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By Dr. Fleming Guttler, M.D., Ph.D., Kennedy Institute, Copenhagen, Denmark
In phenylketonuria the capacity of the liver to convert the amino acid phenylalanine to tyrosine is disturbed. The conversion includes adding an oxygen and a hydrogen atom to the phenylalanine molecule. The process is catalyzed by the enzyme phenylalanine hydroxylase, which is deficient in PKU. The oxygen molecule is coming from the air and the hydrogen molecule from a co-enzyme called BH4.
The Enzyme
The tertiary structure of the enzyme protein has recently been established. The enzyme forms a beautiful tetramer, which means that four enzyme molecules are working together. Each enzyme binds an iron atom just as hemoglobin does in the blood. Similar to hemoglobin, the iron atom in phenylalanine hydroxylase binds the oxygen necessary to form tyrosine. The way that iron, oxygen, BH4 and phenylalanine are bound to the enzyme protein has been established quite recently.2,3 The phenylalanine hydroxylase enzyme consists of three regions: a regulatory region, a region which catalyzes the formation of tyrosine, and a tetramerization region which is responsible for combining the four enzymes.2 The regulatory region binds phenylalanine. This is appropriate. When the phenylalanine concentration is elevated, high amounts of phenylalanine bind to the regulatory region and the enzyme activity becomes high, and when phenylalanine concentrations are low the enzyme activity is low.
The Gene
The enzyme is coded for by two genes located on the chromosomes in the nucleus of the cell. The genetic code, which is responsible for the correct composition of the 452 amino acids (which form the enzyme protein) is transferred from the nucleus into the cytoplasma of the cell where the enzyme protein is synthesized. If there is a mutation in the gene in the nuclei, this mutation is also transported to the cytoplasma and the composition of the amino acids in the protein will be incorrect and the enzyme activity deficient.4,5
The Mutations
The gene is built up by four building blocks named A, C, G, T. For each of the twenty amino acids, which in a very precise order forms the enzyme, there is a particular genetic code consisting of three of the four building blocks. If one of the three building blocks in a code is mutated, the enzyme's synthesis is disturbed and the enzyme is not functioning normally. There are different types of mutations. The mutation may be so severe that the enzyme synthesis stops and no enzyme is produced or the mutation may be milder and some enzyme function is retained. Today we know more than 400 mutations causing various degrees of phenylalanine hydroxylase deficiency.6 Each of us has inherited one gene from our mother and another from our father. The genes may have different mutations and the combinations of these two different mutations is responsible for the degree of phenylalanine hydroxylase deficiency which may be so mild that no dietary treatment is necessary (this is called mild hyperphenylalaninemia). Or the combination may cause one of the more severe phenotypes classified as mild PKU, moderate PKU, and classical PKU.7
If a child has inherited a severe mutation, which completely knocks out enzyme activity (called a "null mutation"), together with a mutation that allows some enzyme activity, it is possible in this child to examine the degree of PKU associated with the mutation, with some enzyme activity left.8
The Genotype and Degree of PKU
In a collaborative study between 11 PKU centers the two mutations in 700 PKU individuals were determined and 136 different mutations were found in total. By looking at the degree of PKU in children with a null mutation and a milder mutation, it was possible to associate the degree of PKU with the gene that had some enzyme production. In that way it has been possible to allocate 105 different mutations to the different degrees of PKU (severe PKU, moderate PKU, mild PKU, and mild hyperphenylalaninemia). By knowing the degree of PKU associated with each of 105 mutations it is possible in a newborn to predict the degree of PKU. This can be done in more than 5000 different combinations of the 105 mutations. The information is very useful for the dietician and the parents taking care of the management of the dietary treatment of their child.8
Genotype and Intelligence Quotient (IQ)
Determination of the two mutant genes has also been performed in 167 pregnant PKU females as part of a collaborative study in the US and Canada. There was a close association between the type of inherited mutations and untreated phenylalanine levels, those with two severe mutations at the highest phenylalanine levels (1600 micromol/L or 27 mg/dl) and those with mild hyperphenylalaninemia at the lowest phenylalanine levels (500 micromol/L or 8 mg/dl). The PKU females were born 20-30 years ago. During those days it was common in the US to stop dietary treatment at 6 years of age. However, some were treated longer. In this group of females there is a significant correlation between the severity of the mutations and the IQ: the more severe the mutations, the lower the IQ. Those who were treated for more than 6 years had higher IQ scores.9 A similar study has been performed in 108 Danish PKU children who so far had been on diet for 10-14 years of age. Their IQ is normal and independent of the severity of their mutations.
Dietary Treatment in Late Diagnosed PKU
There is no doubt that a normal cognitive development (IQ) requires early dietary treatment (within the first three weeks of life) and well controlled phenylalanine levels at least for the first 10 years of life. Is there any beneficial effect of starting dietary treatment in late diagnosed PKU individuals?
A recent study of 59 PKU individuals diagnosed later than 3 months of age and followed for at least 18 years demonstrates that 28 who remained on a phenylalanine restricted diet during the intervening years showed a significant intellectual improvement, with an increase from mean IQ 44 to mean IQ 73. Those who were able to keep a good dietary control had a mean IQ of 82, those with a fair control an IQ of 77, and those with poor control an IQ of 61. The effect of treatment was not associated with the type of mutations and it can be concluded that a PKU individual will benefit substantially by providing a phenylalanine restricted diet even when late-diagnosed and slightly mentally retarded.10
Brain Phenylalanine Levels
It has also quite recently been observed that some individuals have normal intelligence (IQ > 100), in spite of phenylalanine levels > 1200 micromol/L (20 mg/dl) and late and poor treatment or no treatment at all. These individuals apparently have low brain phenylalanine levels. So, some PKU individuals are spared mental retardation because of a mechanism protecting the brain from influx of phenylalanine.11
Pregnancy and PKU
Finally, I will mention the recent results of the Maternal PKU study which is the result of a collaboration between all PKU centers in US and Canada and some PKU centers in Germany. The Kennedy Institute in Denmark is attached to this study. The study began in 1984 and during the intervening years, 572 pregnancies in women with mild hyperphenylalaninemia or PKU and 99 control pregnant females and their outcomes have been evaluated. The main result is that only PKU women treated before and during pregnancy had an offspring outcome similar to females with mild hyperphenylalaninemia and phenylalanine values below 600 micromol/L (10 mg/dl).12
References
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Guttler F, Azen C, Guldberg P, Romstad A, Hanley WB, Levy HL, Matalon R, RouseBM, Trefz F, de la Cruz F, and Koch R. Relationship among genotype, biochemical phenotype, and cognitive performance in females with phenylalanine hydroxylase deficiency. Pediatrics 104 (258-262) 1999.
Koch R, Moseley K, Ning J, Romstad A, Guldberg P. and Guttler F. Long-term beneficial effects of the phenylalanine-restricted diet in late-diagnosed individuals with phenylketonuria. Mol. Genet. Metab. 67(148-155)1999.
Moats RA, Koch R, Moseley K, Guldberg P, Guttler F, Boles RG and Nelson MD Jr. Brain phenylalanine concentration in the management of adults with phenylketonuria. J. Inher. Metab. Dis. 22 (in press) 1999.
Koch R, Azen C, Friedman B, Guttler F, Hanley W, Levy H et al. The international collaborative study of maternal phenylketonuria. Status report 1998. Mental Retard. Develop. Disab. Res. Rev. 5 (117-121) 1999.