The metabolic disorder phenylketonuria (PKU) was first described in 1934 by A. Folling, who was investigating the excretion of ketone bodies in the urine of two mentally retarded children. The reagent in use, acidified FeC^, typically turns a purple-red color with ketones. The urine of these children turned bright blue-green instead. Examination of additional mentally retarded children uncovered eight more who were similar, both clinically and in their urine reactions. Folling subsequently demonstrated that the responsible urinary constituent was phenylpyruvic acid, thus the name phenylketonuria. In addition to mental retardation, affected children share a number of clinical features, including irritability, seizures, eczema, and a "mousy odor." They tend to be more lightly pigmented than others in the same family.
The rarity of this disorder in the general population and the occurrence of more than one affected person in a sibship suggested recessive inheritance. The equal occurrence of males and females and the fact that the parents and other relatives were not affected was consistent with autosomal recessive inheritance.
1. The Biochemistry of PKU
F{0}lling suggested that patients who have PKU have a block in metabolism of phenylalanine, and in 1947 G.A. Jervis proposed that the metabolic error is in the conversion of phenylalanine to tyrosine. He subsequently proved this hypothesis by showing that liver from a PKU patient cannot make the conversion. Thus, PKU was among the earliest examples of a metabolic error, a term proposed by A.E. Garrod in 1909, and it has served as a model for other metabolic defects.
Phenylalanine is efficiently reabsorbed in the kidneys, leading to maintenance of very high blood levels. In normal persons, the blood phenylalanine is ca. 60 |iM. The level in PKU patients can be 40 times higher. This leads to formation and excretion of otherwise rare metabolites of phenylalanine, such as phenylpyruvic acid, phenylacetic acid, and o-hydroxyphenylacetic acid. These metabolites very likely contribute to the clinical features of PKU by interfering with various metabolic and developmental processes.
Phenylalanine hydroxylase (PAH; L-phenylalanine-4-monooxygenase; EC 1.14.16.1) is the enzyme that is ordinarily nonfunctional in PKU. The predicted polypeptide molecular weight is 51,862 daltons, but the enzyme is usually a homotetramer. It catalyzes the oxidation of L-phenylalanine, using tetrahydrobiopterin as a cofactor (Fig. 1). Production of PAH occurs almost entirely in the liver. A large number of PAH alleles are known. Some produce completely inactive PAH; others produce PAH that has reduced or normal activity. Persons whose allelic combinations produce PAH activity <1% of normal have classical PKU. If there is some activity, a mild form of the disorder may result. Heterozygotes, with one normal and one inactive allele, are phenotypically normal even though the total enzymic activity is reduced.
Figure 1. Three enzymes that are important in the conversion of phenylalanine to tyrosine. Additional enzymes and cofactors are required to form BH4. Reduced activity of phenylalanine hydroxylase (PAH) can result from structural modifications of PAH or from deficiency of BH4.
2. Treatment of PKU
Humans cannot make phenylalanine or tyrosine and must depend on diet for these two essential amino acids. The phenylalanine content of foods is in excess of body requirements for protein synthesis, and the surplus is metabolized via tyrosine. This suggested the possibility of reducing the accumulation of phenylalanine by reducing the dietary intake. Such efforts, initiated in 1953 by H. Bickel, ultimately proved successful. The low-phenylalanine diet must be introduced soon after birth of an affected infant in order to avoid permanent damage to the developing central nervous system.
With the development of an effective method of treatment, the urgency of neonatal diagnosis became apparent. In the early 1960s, R. Guthrie developed a microbial test using whole blood from a heel stick. This rapid and inexpensive test made large scale screening possible, and required screening of newborns for elevated blood phenylalanine has become widespread in western countries. Once the central nervous system has matured fully, persons with PKU are somewhat less sensitive to increased blood levels of phenylalanine. This includes not only regulation of protein intake but also avoidance of the artificial sweetener aspartame, which is a dipeptide derivative of aspartic acid and phenylalanine. However, long-term studies have shown benefits of continued treatment of adults.
Although dietary regulation has been highly successful, it is not a perfect treatment. Treated patients show mildly depressed IQ compared to non-PKU controls. One suggested cause is increased dysmyelination with age in the presence of elevated blood phenylalanine. Also, tyrosine is lower in PKU patients, treated or untreated, and this may lead to deficiency of neurotransmitters.
In view of the single gene and its single protein product that are defective in PKU, this disorder would seem a good candidate for gene therapy. It has not been considered a high priority, however, because of the effectiveness of dietary restriction of phenylalanine. When procedures for gene therapy have been sufficiently developed and tested, it is likely that they will be applied to PKU. Introduction of functional PAH genes during the neonatal period should cure the metabolic defect. This, in effect, was the case with one young boy who received a liver transplant for reasons unrelated to his PKU status.
