Phenylketonuria

A number sign (#) is used with this entry because phenylketonuria (PKU) and non-PKU mild hyperphenylalaninemia (HPA) result from mutations in the PAH gene (612349).

Description

Phenylketonuria (PKU) is an autosomal recessive inborn error of metabolism resulting from a deficiency of phenylalanine hydroxylase (PAH; 612349), an enzyme that catalyzes the hydroxylation of phenylalanine to tyrosine, the rate-limiting step in phenylalanine catabolism. If undiagnosed and untreated, phenylketonuria can result in impaired postnatal cognitive development resulting from a neurotoxic effect of hyperphenylalaninemia (Zurfluh et al., 2008).

See Scriver (2007) and Blau et al. (2010) for detailed reviews of PKU.

Clinical Features

Early diagnosis of phenylketonuria, a cause of mental retardation, is important because it is treatable by dietary means. Features other than mental retardation in untreated patients include a 'mousy' odor; light pigmentation; peculiarities of gait, stance, and sitting posture; eczema; and epilepsy (Paine, 1957). Kawashima et al. (1988) suggested that cataracts and brain calcification may be frequently overlooked manifestations of classic untreated PKU. Brain calcification has been reported in dihydropteridine reductase (DHPR) deficiency (261630). Pitt and O'Day (1991) found only 3 persons with cataracts among 46 adults, aged 28 to 71 years, with untreated PKU. They concluded that PKU is not a cause of cataracts. Levy et al. (1970) screened the serum of 280,919 'normal' teenagers and adults whose blood had been submitted for syphilis testing. Only 3 adults with the biochemical findings of PKU were found. Each was mentally subnormal. Normal mentality is very rare among patients with phenylketonuria who have not received dietary therapy.

Evidence of heterogeneity in phenylketonuria was presented by Auerbach et al. (1967) and by Woolf et al. (1968).

Coskun et al. (1990) observed scleroderma in 2 infants with PKU. Improvement in the skin lesions after commencement of a low phenylalanine diet supported the possibility of a causal relationship.

Widespread screening of neonates for phenylketonuria brought to light a class of patients with a disorder of phenylalanine metabolism milder than that in PKU. These patients show serum phenylalanine concentrations well below those in PKU, but still several times the normal. PKU and hyperphenylalaninemia breed true in families (Kaufman et al., 1975), each behaving as an autosomal recessive. Kaufman et al. (1975) studied liver biopsies from patients with HPA and their parents. The patients with HPA had levels of phenylalanine hydroxylase about 5% of normal.

Burgard et al. (1996) found that all patients but one who had predicted in vitro residual enzyme activity greater than 20% had mild PKU, while those with predicted in vitro residual enzyme activity less than 20% were identified as having classical PKU. The authors stated that 'the difficulties of some patients to adjust their blood Phe level according to their target value although they comply with the dietary recommendations might be caused by low residual enzyme activity.' In addition, when considering the R261Q (612349.0006) mutation (a mutation with a considerable amount of residual enzyme activity, which produced higher Phe levels than expected), they hypothesized a negative intraallelic complementation effect as an explanation for higher than expected diagnostic Phe values.

Mildly depressed IQ is common in treated PKU. Griffiths et al. (2000) analyzed IQ scores collected from 57 British children with early-treated classic PKU using variants of the Wechsler intelligence scale for children (WISC) in relation to indicators of dietary control such as serum phenylalanine levels and socioeconomic factors. The authors found that, after correcting for socioeconomic status, phenylalanine control at age 2 was predictive of overall IQ, although early and continuous treatment did not necessarily lead to normalization of overall IQ. Subscale analysis revealed normalized verbal IQ in those children with phenylalanine levels of less than 360 micromol/l during infancy (the recommended UK upper limit), but performance IQ remained depressed.

Weglage et al. (2000) compared 42 PKU patients, aged 10 to 18 years, with 42 diabetic patients matched for sex, age, and socioeconomic status. Patients' groups were compared with a control sample of healthy controls (2,900 individuals) from an epidemiologic study. The Child Behavior Check List, IQ tests, and monitoring of blood phenylalanine concentrations and HBA1 concentrations were used. Weglage et al. (2000) found that internalizing problems such as depressive mood, anxiety, physical complaints, or social isolation were significantly elevated in both PKU and diabetic patients, whereas externalizing problems were not. The 2 patient groups did not differ significantly either in the degree or in the pattern of their psychologic profile.

In a retrospective study from birth in 13 patients with classic PKU, Barat et al. (2002) found greater variation of phenylalanine levels and a higher mean of cumulative variations in the 8 osteopenic patients than in 5 nonosteopenic patients. Barat et al. (2002) suggested that serum phenylalanine variations may contribute to osteopenia in patients with classic PKU.

