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Phenylalanine Hydroxylase Deficiency

[PAH Deficiency. Includes: Hyperphenylalaninemia (HPA), Phenylketonuria (PKU), Variant PKU]

Summary

Disease characteristics.  Phenyalanine hydroxylase (PAH) deficiency results in intolerance to the dietary intake of the essential amino acid phenylalanine and produces a spectrum of disorders including phenylketonuria (PKU), non-PKU hyperphenylalaninemia (non-PKU HPA), and variant PKU. Classic PKU is caused by a complete or near-complete deficiency of phenylalanine hydroxylase activity; without dietary restriction of phenylalanine, most children with PKU develop profound and irreversible mental retardation. Non-PKU HPA is associated with a much lower risk of impaired cognitive development in the absence of treatment. Variant PKU is intermediate between PKU and non-PKU HPA.

Diagnosis/testing.  PAH deficiency can be diagnosed by newborn screening in virtually 100% of cases based upon detection of the presence of hyperphenylalaninemia using the Guthrie microbial or other assays on a blood spot obtained from a heelprick. PKU is diagnosed in individuals with plasma phenylalanine (Phe) concentrations higher than 1000 µmol/L in the untreated state; non-PKU HPA is diagnosed in individuals with plasma Phe concentrations consistently above normal (i.e., >120 µmol/L), but lower than 1000 µmol/L when on a normal diet. Molecular genetic testing of the PAH gene is used primarily for genetic counseling purposes to determine carrier status of at-risk relatives and for prenatal testing.

Management.  Treatment of manifestations: Classic PKU: a low-protein diet and use of a Phe-free medical formula as soon as possible after birth to achieve plasma Phe concentrations of 120-360 µmol/L (2-6 mg/dL) or 40-240 µmol/L (1-4 mg/dL); supplementation with 6R-BH₄ stereoisomer in doses up to 20 mg/kg daily based on individual needs. Non-PKU HPA: It is debated whether those with plasma Phe concentrations consistently below 600 µmol/L (10 mg/dL) require dietary treatment. Prevention of primary manifestations: Same as Treatment of manifestations. Surveillance: regular monitoring of plasma Phe and Tyr concentrations in individuals with classic PKU. Agents/circumstances to avoid: aspartame, an artificial sweetener that contains phenylalanine (or calculate intake of Phe in aspartame and adapt diet accordingly). Testing of relatives at risk: Newborn sibs of an individual with PKU who have not been tested prenatally should have blood concentration of Phe measured shortly after birth (in addition to newborn screening) to allow earliest possible diagnosis and treatment. Other: for women with PAH deficiency, Phe-restricted diet for at least several months prior to conception in order to maintain plasma Phe concentrations between 120 and 360 µmol/L (2-6 mg/dL); after conception, continuous nutritional guidance and weekly or biweekly measurement of plasma Phe concentration to assure adequate energy intake with the proper proportion of protein, fat, and carbohydrates.

Genetic counseling.  PAH deficiency is inherited in an autosomal recessive manner. At conception, each sib of an affected individual has a 25% chance of being affected, a 50% chance of being an asymptomatic carrier, and a 25% chance of being unaffected and not a carrier. Prenatal diagnosis of PAH deficiency is possible in pregnancies at 25% risk either when molecular genetic testing has revealed the disease-causing mutations in the PAH gene in an affected family member, or when linkage analysis has identified informative markers. Prenatal testing is available for pregnancies at risk if the disease-causing mutations have been identified in an affected family member.

Diagnosis

Clinical Diagnosis

Neonates with phenylalanine hydroxylase (PAH) deficiency show no physical signs of hyperphenylalaninemia (HPA).

Testing

For laboratories offering biochemical testing see .

Plasma phenylalanine concentration.  The main route for phenylalanine metabolism is hydroxylation of phenylalanine to tyrosine by phenylalanine hydroxylase (PAH). The diagnosis of primary phenylalanine hydroxylase deficiency (PAH deficiency) is based on the detection of an elevated plasma phenylalanine (Phe) concentration and evidence of normal BH₄ cofactor metabolism. Individuals with PAH deficiency show plasma phenylalanine (Phe) concentrations that are persistently higher than 120 µmol/L (2 mg/dL) in the untreated state [Scriver & Kaufman 2001 , Donlon et al 2004].