The successful treatment of PKU by diet has led to the problem of maternal PKU, which occurs in successfully treated women who are homozygous for PKU and whose offspring therefore are heterozygous. The developing fetus, although genotypically normal (but heterozygous), depends on the mother to regulate blood phenylalanine. The fetus is also highly susceptible to moderate increases in blood phenylalanine. Prior to conception and during pregnancy, a PKU mother must regulate her blood phenylalanine levels rigorously to avoid damage to the fetus. The recommended maximal level is 360 pM.
One consequence of introducing screening was the recognition that some children have levels of phenylalanine that are above normal but that do not reach the levels that lead to pathology and that do not require dietary management. These cases of "hyperphenylalaninemia," defined as plasma levels >120 |iM, are often due to homozygosity for PAH alleles that have low but nonzero enzyme activity. In other instances, the hyperphenylalaninemia is transient in neonates and disappears with time.
3. Other Defects That Interfere with Metabolism of Phenylalanine
In rare instances, high levels of blood phenylalanine are due to deficiencies of PAH activity caused by other loci. The cofactor 5,6,7,8-tetrahydrobiopterin (BH4) is produced from 7,8-dihydrobiopterin (BH2) by the enzyme dihydropteridine reductase (DHPR). The gene that codes for DHPR is on chromosome 4 at 4p15.31. In one form of PKU, loss of activity of this gene leads to absence of BH4 and therefore to inactivity of PAH (Fig. 1). Because BH4 is a cofactor in several other reactions that are important to brain function, dietary management of blood phenylalanine levels does not restore a normal phenotype. Such cases occur in 1 to 2 births per million.
A second rare variant of PKU is due to deficiency of 6-pyruvoyl tetrahydropterin synthase (Fig. 1), one of several enzymes necessary for BH2 synthesis. This enzyme is coded by a gene on chromosome 11 at 11q22.3-q23.3. As in the case of DHPR deficiency, the neurological effects are not prevented by management of diet.
4. Molecular Genetics of PAH
The gene that codes for PAH is on chromosome 12 at 12q24.1. It is some 90 kb long with 13 exons. The messenger RNA is 2,677 nt long, consisting of 472 nt in the 5′ untranslated region, 1,356 nt (452 codons) in the coding sequence, and 849 nt in the 3′ untranslated region. Exons 1 to 12 are relatively small, varying in size from 57 nt to 197 nt. Exon 13 has 890 nt, of which 41 are coding. Introns vary in size from 1.0 kb to 23.5 kb.
Some 200 distinct mutations associated with PKU have been identified in the PAH gene. There are presumed to be many more that are associated with unrecognized hyperphenylalaninemia or that are normal variants. Some normal variants are known, and in a few instances they are polymorphic. Of the mutations that cause PKU, approximately three-fourths are nucleotide substitutions that lead to amino acid substitutions or premature chain termination. Another 25 or more are nucleotide substitutions that interfere with normal splicing of the RNA transcript. A number of small gene deletions have also been identified. A high frequency of mutations is found in exon 7, thought to be due to ascertainment bias rather than mutational hotspots. Almost any change in amino sequence in this region of the PAH molecule is likely to interfere with function.
A relatively small number of these mutations account for the majority of the cases of PKU, and particular mutations may be prevalent only in a limited population. Because several different mutations may occur with relatively high frequencies in a population, most so-called homozygotes are in fact compound heterozygotes, that is, the two alleles involve different mutations. Both, however, are nonfunctional. Comparison of mutations in European and Asian populations shows quite different spectra, indicating that most of the extant mutations arose after separation of the major races.
5. Population Genetics of PKU
The frequency of patients with PKU varies considerably among populations. In Norway, where PKU was first discovered, the frequency is ca. 1 in 14,000 births. Among Northern Europeans in general, the frequency is ca. 1 in 10,000 births, with a higher incidence of 1/4,500 in Ireland. Among U.S. whites, the frequency is 1/8,000. The frequency among African-Americans is ca. 1/50,000. The frequency is very low in Ashkenazi Jews and Asians. A frequency of homozygotes of 1/10,000 corresponds to a gene frequency of 0.01 and a frequency of heterozygotes of 2%.
Investigation of PKU has been aided by the discovery of eight polymorphic DNA variants within the PKU gene. These were originally recognized as restriction fragment length polymorphisms, although now PCR is used for some sites. One of the sites involves different numbers of short tandem repeats, providing several alleles. Altogether, there are about 800 possible combinations (haplotypes) of "alleles" at these sites, of which only about 75 have been observed. Within a population, many fewer are observed, and usually only a few reach substantial frequencies.
The study of mutant-allele—haplotype associations in a variety of populations has provided insight into the origin and spread of the genes in historic and prehistoric periods. For example, the mutation R261Q is widespread in Europe but is always associated with haplotype 1. In general, haplotype 1 has a normal PAH allele. The interpretation is that this particular mutation arose on a haplotype 1 chromosome, and all R261Q mutations are descended from that single mutational event. In contrast, the R408W mutation occurs in northwest Europe on a haplotype 1 background and in eastern Europe on a haplotype 2 background. This suggests that two independent mutations occurred, one in a Celtic population and the other in a Slavic population.
