Skip Navigation

The Use of Mutation Analysis to Anticipate Dietary Requirements in Phenylketonuria

Skip sharing on social media links

The content in this publication was current at the time it was published, but it is not being updated. The publication is provided for historical purposes only.​

By Flemming Güttler, M.D., Ph.D.

In a broad perspective, the identification of the enzyme defect, the development of effective methods for neonatal screening, and the implementation of dietary therapy regimens in phenylketonuria (PKU) are among the major achievements of medical science. Nevertheless, 40 years of experience with diagnosis and treatment of children with PKU have left us with a number of uncertainties regarding the classification of disease phenotypes and the effectiveness of treatment. This abstract reviews the novel suggestion of using molecular genetic analysis to directly assess the metabolic PKU phenotype and discusses how such data might be used to anticipate the treatment requirements for each individual patient.

The Different Phenotypes of PKU

The marker for neonatal detection of PKU is elevated serum levels (hyperphenylalaninemia) of phenylalanine (Phe) caused by reduced activity of the hepatic enzyme phenylalanine hydroxylase (PAH). The degree of enzyme impairment varies greatly among patients and is reflected in the broad continuum of metabolic phenotypes (Güttler, 1980; Scriver, Kaufman, Eisensmith, et al., 1995). Because direct determination of PAH activity through liver biopsies is not feasible, diagnosis and classification of PAH deficiency is done through indirect methods (Güttler, 1980; Scriver, Kaufman, Eisensmith, et al., 1995). One widely used approach is to determine the amount of dietary Phe tolerated while keeping blood Phe levels within the therapeutic range. On the basis of Phe tolerance data, patients can be assigned to one of four arbitrary phenotype categories: classic PKU, moderate PKU, mild PKU, and mild hyperphenylalaninemia (MHP) (Güttler, Guldberg, 1996).

Unfortunately, there are no international guidelines for classification parameters, including age at testing and therapeutic target Phe levels. This effectively hampers comparisons among patients treated at different centers. Meanwhile, however, a number of recent studies have demonstrated the usefulness of an alternative and more universal system for classification of PAH deficiency that is based on PAH mutation genotypes.

PAH Gene Mutations

Mutations in the gene encoding PAH are the ultimate cause of PAH deficiency (Scriver, Kaufman, Eisensmith, et al., 1995). A patient’s PAH mutation genotype refers to the composition of inherited mutant genes (alleles) and is usually determined by identifying the two disease-causing mutations by means of molecular analysis. The ascertainment of PAH mutations is now nearly complete in many patient populations in Europe and the New World. The combined efforts of members of the PAH Mutation Analysis Consortium have raised the number of known PAH mutations to approximately 400. Each of these mutations has a quantitative effect on PAH activity, which provides the molecular basis for the observed spectrum of metabolic phenotypes (Okano, Eisensmith, Güttler, et al., 1991). One further level of complexity is added by the enormous number of possible mutation combinations; the approximately 400 pathogenic PAH alleles known to date can form more than 80,000 heteroallelic genotypes. (A database of mutations by Scriver, Waters, Sarkissian, and colleagues is accessible on the Internet at External Web Site Policy).

Genotype-Based Prediction of Metabolic Phenotype

Hitherto, the major constraints encountered during attempts to establish a correlation between individual mutations and a PAH-deficiency phenotype have been that patients with two identical mutations (homoallelic genotypes) were available for only a minority of mutations, and that patients with two different mutations (heteroallelic genotypes) were not informative. Some of these difficulties have now been circumvented by studying “functionally hemizygous” patients (Guldberg, Mikkelsen, Henriksen, et al., 1995). These patients carry on one chromosome one of several “null” mutations—that is, mutations that produce proteins with no enzyme activity in vivo. The functionally hemizygous constellation is equivalent to the homozygous constellation in the sense that only the enzyme encoded by the non-null allele contributes to the metabolism of Phe. A meta-analysis (Kayaalp, Treacy, Waters, et al., 1997) and a European multicenter study (Guldberg, Rey, Zschocke, et al., 1998) have identified approximately 35 null mutations and have assigned more than 100 different mutations to particular metabolic phenotypes.