Crujeiras et al. (2015) conducted a cross-sectional observational multicenter study that included 156 patients with hyperphenylalaninemia. Prealbumin was reduced in 34.6% of patients (74% with PKU phenotype and 94% below 18 years old), showing an adequate adherence to diet in nearly all patients (96.3%). Selenium was diminished in 25% of patients (95% with PKU phenotype), and 25-OHD in 14%. Surprisingly, folic acid levels were increased in 39% of patients, 66% with classic PKU. Phosphorus and B12 levels were diminished only in patients with low adherence to diet.

Maternal Phenylketonuria

The occurrence of mental retardation in the offspring of homozygous mothers is an example of a genetic disease based on the genotype of the mother. Kerr et al. (1968) demonstrated 'fetal PKU' by administering large amounts of phenylalanine to mother monkeys. The offspring had reduced learning ability. They pointed out that the damage is aggravated by the normal placental process which functions to maintain higher levels of amino acids in the fetus than in the mother. Huntley and Stevenson (1969) and Hanley et al. (1987) reviewed the subject of PKU embryofetopathy, also known as the maternal PKU syndrome.

Huntley and Stevenson (1969) described 2 sisters with PKU who had a total of 28 pregnancies. Sixteen ended in spontaneous first-trimester abortion. The fetus in each of the 12 pregnancies carried to term had intrauterine growth retardation and microcephaly and 9 of the 12 term infants had cardiac malformations as well.

Superti-Furga et al. (1991) reported the maternal PKU syndrome in cousins, caused by mild unrecognized PKU in their mothers, who were homozygous for the arg261-to-gln mutation (612349.0006).

Usha et al. (1992) found 3 children with PKU embryofetopathy among the offspring of a Bedouin woman who was not recognized to have PKU until the birth of the third affected child. She had an apparently normal phenotype except for pigment dilution of the hair, which was more lightly colored than expected for the family and ethnic norms. She was not mentally retarded. One of the affected offspring had died of congenital heart disease at the age of 4 months.

Fisch et al. (1993) suggested that surrogate motherhood should be recommended as alternative management of PKU in women who wish to have children, i.e., in vitro fertilization using the parental gametes, followed by implantation of the pre-embryo in a surrogate mother.

Levy et al. (1996) compared MRI results of 5 children (age range: 8 months to 17 years) whose mothers had classic PKU and were not under metabolic control (plasma phenylalanine = 1,260 micromoles per liter) during at least the first 2 trimesters of pregnancy to MRI results of 2 sibs aged 9 and 11 years whose mother had classic PKU but whose plasma phenylalanine levels were generally below 360 micromoles per liter during both pregnancies. The MRI results showed a tendency for corpus callosum hypoplasia in those children whose mothers were not in metabolic control during their pregnancies. All children studied (even those with mothers in metabolic control) displayed some residual developmental/behavioral effects such as hyperactivity.

Rouse et al. (1997) reported a collaborative study of maternal PKU offspring. The cohort of offspring were examined for malformations, including congenital heart disease, craniofacial abnormalities, microcephaly, intrauterine and postnatal growth retardation, other major and minor defects, and early abnormal urologic signs. The mothers were grouped according to their mean phenylalanine levels during critical gestational weeks and average for phenylalanine exposure throughout the pregnancy. The frequency of congenital abnormalities increased with increasing maternal phenylalanine levels. Significant relationships included average phenylalanine levels at weeks 0 to 8 with congenital heart disease (P = 0.001); average phenylalanine at weeks 8 to 12 with brain, fetal, and postnatal growth retardation, wide nasal bridge, and anteverted nares; and average phenylalanine exposure during the entire pregnancy with neurologic signs. Although 14% of infants had congenital heart disease, none of the congenital heart disease occurred at the lower range of the maternal phenylalanine levels. At the lowest levels of phenylalanine, there were 3 infants (6%) with microcephaly, 2 (4%) with postnatal growth, and none with intrauterine growth retardation, in contrast to 85%, 51%, and 26%, respectively, with phenylalanine levels in the highest range. These data supported the concept that women with PKU should begin a low phenylalanine diet to achieve phenylalanine levels of less than 360 micromole/liter prior to conception and maintain this throughout the pregnancy.

Waisbren et al. (2000) studied 149 children of women with PKU and 33 children of women with mild hyperphenylalaninemia at 4 years of age. Children were stratified by the timing of maternal metabolic control at 0 to 10 weeks', 10 to 20 weeks', or after 20 weeks' gestation. Scores of a General Cognitive Index decreased as weeks to maternal metabolic control increased. Offspring of women who had metabolic control prior to pregnancy had a mean score of 99. Forty-seven percent of offspring whose mothers did not have metabolic control by 20 weeks' gestation had a General Cognitive Index score 2 standard deviations below the norm. Overall, 30% of children born to mothers with PKU had social and behavioral problems.