For a description of two different systems of nomenclature, see Clinical Description .

Newborn screening.  PAH deficiency is most commonly diagnosed upon routine screening of newborns. PAH deficiency can be detected in virtually 100% of cases by newborn screening utilizing the Guthrie card bloodspot obtained from a heelprick. Newborn screening for HPA has been routine throughout North America [National Newborn Screening Status Report (pdf)] and the UK since the mid-1960s and in most other developed countries since the early 1970s [Scriver 1998 , Levy 1999]. The test became routine because of the excellent prognosis for individuals with PAH deficiency who are treated early and the high risk for severe and irreversible brain damage for individuals who are not treated. In most countries a parental right of refusal for this test exists; however, this right is exercised only in rare circumstances.

• Initial test.  The initial test yields a significant number of false positive test results (imperfect specificity of the test) leading to follow-up tests and sometimes stressful experiences for parents. A false positive elevated Phe concentration (hyperphenylalaninemia) may result from a blood spot that is too thick, a sample that is improperly prepared, or combinations of the following: liver immaturity, protein overload (in newborns who are fed cow's milk), and possible heterozygosity for phenylalanine hydroxylase deficiency in premature babies [Hennermann et al 2004].

  Infants whose initial test results exceed the threshold concentration of Phe must be tested a second time.

• Second test.  If the second test confirms hyperphenylalaninemia, further tests are performed to distinguish those infants with PAH deficiency from the approximately 2% of infants with hyperphenylalaninemia who have impaired synthesis or recycling of tetrahydrobiopterin (BH₄) (see Differential Diagnosis) [Scriver & Kaufman 2001 , Donlon et al 2004]. Molecular genetic testing of the PAH gene in these infants can be used to confirm PAH deficiency [Guttler & Guldberg 2000].

A concern is the reliability of current tests to accurately measure Phe concentrations in infants less than 24 hours old, since hyperphenylalaninemia (HPA) manifests itself as a time-dependent increase of Phe concentration in the blood [Rouse et al 1997].

Three methods of newborn screening are currently in use:

• Guthrie card bacterial inhibition assay (BIA), a time-tested, inexpensive, simple, and reliable test

• Fluorometric analysis, a reliable quantitative and automated test which produces fewer false positive test results than the BIA

• Tandem mass spectrometry (MS), which has the same benefits as fluorometric analysis, can also measure tyrosine concentration, and can be useful in interpreting Phe concentration. Tandem mass spectrometry can be used to identify numerous other metabolic disorders on the same sample [NIH Consensus Development Panel 2000]. Cost-effectiveness analysis, with economic modeling, suggests that to replace existing methods of newborn screening for PKU with tandem MS would not be justified, unless the intent is to capture additional metabolic disorders, notably MCAD deficiency [Pandor et al 2004].

Determination of carrier status.   Biochemical analysis can be used to determine carrier status if molecular genetic testing is not possible. Biochemical analysis relies on analysis of the plasma Phe concentration and Phe/Tyr ratio. Of note: this test must take into account circadian variation (i.e., it must be performed before noon after a normal breakfast) and variations in a woman's menstrual cycle; it is not accurate during pregnancy. When these variations are taken into account, the probability of heterozygote misclassification is 0.01 or less [Rosenblatt & Scriver 1968 , Gold et al 1974].

Molecular Genetic Testing

GeneReviews designates a molecular genetic test as clinically available only if the test is listed in the GeneTests Laboratory Directory by either a US CLIA-licensed laboratory or a non-US clinical laboratory. GeneTests does not verify laboratory-submitted information or warrant any aspect of a laboratory's licensure or performance. Clinicians must communicate directly with the laboratories to verify information. —ED.

Gene.   PAH is the only gene associated with phenylalanine hydroxylase deficiency [Scriver & Kaufman 2001].

Clinical uses

• Confirmatory diagnostic testing
• Prognostication
• Carrier testing
• Prenatal diagnosis
• Preimplantation genetic diagnosis

Clinical testing

• Targeted mutation analysis.  A panel of four to 15 common point mutations and very small deletions has a detection rate of approximately 30%-50%. Alleles may be population related.