In the European multicenter study, the degree of concordance between predicted versus observed phenotypes was tested in 651 patients for whom exact data on genotypes and metabolic phenotypes were available. The observed phenotype matched the predicted phenotype in 562 of the cases (86 percent), and in only 10 of the cases (1.5 percent) was the observed phenotype more than one phenotype category away from that expected. Notably, there was virtually complete association between genotype and phenotype in the group of individuals with MHP. This group is particularly informative because patients in the group are not being treated and thus can be classified solely on the basis of serum Phe values, with no interference from dietary therapy regimens. A substantial fraction of genotype-phenotype inconsistencies may be due to phenotype “misclassifications” related to differences in criteria and methods used for phenotype assessment (Guldberg, Rey, Zschocke, et al., 1998).

Genotype Related to Outcome and Dietary Requirements

The relationships among genotype, biochemical phenotype, and cognitive performance were studied in 199 PAH-deficient females enrolled in the Maternal PKU Collaborative Study (Güttler, Azen, Guldberg, et al., 1999). Most of them had been treated only for the first 6 years of life. Based on their PAH mutation genotype, the patients were assigned to one of the four classes of severity. Genotype severity was significantly related not only to untreated blood Phe levels but also to cognitive development in terms of intelligence quotient (IQ). Patients with genotypes indicating classic or moderate PKU showed IQ scores of 83 and 84, respectively, whereas the IQ score was 96 in females with a mild PKU genotype. Those who were treated for more than 6 years showed IQ scores 10 points above average for their group (Güttler, Azen, Guldberg, et al., 1999).

Preliminary data from a study of 108 Danish patients with PKU who have been on dietary therapy for between 10 and 14 years show that their median IQ is normal and not dependent on genotype. The results from these two studies demonstrate the importance of maintaining dietary therapy, at least until somewhere between the ages of 10 and 14 years.


DNA analysis may provide a new and powerful tool for refining PKU diagnosis and anticipating dietary requirements in PAH deficiency. PAH mutations may be determined by analysis of DNA extracted from blood deposited on a Guthrie card, and thus a genetic diagnosis can be made immediately after birth, with no further examination of the child. Although still not simple enough to be performed at all screening centers, methods for detection of PAH mutations are now at a stage where genetic diagnosis becomes feasible in the routine diagnosis and management of PKU. One of the most beneficial applications of mutation analysis may be the identification of patients for whom dietary treatment beyond 6 years of age is an absolute requirement for the prevention of mental retardation.


  • Guldberg P, Mikkelsen I, Henriksen KF, Lou HC, Güttler F. In vivo assessment of mutations in the phenylalanine hydroxylase gene by phenylalanine loading: characterization of seven common mutations. Eur J Pediatr 1995;154:551-6.
  • Guldberg P, Rey F, Zschocke J, Romano V, Francois B, Michiels L, et al. 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 1998;63:71-9.
  • Güttler F. Hyperphenylalaninemia: diagnosis and classification of the various types of phenylalanine hydroxylase deficiency in childhood. Acta Paediatr Scand Suppl 1980;280:1-80.
  • Güttler F, Azen C, Guldberg P, Romstad A, Hanley WB, Levy HL, et al. Relationship among genotype, biochemical phenotype, and cognitive performance in females with phenylalanine hydroxylase deficiency. Report from the Maternal PKU Collaborative Study. Pediatrics 1999;104:258-62.
  • Güttler F, Guldberg P. The influence of mutations on enzyme activity and phenylalanine tolerance in phenylalanine hydroxylase deficiency. Eur J Pediatr 1996;155(Suppl. 1):S6-10.
  • Kayaalp E, Treacy E, Waters PJ, Byck S, Nowacki P, Scriver CR. Human PAH mutation and hyperphenylalaninemia phenotypes: a metanalysis of genotype-phenotype correlations. Am J Hum Genet 1997;61:1309-17.
  • Okano Y, Eisensmith RC, Güttler F, Lichter-Konecki U, Konecki DS, Trefz FK, et al. Molecular basis of phenotypic heterogeneity in phenylketonuria. N Engl J Med 1991;324:1232-8.
  • Scriver CR, Kaufman S, Eisensmith RC, Woo SLC. The hyperphenylalaninemias. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The metabolic and molecular bases of inherited disease. New York: McGraw-Hill; 1995. p. 1015-75.
  • Scriver CR, Waters PJ, Sarkissian C, Ryan S, Prevost L, Cote D, et al. PAHdb: a locus-specific knowledgebase. Hum Mutat 2000;15:99-104.

Back to Abstracts

first | previous | next | last

BOND National Institues of Health Home Home Division of Intramural Population Health Research