Rouse et al. (2000) studied a cohort of 354 women with PKU, followed up weekly with diet records, blood phenylalanine levels, and sonograms obtained at 18 to 20 and 32 weeks' gestation. At birth, 413 offspring were examined; they were followed up at 3 months, 6 months, and then annually. Bayley Mental Developmental Index and Psychomotor Developmental Index tests were given at 1 and 2 years. Congenital heart defects were found in 31 offspring; of these, 17 also had microcephaly. Mean phenylalanine levels at 4 to 8 weeks' gestation predicted congenital heart defects (P less than 0.0001). An infant with a congenital heart defect had a 3-fold risk of having microcephaly when the mother had higher phenylalanine levels. No direct relationship to the specific PAH mutation was found. None of the women whose offspring had congenital heart defects had blood phenylalanine levels in control during the first 8 weeks of gestation. Rouse et al. (2000) concluded that women with PKU need to be well controlled on a low phenylalanine diet before conception and throughout pregnancy.

Levy et al. (2001) reported on 416 offspring from 412 maternal PKU pregnancies that produced live births and compared them to 100 offspring from 99 control pregnancies. Thirty-four of the 235 offspring (14%; 95% confidence interval, 10.2 to 19.6%) from pregnancies in maternal PKU patients with a basal phenylalanine level of greater than 900 micromolar and not in metabolic control (defined as phenylalanine level less than or equal to 600 micromolar) by the eighth gestational week had congenital heart disease compared with 1 control offspring with congenital heart disease. One of the children among 50 from mothers with non-PKU mild hyperphenylalaninemia also had congenital heart disease. Coarctation of the aorta and hypoplastic left heart syndrome were overrepresented.

Other Features

Brumm et al. (2010) reviewed studies of psychiatric symptoms and disorders in patients with PKU. Those with untreated PKU tended to have severe behavioral disturbances, including psychotic disorders, autistic features, hyperactivity, and aggression, as well as self-mutilation. Among early-treated children and adolescents, discontinuation of treatment was associated with attention-deficit disorder and decreased social competence. Children who continued treatment had fewer behavioral problems. However, most tended to be less happy and confident. Even adults who had early treatment had higher rates of depression, anxiety-related disorders, and social introversion compared to the normal population. In general, the severity of problems correlated with the timing and degree of exposure to increased blood levels of phenylalanine. Brumm et al. (2010) stated that mechanisms of psychiatric disorders in PKU most likely result from a combination of neurotransmitter imbalance, myelination defects, and the stress of living with a chronic illness.

Gentile et al. (2010) reviewed studies of psychosocial aspects of PKU and concluded that even treated individuals have hidden disabilities resulting from poor executive function, decreased mental processing speed, and psychosocial problems. These included difficulties in forming interpersonal relationships, achieving autonomy, attending educational goals, and having healthy emotional development. The most important way to reduce these problems is strict metabolic control throughout life, with particular importance on the first year of life.

Biochemical Features

Normal blood phenylalanine levels are 58 +/- 15 micromoles/liter in adults, 60 +/- 13 micromoles/liter in teenagers, and 62 +/- 18 micromoles/liter (mean +/- SD) in childhood. In the newborn, the upper limit of normal is 120 micromoles/liter (2 mg/dl) (Scriver et al., 1985; Gregory et al., 1986). In untreated classical PKU, blood levels as high as 2.4 mM/liter can be found.

Bowden and McArthur (1972) found that phenylpyruvic acid inhibits pyruvate decarboxylase in brain but not in liver. They suggested that this accounts for the defect in formation of myelin and mental retardation in this disease.

In the liver of a fetus aborted after prenatal DNA diagnosis of PKU, Ledley et al. (1988) found no detectable phenylalanine hydroxylase enzymatic activity or immunoreactive protein, although both were found in control specimens of similar gestational age. Both the size and the amount of phenylalanine hydroxylase mRNA were normal. The findings confirmed the genetic diagnosis of PKU in the fetus and indicated that the mutations affected translation or stability of the protein.

Tolerance to dietary phenylalanine and therefore the clinical severity of PKU have been presumed to be the consequence of the rate of conversion of phenylalanine into tyrosine. However, in a study of 7 classic PKU patients, van Spronsen et al. (1998) found that although the in vivo hydroxylation of phenylalanine into tyrosine was decreased, there was no significant correlation between the in vivo hydroxylation rates and the tolerances.

Kaufman (1999) described the derivation of a quantitative model of phenylalanine metabolism in humans. The model was based on the kinetic properties of pure recombinant human PAH and on estimates of the in vivo rates of phenylalanine transamination and protein degradation. Calculated values for the steady-state concentration of blood phenylalanine, rate of clearance of phenylalanine from the blood after an oral load of the amino acid, and dietary tolerance of phenylalanine all agreed with data from normal as well as from phenylketonuric patients and obligate heterozygotes. Kaufman (1999) suggested that these calculated values may help in the decision about the degree of restriction of phenylalanine intake that is necessary to achieve a satisfactory clinical outcome in patients with classic PKU and in those with milder forms of the disease.

