledley FD (1987) Structure and expression of human dihydropteridine reductase. Proc Natl Acad Sci USA 84: 3329–3333
Myers RM, Maniatis T, Lerman LS (1987) Detection and localization of single base changes by denaturing gradient gel electrophoresis. Methods Enzymol 155: 501–527
Özalp I, Coşkun T, Tokatli A, Tokol S, Özgüc M, Koksal G, Erdem G, Yurdakok M (1995) Neonatal PKU screening in Turkey: 7 years experience in a developing country. Screening 4: 139–147
Romstad A, Guldberg P, Blau N, Güttler F (1999) Single-step mutation analysis of the 6-pyruvoyltetrahydropterin synthase gene in patients with hyperphenylalaninemia. Clin Chem 45: 2102–2108
Rychlik W, Rhoads RE (1989) A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA. Nucleic Acids Res 17: 8543–8551
Scriver CR, Kaufman S, Eisensmith RC, Woo SLC (1995) The hyperphenylalaninemias. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds) The metabolic and molecular bases of inherited disease. McGraw-Hill, New York, pp 1015–1075
Scrutton NS, Berry A, Perham RN (1990) Redesign of the coenzyme specificity of a dehydrogenase by protein engineering. Nature 343: 38–43
Sheffield VC, Cox DR, Lerman LS, Myers RM (1989) Attachment of a 40-base-pair G+C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes. Proc Natl Acad Sci USA 86: 232–236
Smith I, Brenton D (1996) Hyperphenylalaninemias. In: Fernandes J, Saudubray JM, van den Berghe G (eds) Inborn metabolic diseases: diagnosis and treatment. Springer-Verlag, Berlin, pp 147–160
Smith I, Hyland K, Kendall B (1985) Clinical role of pteridine therapy in tetrahydrobiopterin deficiency. J Inherit Metab Dis 8: 39–45
Smooker PM, Howells DW, Cotton RGH (1993) Identification and in vitro expression of mutations causing dihydropteridine reductase deficiency. Biochemistry 32: 6443–6449
Smooker PM, Gough TJ, Cotton RGH, Alliaudi C, de Sanctis L, Dianzani I (1999) A series of mutations in the dihydropteridine reductase gene resulting in either abnormal RNA splicing or DHPR protein defects. Hum Mutat 13: 503–504
Su Y, Varughese KI, Xuong NH, Bray TL, Roche DJ, Whiteley JM (1993) The crystallographic structure of a human dihydropteridine reductase NADH binary complex expressed in Escherichia coli by a cDNA constructed from its rat homologue. J Biol Chem 268: 26836–26841
Tuncbilek E, Koc I (1994) Consanguineous marriage in Turkey and its impact on fertility and mortality. Ann Hum Genet 58: 321–329
Varughese KI, Skinner MM, Whiteley JM, Matthews DA, Xuong NH (1992) Crystal structure of rat liver dihydropteridine reductase. Proc Natl Acad Sci USA 89: 6080–6084
Varughese KI, Xuong NH, Kiefer PM, Matthews DA, Whiteley JM (1994) Structural and mechanistic characteristics of dihydropteridine reductase: a member of the Tyr-(Xaa)3-Lys-containing family of reductases and dehydrogenases. Proc Natl Acad Sci USA 91: 5582–5586
Wierenga RK, De Maeyer MCH, Hol WGJ (1985) Interaction of pyrophosphate moieties with α-helixes in dinucleotide binding proteins. Biochemistry 24: 1346–1357
Wierenga RK, Terpstra P, Hol WGJ (1986) Prediction of the occurrence of the ADP-binding βαβ-fold in proteins, using an amino acid sequence fingerprint. J Mol Biol 187: 101–107
Yilmaz E, Cali F, Romano V, Özalp I, Cosşkun T, Tokatli A, Kalkanoğlu HS, Özgüc M (2000) Molecular basis of mild hyperphenylalaninemia in Turkey. J Inherit Metab Dis 23: 523–525
Zhang HP, Yang N, Armarego WLF (1996) In vitro mutagenesis of human dihydropteridine reductase at the active site and at altered sites found in the reductase of deficient children. Pteridines 7: 126–136
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Romstad, A., Kalkanoğlu, H.S., Coşkun, T. et al. Molecular analysis of 16 Turkish families with DHPR deficiency using denaturing gradient gel electrophoresis (DGGE). Hum Genet 107, 546–553 (2000). https://doi.org/10.1007/s004390000407
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/s004390000407