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.
Competing interests: None declared
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References
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): 8–12
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):158–163
Dipple KM, McCabe ER (2000) Phenotypes of patients with “simple” Mendelian disorders are complex traits: thresholds, modifiers and system dynamics. Am J Hum Genet 66:1729–1735
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):116–121
Eiken HG, Knappskog PM, Apold J (1993) Restriction enzyme-based assays for complete genotyping of phenylketonuria patients. Dev Brain Dysfunct 6:53–59
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 101:16903–16908
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 4:69–89
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):154–159
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):142–148
Guttler F, Guldberg P (2000) Mutation analysis anticipates dietary requirements in phenylketonuria. Eur J Pediatr 159:S150–S153
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):880–881
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 63:71–79
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):165–171
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 61:1309–1317
Kim SW, Jung J, Oh HJ (2006) Structural and functional analyses of mutations of the human phenylalanine hydroxylase gene. Clin Chim Acta 365:279–297
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):389–393
Kure S, Hou DC, Ohura T et al (1999) Tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency. J Pediatr 135(3):375–378
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):602–612
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):544–550
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 370:504–510
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 92:287–291
Muntau AC, Röschinger W, Habich M et al (2002) Tetrahydrobiopterin as an alternative treatment for mild phenylketonuria. N Engl J Med 347(26):2122–2132
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–S16
Pey AL, Pérez B, Desviat LR et al (2004) Mechanisms underlying responsiveness to tetrahydrobiopterin in mild phenylketonuria mutations. Hum Mutat 24(5):388–399
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–S92
Scriver CR, Waters PJ (1999) Monogenic traits are not simple. Trends Genet 15:267–272
Scriver CR et al (2003) PAHdb 2003: What a locus-specific knowledgebase can do. Hum Mutat 21:333–344 (http://www.pahdb.mcgill.ca)
Scriver CR (2007) The PAH gene, phenylketonuria, and a paradigm shift. Hum Mutat 28:831–845
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)
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):2628–2641
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
Stojiljkovic M, Jovanovic J, Djordjevic M et al (2006) Molecular and phenotypic characteristics of phenylketonuria patients in Serbia and Montenegro. Clin Genet 70:151–155
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):161–167
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):294–299
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):81–83
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 86–100
Trefz FK, Scheible D, Götz H, Frauendienst-Egger G (2009a) Significance of genotype in tetrahydrobiopterin-responsive phenylketonuria. J Inherit Metab Dis 32(1):22–26
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 154:700–707
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):27–31
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):4–17
Zschocke J (2003) Phenylketonuria mutations in Europe. Hum Mutat 21:345–356
Zurflüh MR, Zschocke J, Lindner M et al (2008) Molecular genetics of tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency. Hum Mutat 29:167–175
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This work has been funded by the Ministry of Education and Science, Republic of Serbia, grant No. III 41004.
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Communicated by: Nenad Blau
Synopsis
Synopsis
Analysis of Serbian PKU patients showed that Phe tolerance was a better parameter for phenotypic classification of patients carrying p.L48S mutation and the genotype study identified significant number of individuals that may respond to BH4 supplementation therapy.
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Djordjevic, M. et al. (2012). Molecular Genetics and Genotype-Based Estimation of BH4-Responsiveness in Serbian PKU Patients: Spotlight on Phenotypic Implications of p.L48S. In: Zschocke, J., Gibson, K.M., Brown, G., Morava, E., Peters, V. (eds) JIMD Reports – Case and Research Reports, 2012/6. JIMD Reports, vol 9. Springer, Berlin, Heidelberg. https://doi.org/10.1007/8904_2012_178
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DOI: https://doi.org/10.1007/8904_2012_178
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