It has been postulated that the significant incidence of learning disabilities in treated patients with PKU may be due, in part, to reduced production of neurotransmitters as a result of deficient tyrosine transport across the neuronal cell membrane. In a study of hypotyrosinemia in a PKU population, Hanley et al. (2000) found that the mean nonfasting plasma tyrosine was 41.1 micromol/L in 99 classic PKU patients, 53.3 micromol/L in 26 mild (atypical) PKU patients, and 66.6 micromol/L in 35 non-PKU mild hyperphenylalaninemia patients. This compared to nonfasting plasma tyrosine levels of 64.0 micromol/L in 102 non-PKU subjects in their hospital biochemistry database, 69.1 micromol/L in 58 volunteers in private office practice, and 64 to 78.8 micromol/L in infants, children, and adolescents in a literature review. The data supported previous findings that plasma tyrosine levels are low in PKU.

Leuzzi et al. (2000) assessed brain Phe concentration by in vivo proton magnetic resonance spectroscopy in 10 off-diet PKU patients, aged 15.5 to 30.5 years. An abnormal concentration of brain Phe was detected in all patients, but there was wide interindividual variability of concurrent plasma Phe. In late-detected subjects, brain Phe concentration correlated with clinical phenotype better than did plasma Phe. White-matter alterations were found in all patients.

Koch et al. (2000) referred to preliminary reports suggesting that the occasional untreated person with PKU with normal intellect has elevated blood phenylalanine but low brain phenylalanine levels, They measured blood phenylalanine levels and used MRI/MRS to measure brain phenylalanine content in 29 individuals with PKU, 4 carriers of phenylalanine hydroxylase mutations, and 5 controls. For each individual with PKU, the authors also noted IQ, mutations, whether or not a restricted diet was followed, and age at diagnosis. Koch et al. (2000) concluded that MRI/MRS measurements of brain phenylalanine content may be of value in recommending appropriate blood phenylalanine concentrations for treatment of adults.

Weglage et al. (2002) investigated 4 pairs of sibs with classical PKU using in vivo NMR spectroscopy in the course of an oral phenylalanine load (100 mg/kg body weight). Patients' brain phenylalanine concentrations were different in spite of similar blood levels. Interindividual variations of the apparent transport Michaelis constant ranged from 0.10 to 0.84 mmol/L. Sibs with lower values for the apparent transport constant, higher values for the ratio of the maximal transport velocity over the intracerebral consumption rate, and higher concurrent brain phenylalanine levels showed a lower IQ and a higher degree of cerebral white matter abnormalities. Weglage et al. (2002) concluded that blood-brain barrier transport characteristics and the resultant brain phenylalanine levels are causative factors for the individual clinical outcome in PKU.

To determine whether impairments of cerebral metabolism may play a role in acute phenylalanine neurotoxicity, Pietz et al. (2003) studied 11 adult early-treated PKU patients and 10 healthy controls for changes in concentrations of cerebral metabolites using noninvasive quantitative phosphorus-31 MRS. In adult patients, derived ADP concentration and phosphorylation potential were increased by 11% and 22%, respectively; peak areas of inorganic phosphate and phospholipids were decreased by 22% and 8%, respectively. ADP correlated with concurrent plasma (r = 0.65) and brain (r = 0.55) phenylalanine levels. PKU patients showed slowing of EEG background activity, a sign of impaired brain function, 24 hours after oral phenylalanine challenge. Pietz et al. (2003) concluded that there were subtle abnormalities of cerebral energy metabolism and encouraged more clinical studies on the relationship of imbalances of high energy phosphates and cerebral energy metabolism to acute phenylalanine neurotoxicity.

Inheritance

Classical PKU is inherited in a strictly autosomal recessive manner and is the result of mutations in the PAH gene. Most variation in classical PKU is due to heterogeneity in the mutant alleles with many patients being compound heterozygotes rather than homozygotes for one particular mutant allele. Bartholome et al. (1984) concluded that examples of parent (usually mother)-to-child transmission of hyperphenylalaninemia are likely to be due to compound heterozygosity for PKU and HPA in either the parent or the child or both.

Mapping

Using a cDNA probe for human phenylalanine hydroxylase to analyze human-mouse hybrid cells by Southern hybridization, Lidsky et al. (1984) showed that the PAH locus is on chromosome 12 and presumably on the distal part of 12q because in hybrids containing translocated chromosome 12, it segregated with PEPB (12q21) and not with TPI (12p13). Since in family studies concordance of segregation between a mutant PAH gene and PKU was found (Woo et al., 1983), one can state that the 'PKU locus' is on chromosome 12. By in situ hybridization, the assignment of the PAH locus was narrowed to chromosome 12q22-q24.1 (Woo et al., 1984).