• Mutation scanning.  Mutation scanning detects virtually all point mutations in the PAH gene. Mutation scanning by denaturing high-performance liquid chromatography, a fast and very efficient method to detect locus-specific point mutations, has shown its relevance for detection of PKU-causing alleles [Brautigam et al 2003].

• Sequence analysis.  Sequencing of all 13 exons has a mutation detection rate of about 99%.

• Duplication/deletion analysis.  Comparative multiplex dosage analysis may be useful in detecting large duplications or deletions when no mutations have been identified by mutation scanning or sequence analysis. This technique has been used to detect abnormal dosage in 20% of uncharacterized PKU alleles [Gable et al 2003] and therefore duplications and deletions may account for up to 3% of mutations [Kozak et al 2006].

• Linkage analysis.  For families in which only one or neither PAH mutation has been identified, linkage analysis may be an option for carrier testing and prenatal diagnosis. Linkage studies are based upon accurate clinical diagnosis of PAH deficiency in the affected family member(s) and accurate understanding of the genetic relationships in the family. Samples from multiple family members, including a sample from at least one affected individual, are required to perform linkage analysis. The markers used for linkage are highly informative and are both intragenic and flanking to the PAH locus; thus they can be used with greater than 99% accuracy in families with PAH deficiency.

Table 1 summarizes molecular genetic testing for this disorder.

Table 1. Molecular Genetic Testing Used in Phenylalanine Hydroxylase Deficiency Test Method Mutations Detected Mutation Detection Frequency  1 Test Availability Targeted mutation analysis 4-15 common PAH mutations 30%-50% Clinical Mutation scanning Common and private PAH sequence variants 99% Sequencing analysis Common and private PAH sequence variants 99%  2 Duplication/deletion analysis  3 PAH deletions/duplications Rare

1.  Proportion of affected individuals with a mutation(s) as classified by gene/locus, phenotype, population group, genetic mechanism, and/or test method 2. The mutation detection frequency may be lower than 99% if not all of the PAH exons are included in the sequence analysis. 3. Using multiplex ligation-dependent probe amplification (MPLA)

Interpretation of test results.  For issues to consider in interpretation of sequence analysis results, click here.

Testing Strategy

To establish the diagnosis in a proband.  Once the initial screen is positive, diagnostic tests must be performed:

• Blood for amino acid analysis permits confirmation of hyperphenylalaninemia and the exclusion of the diagnoses of hepatic insufficiency or other aminoacidopathy.
• To exclude hyperphenylalaninemia as a secondary consequence of an inborn error of biopterin metabolism, a urine sample to measure pterins and a blood sample to assay dihydropteridine reductase activity are collected (see Differential Diagnosis). Once these blood samples have been collected, a low phenylalanine diet is started.
• Although molecular genetic testing is not necessary for diagnosis or treatment in the immediate newborn period, it is often performed for genotype/phenotype correlation.

Genetically Related (Allelic) Disorders

No other phenotypes are known to be associated with mutations in the PAH gene.

Clinical Description

Natural History

Phenylalanine in excess can be toxic to brain and cognitive development; thus any degree of hyperphenylalaninemia could be called a 'phenylketonuric' phenotype and would be a risk factor to be managed accordingly [Smith 1994]. However, because the risk also appears to vary relative to the degree of hyperphenylalaninemia, various classification schemes have emerged.

The initial classification scheme proposed by Kayaalp et al (1997) was meant to keep the nomenclature simple. This system uses the following terms:

• Phenylketonuria (PKU) is the most severe of the three types and in an untreated state is associated with plasma Phe concentrations greater than 1000 µmol/L and a dietary Phe tolerance of less than 500 mg/day. PKU is associated with a high risk of severely impaired cognitive development.

• Non-PKU hyperphenylalaninemia (non-PKU HPA) is associated with plasma Phe concentrations consistently above normal (i.e., >120 µmol/L) but lower than 1000 µmol/L when an individual is on a normal diet. Individuals with non-PKU HPA have a much lower risk of impaired cognitive development in the absence of treatment.

• Variant PKU includes those individuals who do not fit the description for either PKU or non-PKU HPA.

The classification scheme proposed by Guldberg et al (1998) subdivides PAH deficiency into the following four categories:

• Classic PKU is caused by a complete or near-complete deficiency of PAH activity. Affected individuals tolerate less than 250-350 mg of dietary phenylalanine per day to keep plasma concentration of Phe at a safe level of no more than 300 µmol/L (5 mg/dL). Without dietary treatment most individuals develop profound, irreversible mental retardation.

