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Molecular Genetics and Genotype-Based Estimation of BH4-Responsiveness in Serbian PKU Patients: Spotlight on Phenotypic Implications of p.L48S

  • Maja Djordjevic
  • Kristel Klaassen
  • Adrijan Sarajlija
  • Natasa Tosic
  • Branka Zukic
  • Bozica Kecman
  • Milena Ugrin
  • Vesna Spasovski
  • Sonja Pavlovic
  • Maja Stojiljkovic
Research Report
Part of the JIMD Reports book series (JIMD, volume 9)

Abstract

Phenylketonuria (PKU) is caused by mutations in the gene encoding phenylalanine hydroxylase (PAH) enzyme. Here, we report the updated spectrum of PAH mutations in 61 Serbian PKU patients. By using both DGGE/DNA sequencing and PCR-RFLP, we identified 26 disease-causing mutations (detection rate 99%). The most frequent ones were p.L48S (31%), p.R408W (16.4%), p.P281L (6%), p.E390G (5.2%), and p.I306V (5.2%). Homozygosity value indicated high heterogeneity of Serbian population.

To overcome possible pitfalls of patients’ phenotypic classification, we used two parameters: pretreatment/maximal phenylalanine blood concentration and Phe tolerance. The two phenotypes did not match only for patients with p.L48S. Therefore, we used Mann-Whitney statistical test to compare pretreatment/maximal blood Phe concentration and Phe tolerance detected in patients with p.[L48S];[null] and p.[missense];[null] genotypes. For patients with p.L48S, our results implied that Phe tolerance is a better parameter for phenotypic classification. Also, Fisher’s exact test was used to compare p.L48S effect on phenotype of homozygous and functionally hemizygous patients. Our findings showed that effect of p.L48S was altered in functional hemizygotes. Moreover, phenotypic inconsistency found in homozygotes suggested that interallelic complementation and/or additional factors play a role in genotype-phenotype correlation.

Since BH4-supplementation therapy is not available in Serbia, we made the first estimation of its potential benefit based on patients’ genotypes. In the analyzed cohort, the total frequency of BH4-responsive mutations was 52.6%. Furthermore, we found a significant number of genotypes (26.2% BH4-responsive and 51% probably BH4-responsive) that may respond to BH4 therapy. This led us to a conclusion that BH4-supplementation therapy could bring benefit to Serbian PKU patients.

Notes

Acknowledgments

This work has been funded by the Ministry of Education and Science, Republic of Serbia, grant No. III 41004.