For information on early mapping studies, see HISTORY.

Molecular Genetics

The first PKU mutation identified in the PAH gene was a single base change (GT-to-AT) in the canonical 5-prime splice donor site of intron 12 (612349.0001). Gene transfer and expression experiments demonstrated that the splice donor site mutation resulted in abnormal PAH mRNA processing and loss of PAH activity (DiLella et al., 1986).

Eisensmith and Woo (1992) reviewed mutations and polymorphisms in the human PAH gene. About 50 of the mutations were single-base substitutions, including 6 nonsense mutations and 8 splicing mutations, with the remainder being missense mutations. Of the missense mutations, 12 apparently resulted from the methylation and subsequent deamination of highly mutagenic CpG dinucleotides. Recurrent mutations had been observed at several sites, producing associations with different haplotypes in different populations. Studies of in vitro expression showed significant correlations between residual PAH activity and severity of the disease phenotype.

For more detailed information on the molecular genetics of PKU and non-PKU hyperphenylalaninemia, see 612349.

Genotype/Phenotype Correlations

For information on genotype/phenotype correlations in PKU and non-PKU hyperphenylalaninemia, see 612349.

Pathogenesis

Waters et al. (2000) characterized 4 PKU-associated PAH mutations that change an amino acid distant from the enzyme active site. Using 3 complementary in vitro protein expression systems and 3D structural localization, Waters et al. (2000) demonstrated a common mechanism, i.e., PAH protein folding is affected, causing altered oligomerization and accelerated proteolytic degradation, leading to reduced cellular levels of this cytosolic protein. Enzyme-specific activity and kinetic properties are not adversely affected, implying that the only way these mutations reduce enzyme activity within cells in vivo is by producing structural changes which provoke the cell to destroy the aberrant protein. The mutations were chosen because of their associations with a spectrum of in vivo hyperphenylalaninemia among patients. Waters et al. (2000) concluded that their in vitro data suggests that interindividual differences in cellular handling of the mutant but active PAH proteins contributes to the observed variability of phenotypic severity.

Most PAH missense mutations impair enzyme activity by causing increased protein instability and aggregation. Gjetting et al. (2001) described an alternative mechanism by which some PAH mutations may render phenylalanine hydroxylase defective. They used database searches to identify regions in the N-terminal domain of PAH with homology to the regulatory domain of prephenate dehydratase (PDH), the rate-limiting enzyme in the bacterial phenylalanine biosynthesis pathway. Naturally occurring N-terminal PAH mutations are distributed in a nonrandom pattern and cluster within residues 46-48 (amino acids GAL) and 65-69 (amino acids IESRP), 2 motifs highly conserved in PDH. To examine whether N-terminal PAH mutations affect the ability of PAH to bind phenylalanine at the regulatory domain, wildtype and 5 mutant forms (including G46S, 612349.0055; A47V, 612349.0056; and I65T, 612349.0063) of the N-terminal domain (residues 2-120) of human PAH were expressed as fusion proteins in E. coli. Binding studies showed that the wildtype form of this domain specifically binds phenylalanine, whereas all mutations abolished or significantly reduced this phenylalanine-binding capacity. The data suggested that impairment of phenylalanine-mediated activation of PAH may be an important disease-causing mechanism of some N-terminal PAH mutations.

Most missense mutations found in PKU result in misfolding of the phenylalanine hydroxylase protein, increased protein turnover, and loss of enzymatic function. Pey et al. (2007) studied the prediction of the energetic impact on PAH native-state stability of 318 PKU-associated missense mutations, using the protein-design algorithm FoldX. For the 80 mutations for which expression analyses had been performed in eukaryotes, in most cases they found substantial overall correlation between the mutational energetic impact and both in vitro residual activities and patient metabolic phenotype. This finding confirmed that the decrease in protein stability is the main molecular pathogenic mechanism in PKU and the determinant for phenotypic outcome. Metabolic phenotypes had been shown to be better predicted than in vitro residual activities, probably because of greater stringency in the phenotyping process. All the remaining 238 PKU missense mutations compiled in the PAH locus knowledgebase (PAHdb) were analyzed, and their phenotypic outcomes were predicted on the basis of the energetic impact provided by FoldX. Residues in exons 7-9 and in interdomain regions within the subunit appeared to play an important structural role and constitute hotspots for destabilization.