• Moderate PKU.  Affected individuals tolerate 350-400 mg of dietary phenylalanine per day.

• Mild PKU.  Affected individuals tolerate 400-600 mg of dietary phenylalanine per day.

• Mild hyperphenylalaninemia (MHP).  Affected infants have plasma Phe concentrations lower than 600 µmol/L (10 mg/dL) on a normal diet. A study by Weglage et al (2001) confirmed the hypothesis of Levy et al (1971) that such individuals may not need dietary treatment. Thirty-one individuals with HPA who were never treated and whose plasma Phe concentrations did not exceed 600 µmol/L had normal cognitive neuropsychological development.

Untreated children with persistent severe hyperphenylalaninemia (i.e., PKU) show impaired brain development. Signs and symptoms include microcephaly, epilepsy, severe mental retardation, and behavior problems. The excretion of excessive phenylalanine and its metabolites can create a musty body odor and skin conditions such as eczema. The associated inhibition of tyrosinase is responsible for decreased skin and hair pigmentation. Affected individuals also have decreased myelin formation and dopamine, norepinephrine, and serotonin production. Further problems can emerge later in life and include exaggerated deep tendon reflexes, tremor, and paraplegia or hemiplegia [Pietz et al 1998 , Williams 1998 , Perez-Duenas et al 2005].

Individuals treated late or never treated may develop severe behavioral or psychiatric problems (depression, anxiety, phobias) in the third or fourth decade [Hanley 2004]. A few case reports have also described untreated individuals with PKU with normal intelligence who were diagnosed in adulthood as a result of a sudden and severe psychiatric deterioration [Weglage et al 2000].

The changes that occur in adolescence and adulthood as a result of high plasma Phe concentrations are both chronic and acute. The important chronic effects of noncompliance are neuropsychological and include a decrease in cognitive function as well as structural changes visible on MRI. The acute toxic effects are initially neurophysiological. They affect neurotransmitter production and can be reflected in electroencephalographic changes. These changes are reversible.

Osteopenia.  Numerous studies indicate that individuals with PKU have a high incidence of osteopenia (as measured by DEXA) [Zeman et al 1999 , Perez-Duenas et al 2002]. Abnormalities in bone density are more likely to occur in those who are poorly compliant with treatment than those who are compliant. Although adolescence is an important time to accrue bone mass, adolescents with PKU are often not compliant with diets. However, Schwahn et al (1998) found osteopenia in an individual diagnosed late who had not received an artificial diet, suggesting that a component of abnormal bone metabolism may be a result of the disease itself.

Genotype-Phenotype Correlations

Phenylalanine hydroxylase deficiency is a 'multifactorial disorder' in that both environment (dietary intake of Phe) and genotype (mutation of the PAH gene) are necessary causal components of disease [Scriver & Waters 1999]. Because each individual has a personal genome, even those with similar mutant PAH genotypes may not have similar 'PKU' phenotypes. As explained elsewhere [Scriver 2002], PAH genotype may not be a robust predictor of phenotype, and to evaluate and treat the individual (the actual phenotype), rather than the phenotype predicted from genotype, is the correct approach.

Variability of metabolic phenotypes in PAH deficiency is caused primarily by different mutations within the PAH gene [Kayaalp et al 1997 , Guldberg et al 1998]. DiSilvestre et al (1991) found that genotype does predict biochemical phenotype (i.e., by Phe loading tests) but does not predict clinical phenotype (i.e., occurrence of mental retardation). PAH deficiency is therefore a 'complex' disorder at the cognitive and metabolic levels [Scriver & Waters 1999]. It is becoming more difficult to assess clinical phenotypes given that most individuals with PAH deficiency in developed countries are treated successfully.