References

  1. Aguado C, Perez B, Garcia MJ, Belanger-Quintana A, Martinez-Pardo M, Ugarte M, Desviat LR (2007) BH4 responsiveness associated to a PKU mutation with decreased binding affinity for the cofactor. Clin Chim Acta 380(1–2): 8–12CrossRefPubMedGoogle Scholar
  2. Blau N, Bélanger-Quintana A, Demirkol M, Feillet F, Giovannini M, MacDonald A, Trefz FK, van Spronsen FJ (2009) Optimizing the use of sapropterin (BH(4)) in the management of phenylketonuria. Mol Genet Metab 96(4):158–163CrossRefPubMedGoogle Scholar
  3. Dipple KM, McCabe ER (2000) Phenotypes of patients with “simple” Mendelian disorders are complex traits: thresholds, modifiers and system dynamics. Am J Hum Genet 66:1729–1735CrossRefPubMedPubMedCentralGoogle Scholar
  4. Dobrowolski SF, Heintz C, Miller T et al (2011) Molecular genetics and impact of residual in vitro phenylalanine hydroxylase activity on tetrahydrobiopterin responsiveness in Turkish PKU population. Mol Genet Metab 102(2):116–121CrossRefPubMedGoogle Scholar
  5. Eiken HG, Knappskog PM, Apold J (1993) Restriction enzyme-based assays for complete genotyping of phenylketonuria patients. Dev Brain Dysfunct 6:53–59Google Scholar
  6. Erlandsen H, Pey AL, Gamez A et al (2004) Correction of kinetic and stability defects by tetrahydrobiopterin in phenylketonuria patients with certain phenylalanine hydroxylase mutations. Proc Natl Acad Sci USA 101:16903–16908CrossRefPubMedGoogle Scholar
  7. Fenech M, El-Sohemy A, Cahill L et al (2011) Nutrigenetics and nutrigenomics: viewpoints on the current status and applications in nutrition research and practice. J Nutrigenet Nutrigenomics 4:69–89CrossRefPubMedPubMedCentralGoogle Scholar
  8. Giannattasio S, Dianzani I, Lattanzio P et al (2001) Genetic heterogeneity in five Italian regions: analysis of PAH mutations and minihaplotypes. Hum Hered 52(3):154–159CrossRefPubMedGoogle Scholar
  9. Groselj U, Tansek MZ, Kovac J, Hovnik T, Podkrajsek KT, Battelino T (2012) Five novel mutations and two large deletions in a population analysis of the phenylalanine hydroxylase gene. Mol Genet Metab 106(2):142–148CrossRefPubMedGoogle Scholar
  10. Guttler F, Guldberg P (2000) Mutation analysis anticipates dietary requirements in phenylketonuria. Eur J Pediatr 159:S150–S153CrossRefPubMedGoogle Scholar
  11. Guldberg P, Guttler F (1994) “Broad-range” DGGE for single-step mutation scanning of entire genes: the human phenylalanine hydroxylase gene. Nucleic Acids Res 22(5):880–881CrossRefPubMedPubMedCentralGoogle Scholar
  12. Guldberg P, Rey F, Zschocke J et al (1998) 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 63:71–79CrossRefPubMedPubMedCentralGoogle Scholar
  13. Karacic I, Meili D, Sarnavka V et al (2009) Genotype-predicted tetrahydrobiopterin (BH4)-responsiveness and molecular genetics in Croatian patients with phenylalanine hydroxylase (PAH) deficiency. Mol Genet Metab 97(3):165–171CrossRefPubMedGoogle Scholar
  14. Kayaalp E, Treacy E, Waters PJ, Byck S, Nowacki P, Scriver CR (1997) Human phenylalanine hydroxylase mutations and hyperphenylalaninemia phenotypes: A metanalysis of genotype-phenotype correlations. Am J Hum Genet 61:1309–1317CrossRefPubMedPubMedCentralGoogle Scholar
  15. Kim SW, Jung J, Oh HJ (2006) Structural and functional analyses of mutations of the human phenylalanine hydroxylase gene. Clin Chim Acta 365:279–297CrossRefPubMedGoogle Scholar
  16. Konecki DS, Schlotter M, Trefz FK, Lichter-Konecki U (1991) The identification of two mis-sense mutations at the PAH gene locus in a Turkish patient with phenylketonuria. Hum Genet 87(4):389–393PubMedGoogle Scholar
  17. Kure S, Hou DC, Ohura T et al (1999) Tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency. J Pediatr 135(3):375–378CrossRefPubMedGoogle Scholar
  18. Leandro J, Leandro P, Flatmark T (2011) Heterotetrameric forms of human phenylalanine hydroxylase: co-expression of wild-type and mutant forms in a bicistronic system. Biochim Biophys Acta 1812(5):602–612CrossRefPubMedGoogle Scholar
  19. Leandro J, Nascimento C, de Almeida IT, Leandro P (2006) Co-expression of different subunits of human phenylalanine hydroxylase: evidence of negative interallelic complementation. Biochim Biophys Acta 1762(5):544–550CrossRefPubMedGoogle Scholar
  20. Levy HL, Milanowski A, Chakrapani A et al (2007a) Efficacy of sapropterin dihydrochloride (tetrahydrobiopterin, 6R-BH4) for reduction of phenylalanine concentration in patients with phenylketonuria: a phase III randomised placebo-controlled study. Lancet 370:504–510CrossRefPubMedGoogle Scholar
  21. Levy H, Burton B, Cederbaum S, Scriver C (2007b) Recommendations for evaluation of responsiveness to tetrahydrobiopterin (BH(4)) in phenylketonuria and its use in treatment. Mol Genet Metab 92:287–291CrossRefPubMedGoogle Scholar
  22. Muntau AC, Röschinger W, Habich M et al (2002) Tetrahydrobiopterin as an alternative treatment for mild phenylketonuria. N Engl J Med 347(26):2122–2132CrossRefPubMedGoogle Scholar