Using recombinant proteins expressed in E. coli, Gersting et al. (2008) characterized 10 BH4-responsive PAH mutations, including arg408 to trp (R408W; 612349.0002) and tyr414 to cys (Y414C; 612349.0017). Residual activity was generally high, but allostery was disturbed in almost all variants, suggesting altered protein conformation. This hypothesis was confirmed by reduced proteolytic stability, impaired tetramer assembly or aggregation, increased hydrophobicity, and accelerated thermal unfolding, which primarily affected the regulatory domain, in most variants. Three-dimensional modeling revealed that the misfolding was communicated throughout the protein. Gersting et al. (2008) concluded that global conformational changes in PAH hinder the molecular motions essential for enzyme function.

Diagnosis

Matalon et al. (1977) reported high levels of phenylalanine hydroxylase in placenta and suggested use of placental biopsy in prenatal diagnosis.

Woo (1983) identified a DNA restriction polymorphism detected by a phenylalanine hydroxylase cDNA probe and tentatively demonstrated the feasibility of carrier detection and prenatal diagnosis, using the haplotypes defined by the DNA polymorphism.

By the use of RFLPs related to the phenylalanine hydroxylase gene, Lidsky et al. (1985) achieved prenatal diagnosis of a PKU homozygote and a PKU heterozygote. Riess et al. (1987) described experience with prenatal diagnosis of PKU by RFLP analysis. They pointed out that in those cases in which the affected child had died but a phenotypically normal brother or sister is available for investigation, full genetic predictability could be obtained only if this child proved to be homozygously healthy in the phenylalanine-loading heterozygote test.

DiLella et al. (1988) showed that the 2 mutant alleles of PAH common among Caucasians of northern European ancestry can be detected by direct analysis of genomic DNA after specific amplification of a DNA fragment by PCR. The results suggested that it is technically feasible to develop a program for carrier detection of the genetic trait in a population of individuals without a family history of PKU.

Ramus et al. (1992) used PCR amplification of the low levels of mRNA resulting from illegitimate transcription of the PAH gene in fibroblasts and Epstein-Barr virus-transformed lymphocytes to detect mutations in patients with PKU.

Taking advantage of the 'illegitimate' transcription of the PAH gene in circulating lymphocytes, Abadie et al. (1993) succeeded in making the DNA diagnosis of phenylketonuria. Furthermore, they identified 3 novel mutations in 2 patients.

Kalaydjieva et al. (1991) identified 3 silent mutations in the PAH gene, in codons 232, 245, and 385, linked to specific RFLP haplotypes in several Caucasian populations. All 3 mutations created a new restriction site and were easily detected on PCR-amplified DNA. The combined analysis of these markers and 1 or 2 PKU mutations formed a simple panel of diagnostic tests with full informativeness in a large proportion of PKU families.

Forrest et al. (1991) used a modification of the chemical cleavage of mismatch (CCM) method to identify mutations in PAH in PKU. They stated that 'judicious choice of probes gives the CCM method the potential to detect close to 100% of single-base mutations.'

Clinical Management

Dietary Treatment

Phenylketonuria is treatable by a low phenylalanine diet. In treated patients, severe white matter abnormalities are predominantly associated with blood phenylalanine levels above 15 mg per deciliter (Thompson et al., 1993). Ullrich et al. (1994) performed MRI on 15 adolescents with good dietary control (phenylalanine levels below 10 mg per deciliter). Ten of these patients had a normal cranial MRI whereas 4 showed mild changes of the signal intensity of the white matter on T2-weighted images confined to the parietooccipital region. The affected and unaffected patients could not be distinguished by age, sex, or mean blood phenylalanine concentrations.

From studies in 4 women, Rohr et al. (1987) concluded that fetal damage from maternal PKU can be largely and perhaps entirely prevented by dietary therapy, but that therapy must begin before conception for the best chance of a normal infant. Drogari et al. (1987) presented evidence suggesting that only a diet restricting phenylalanine intake started before conception is likely to prevent fetal damage.

In a report of preliminary results from the North American Maternal PKU Study, Hanley et al. (1996) suggested that early and adequate dietary treatment during pregnancy may provide some protection to the fetus for later intellectual development. The German Maternal PKU Study had followed 43 pregnancies (Cipcic-Schmidt et al., 1996). For minimizing risks of ill effects, preconceptional dietary control was strongly recommended.

Brenton and Lilburn (1996) reported that by November 1994, 39 pregnancies had been completed in PKU mothers. Dietary control was post-conception in 6; 2 of these offspring died of congenital heart disease and another needed surgery for coarctation. There were no heart defects in the 34 offspring of the 33 pregnancies following preconception diet controlled by Guthrie assays of maternal Phe 3 times weekly. Excessively high and low values occurred intermittently in many pregnancies, both of which may adversely affect the fetus.

A multicenter follow-up study (Holtzman et al., 1986) presented evidence that treatment of PKU should be continued beyond age 8 years.

Weglage et al. (1999) reported results of testing of IQ, fine motor abilities, and sustained and selective attention in 10 boys and 10 girls with early-treated phenylketonuria and 20 healthy controls matched for age, sex, and IQ; the individuals were tested twice, at mean ages of 11 and 14 years. At the first test, examination showed significant blood phenylalanine-correlated neuropsychologic deficits in PKU patients. In spite of raised blood phenylalanine concentrations during the following 3 years, the repeated measurements revealed a significant decrease in patients' deficits compared to controls. Clinical-neurologic status of patients and controls was normal at both test times. The results indicated decreased vulnerability of PKU patients with respect to their neuropsychologic functioning against elevated phenylalanine levels on aging.

Greeves et al. (2000) examined the effect of diet relaxation after the age of 8 years in 125 Northern Irish children with PKU or non-PKU hyperphenylalaninemia, correlating verbal, performance, and overall IQ at ages 8, 14, and 18 with the predicted residual enzyme activity conferred by their genotype. Multiple regression analysis demonstrated a significant reduction in verbal and overall IQ between the ages of 8 and 14 or 18, with a greater reduction in those with a lower predicted residual enzyme activity. This study also showed that patients with residual enzyme activities of 25% or more were more likely to maintain or gain IQ points after dietary relaxation than those patients with lower enzyme activities. These data suggested that continued dietary control in this latter group, as defined by genotype, may prove beneficial.

Recognizing that a low phenylalanine diet is also low in the long-chain polyunsaturated fatty acids (LCPUFA) necessary for cell membrane formation and normal brain and visual development, Agostoni et al. (2000) examined the effects of a 12-month supplementation of LCPUFA on fatty acid composition of erythrocyte lipids and visual evoked potentials in children with well-controlled PKU. The children who received supplementation showed a significant increase in docosahexaenoic acid (DHA) levels of erythrocyte lipids and improved visual function, as measured by a decreased P100 wave latency.

Huijbregts et al. (2002) sought to answer whether there is an effect of dietary interventions that induce relatively small changes in phenylalanine concentration on neuropsychologic outcome of early and continuously treated PKU patients and whether there are differences in effect for PKU children versus adolescents. Huijbregts et al. (2002) sought short-term dietary intervention of 1 to 2 weeks and compared this for patients whose phenylalanine concentrations increased versus those whose phenylalanine concentrations decreased. Huijbregts et al. (2002) found that relatively small fluctuations in phenylalanine concentration influenced neuropsychologic task performance of PKU patients. Patients whose phenylalanine concentrations had decreased by the second assessment showed generally more improvement than controls. Patients whose phenylalanine concentrations had increased showed minimal improvement or deterioration of task performance. The strongest effects were observed when sustained attention and manipulation of working memory content were required.

Koch et al. (2002) reported the follow-up studies of 125 children who were a part of the original cohort for short-term versus long-term treatment of PKU with diet. Seventy of the 125 children were located and evaluated in adulthood. Mental problems, including phobias and depression, were reported in 41% of those off diet and 22% of continuers. The 'on diet' group had only 2 reported episodes of transient depression not requiring psychiatric care. The neurologic signs related primarily to increased or decreased muscle tone and deep tendon reflex changes. The group who remained on a phenylalanine-restricted diet had fewer problems overall than the discontinued group (P = 0.02).

Singh et al. (2014) reported updated recommendations for the nutritional management of phenylalanine hydroxylase deficiency. Their paper was accompanied by an American College of Medical Genetics practice guideline authored by Vockley et al. (2014), which updated phenylalanine hydroxylase deficiency diagnosis and management, including the use of sapropterin dihydrochloride to achieve improved metabolic control and/or increased protein tolerance in patients who respond.

Sapropterin (Tetrahydrobiopterin)-Responsive PKU

At least half of patients with phenylketonuria have a mild clinical phenotype. Muntau et al. (2002) explored the therapeutic efficacy of tetrahydrobiopterin for the treatment of mild phenylketonuria. Tetrahydrobiopterin significantly lowered blood phenylalanine levels in 27 of 31 patients with mild hyperphenylalaninemia (10 patients) or mild phenylketonuria (21 patients). Phenylalanine oxidation was significantly enhanced in 23 of these 31 patients. Conversely, none of the 7 patients with classic phenylketonuria had a response to tetrahydrobiopterin. Long-term treatment with tetrahydrobiopterin in 5 children increased daily phenylalanine tolerance, allowing them to discontinue their restricted diets. Mutations connected to tetrahydrobiopterin responsiveness were predominantly in the catalytic domain of the PAH protein and were not directly involved in cofactor binding. Muntau et al. (2002) concluded that responsiveness could not consistently be predicted on the basis of genotype, particularly in compound heterozygotes.