  23. Pérez B, Desviat LR, Gómez-Puertas P, Martínez A, Stevens RC, Ugarte M (2005) Kinetic and stability analysis of PKU mutations identified in BH4-responsive patients. Mol Genet Metab 86:S11–S16CrossRefPubMedGoogle Scholar
  24. Pey AL, Pérez B, Desviat LR et al (2004) Mechanisms underlying responsiveness to tetrahydrobiopterin in mild phenylketonuria mutations. Hum Mutat 24(5):388–399CrossRefPubMedGoogle Scholar
  25. Rivera I, Mendes D, Afonso  et al (2011) Phenylalanine hydroxylase deficiency: molecular epidemiology and predictable BH4-responsiveness in South Portugal PKU patients. Mol Genet Metab 104:S86–S92CrossRefPubMedGoogle Scholar
  26. Scriver CR, Waters PJ (1999) Monogenic traits are not simple. Trends Genet 15:267–272CrossRefPubMedGoogle Scholar
  27. Scriver CR et al (2003) PAHdb 2003: What a locus-specific knowledgebase can do. Hum Mutat 21:333–344 (http://www.pahdb.mcgill.ca)CrossRefPubMedGoogle Scholar
  28. Scriver CR (2007) The PAH gene, phenylketonuria, and a paradigm shift. Hum Mutat 28:831–845CrossRefPubMedGoogle Scholar
  29. Scriver CR, Levy H, Donlon J (2008) Hyperphenylalaninemia: phenylalanine hydroxylase deficiency. In: Valle D, Beaudet AL, Vogelstein B, Kinzler KW, Antonarakis S, Ballabio A (eds) Scriver CR, Childs B, Sly WS (eds emeritus) The online metabolic and molecular basis of inherited disease. McGraw-Hill, New York. Online Chapter 77 (www.ommbid.com)
  30. Staudigl M, Gersting SW, Danecka MK et al (2011) The interplay between genotype, metabolic state and cofactor treatment governs phenylalanine hydroxylase function and drug response. Hum Mol Genet 20(13):2628–2641CrossRefPubMedGoogle Scholar
  31. Sterl E, Paul K, Paschke E, et al (2012) Prevalence of tetrahydrobiopterine (BH4)-responsive alleles among Austrian patients with PAH deficiency: comprehensive results from molecular analysis in 147 patients. J Inherit Metab Dis. doi:  10.1007/s10545-012-9485-y CrossRefPubMedGoogle Scholar
  32. Stojiljkovic M, Jovanovic J, Djordjevic M et al (2006) Molecular and phenotypic characteristics of phenylketonuria patients in Serbia and Montenegro. Clin Genet 70:151–155CrossRefPubMedGoogle Scholar
  33. Stojiljkovic M, Stevanovic A, Djordjevic M et al (2007) Mutations in the PAH gene: a tool for population genetic study. Arch Biol Sci 59(3):161–167CrossRefGoogle Scholar
  34. Stojiljkovic M, Pérez B, Desviat LR, Aguado C, Ugarte M, Pavlovic S (2009) The Missense p.S231F phenylalanine hydroxylase gene mutation causes complete loss of enzymatic activity in vitro. Protein J 28(6):294–299CrossRefPubMedGoogle Scholar
  35. Stojiljkovic M, Zukic B, Tosic N et al (2010) Novel transcriptional regulatory element in the phenylalanine hydroxylase gene intron 8. Mol Genet Metab 101(1):81–83CrossRefPubMedGoogle Scholar
  36. Trefz FK, Schmidt H, Bartholome K, Mahle M, Mathis P, Pecht G (1985) Differential diagnosis and significance of various hyperphenylalaninemias. In: Bickel H, Wachtel U (eds) Inherited diseases of amino acid metabolism. Thieme, Stuttgart, pp 86–100Google Scholar
  37. Trefz FK, Scheible D, Götz H, Frauendienst-Egger G (2009a) Significance of genotype in tetrahydrobiopterin-responsive phenylketonuria. J Inherit Metab Dis 32(1):22–26CrossRefPubMedGoogle Scholar
  38. Trefz FK, Burton BK, Longo N et al (2009b) Efficacy of sapropterin dihydrochloride in increasing phenylalanine tolerance in children with phenylketonuria: a phase III, randomized, double-blind, placebo-controlled study. J Pediatr 154:700–707CrossRefPubMedGoogle Scholar
  39. van Sponsen FJ, van Rijn M, Dorgelo B et al (2009) Phenylalanine tolerance can already reliably be assessed at the age of 2 years in patients with PKU. J Inherit Metab Dis 32(1):27–31CrossRefGoogle Scholar
  40. Waters PJ, Parniak MA, Nowacki P, Scriver CR (1998) In vitro expression analysis of mutations in phenylalanine hydroxylase: linking genotype to phenotype and structure to function. Hum Mutat 11(1):4–17CrossRefPubMedGoogle Scholar
  41. Zschocke J (2003) Phenylketonuria mutations in Europe. Hum Mutat 21:345–356CrossRefPubMedGoogle Scholar
  42. Zurflüh MR, Zschocke J, Lindner M et al (2008) Molecular genetics of tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency. Hum Mutat 29:167–175CrossRefPubMedGoogle Scholar

Copyright information

© SSIEM and Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Maja Djordjevic
    • 2
  • Kristel Klaassen
    • 1
  • Adrijan Sarajlija
    • 2
  • Natasa Tosic
    • 1
  • Branka Zukic
    • 1
  • Bozica Kecman
    • 2
  • Milena Ugrin
    • 1
  • Vesna Spasovski
    • 1
  • Sonja Pavlovic
    • 1
  • Maja Stojiljkovic
    • 1
  1. 1.Institute of Molecular Genetics and Genetic EngineeringUniversity of BelgradeBelgradeSerbia
  2. 2.Mother and Child Healthcare Institute “Dr Vukan Cupic”, School of MedicineUniversity of BelgradeBelgradeSerbia

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