Lassker et al. (2002) reported 2 new patients with tetrahydrobiopterin-responsive PKU who carried missense mutations in the PAH gene. Both patients showed no effect of tetrahydrobiopterin at 7.5 mg/kg/day on plasma phenylalanine levels in the newborn period, and the authors suggested that a normal neonatal tetrahydrobiopterin test does not necessarily exclude tetrahydrobiopterin responsiveness in all such patients.

Matalon et al. (2004) found that 21 of 36 (58.3%) PKU patients responded favorably to oral tetrahydrobiopterin (BH4) supplementation. A single dose of 10 mg/kg resulted in a mean decrease of greater than 30% in blood phenylalanine levels. Patients who responded were found to have mutations in the PAH gene within the catalytic, regulatory, oligomerization, and BH4-binding domains.

Steinfeld et al. (2004) reported 2 unrelated infants with PKU who responded favorably to daily BH4 supplementation. They no longer needed dietary restriction and showed normal development after 2 years. One of the patients was homozygous for a mild PAH mutation (Y414C; 612349.0017). No side effects were observed.

Keil et al. (2013) reported the follow-up of 147 patients treated with sapropterin dihydrochloride for up to 12 years: 41.9% had mild hyperphenylalaninemia, 50.7% mild PKU, and 7.4% classic PKU. Median phenylalanine (Phe) tolerance increased 3.9 times with BH4/sapropterin therapy, compared with dietary treatment, and median Phe blood concentrations were within the therapeutic range in all patients. Compared with diet alone, improvement in quality of life was reported in 49.6% of patients, improvement in adherence to diet in 47% of patients, and improvement in adherence to treatment in 63.3% of patients. No severe adverse events were reported. Keil et al. (2013) concluded that their data documented a long-term beneficial effect of orally administered BH4/sapropterin in responsive PKU patients by improving metabolic control, increasing daily tolerance for dietary Phe intake, and for some, by improving dietary adherence and quality of life.

For more detailed information on genotype/phenotype correlations in tetrahydrobiopterin-responsive PKU, see 612349

Other Treatments

Hoskins et al. (1980) showed that the plant enzyme phenylalanine ammonia lyase (PAL; EC 4.3.1.5) will survive in the gut long enough to deplete the phenylalanine derived from food protein and so reduce the rise in blood phenylalanine that otherwise occurs after a protein meal. Preliminary studies suggested that it may have a place in the treatment of PKU.

Sarkissian et al. (1999) described experiments on a mouse model using a different modality for treatment of PKU compatible with better compliance using ancillary PAL to degrade phenylalanine, the harmful nutrient of PKU; in this treatment, PAL acts as a substitute for the enzyme phenylalanine monooxygenase, which is deficient in PKU. PAL, a robust enzyme without need for a cofactor, converts phenylalanine to trans-cinnamic acid, a harmless metabolite. Sarkissian et al. (1999) described (i) an efficient recombinant approach to produce large quantities of PAL enzyme using a construct of the PAL gene from Rhodosporidium toruloides and expressing it in a strain of E. coli; (ii) testing of PAL in orthologous mouse with hyperphenylalaninemia induced by N-ethyl-N-nitrosourea (ENU) mutation; and (iii) proofs of principle (PAL reduces hyperphenylalaninemia), both pharmacologic (with a clear dose-response effect) and physiologic (protected enteral PAL is significantly effective against hyperphenylalaninemia). They concluded that the appropriate dosage of orally administered PAL, perhaps in combination with a controlled and modestly low protein diet, should effectively control the phenylalanine pool size through its effect on the gastrointestinal tract. These findings opened a new avenue to the treatment of this classic genetic disorder.

Stegink et al. (1989) tested the effect of aspartame (N-L-alpha-aspartyl-L-phenylalanine methyl ester--a widely used dipeptide sweetener) on phenylalanine concentrations in persons heterozygous for PKU. They found moderate elevations in phenylalanine levels above baseline for heterozygotes for PKU (2.3-4.7 micromoles, 30-45 minutes after ingestion of a 12-ounce beverage).

Liver transplantation is not a usual therapy for PKU because of the usually good results achieved with early dietary restriction and because liver disease is not part of the clinical picture of PKU. Vajro et al. (1993) reported that orthotopic liver transplantation in a 10-year-old boy with PKU and concomitant, unrelated end-stage liver disease cured the PKU.

Eisensmith and Woo (1996) reviewed the current state of gene therapy for phenylketonuria. Of the 3 basic steps required, 2 have been accomplished: a cDNA clone expressing human phenylalanine hydroxylase and a phenylalanine hydroxylase-deficient animal model have been developed, while vectors for efficient gene transfer in vivo have yet to be developed. Retroviral vectors, while effective in vitro, have a low transduction efficiency in vivo. Similarly, DNA/protein complexes have not been efficiently transduced in vivo. Recombinant adenoviral vectors, although completely successful in the short term, did not persist beyond a few weeks due to an immune response against the adenoviral vector.

Population Genetics