Some untreated individuals with PAH deficiency and PAH mutations that usually confer classic PKU have elevated plasma Phe concentration but normal intelligence. Some sibs with the same genotype have different clinical and metabolic phenotypes. The mechanisms that cause dissimilarities in pathogenesis at the level of the brain in spite of comparable plasma Phe concentrations are still unclear. These atypical individuals have significantly lower brain Phe concentrations than do individuals with similar blood Phe concentrations. Moller et al (1998) suggest that different brain Phe concentrations in individuals with similar blood Phe concentrations are the result of individual variations in the kinetics of Phe uptake and distribution at the blood-brain barrier. Trefz et al (2000) speculate that these atypical individuals may be protected by a second catalytic variant affecting an amino acid transporter. Pietz et al (1999) have demonstrated that loading individuals who have classic PKU with large neutral amino acids decreases Phe uptake into the brain at the transporter and improves neurophysiological parameters.

An emerging theory that attempts to explain the variations in plasma Phe concentrations in individuals with the same genotypes [Scriver & Waters 1999 , Waters et al 2000 , Waters et al 2001] suggests that some missense mutations affect protein folding, thus altering the oligomerization of the nascent PAH protein. This process is likely influenced by an individual's genetic background, including, potentially, differences in the quality and quantity of chaperones and proteases.

Despite all the different factors influencing phenylalanine hydroxylase deficiency phenotypes, the specific PAH genotype is the main determinant of metabolic phenotype in most cases. In compound heterozygotes with functional hemizygosity (null/missense paired alleles), the less severe of the two PAH mutations determines disease severity. When two mutations with similar severity are present, the phenotype may be milder than predicted by either of the mutations [Kayaalp et al 1997 , Guldberg et al 1998 , Waters et al 1998].

Many physicians are now advocating genotyping all newborns identified with hyperphenylalaninemia to better anticipate dietary needs [Guttler & Guldberg 2000 , Zschocke & Hoffman 2000 , NIH Consensus Development Panel 2000]. Zschocke & Hoffman (2000) even suggest that individuals identified as having mild hyperphenylalaninemia associated with known 'mild' mutations be treated as such with relaxation of their follow-up control analyses at a relatively early stage. Greeves et al (2000) suggest that genotype can help predict the likelihood of intellectual change if or when individuals relax their dietary restrictions later in life.

Genotype-phenotype correlation is also seen with a large number of mutations that are BH₄ responsive. Therefore, genotyping may help to predict which individuals will respond to BH₄ supplementation and what the response will be. However, there are mutations that show inconsistency from person to person (see BH₄ Databatases).

Prevalence

Since the appearance of universal newborn screening, symptomatic classic PKU is infrequently seen. Its predicted incidence in screened populations of fewer than one in a million live births reflects those children not detected by newborn screening.

Historical Perspective

Hyperphenylalaninemia (HPA) has been called the epitome of human biochemical genetics. In 1934, Asjbørn Følling recognized that a certain type of mental retardation was caused by elevated levels of phenylalanine in body fluids. He identified the disease as an autosomal recessive condition. In the 1940s, Lionel Penrose, who had recognized 'phenylketonuria' (PKU) to be the first form of mental retardation with a chemical explanation, introduced the idea that PKU was not randomly distributed in human populations and could be treatable. In the mid-1950s, it was demonstrated that individuals with PKU had a deficiency of hepatic cytosolic phenylalanine hydroxylase (PAH) enzyme activity.

Next it was shown that affected individuals responded to dietary restriction of the essential nutrient phenylalanine. By the 1960s, a microbial inhibition assay was used for mass screening of newborns, providing early diagnosis and access to successful treatment.

In the 1970s, it was discovered that not all HPA was PKU. Some forms of HPA were caused by disorders of synthesis and recycling of the cofactor (tetrahydrobiopterin, or BH₄) involved in the Phe hydroxylation reaction. During the 1980s, the human PAH gene was mapped and cloned, and the first mutations identified. In the 1990s, in vitro expression analysis was being used to study the effects of different PAH alleles on enzyme function and the crystal structure of PAH was elucidated.

HPA is treatable. Affected individuals can lead normal lives. Continuous efforts are made to improve the taste and convenience of the current synthetic dietary supplements [Rohr et al 2001]. Research to improve the current treatment with restrictive phenylalanine diets, supplemented by medical formula, is ongoing (see Management).

Differential Diagnosis

For current information on availability of genetic testing for disorders included in this section, see GeneTests Laboratory Directory. —ED.

Hyperphenylalaninemia (HPA) may also result from the impaired synthesis or recycling of tetrahydrobiopterin (BH₄), the cofactor in the phenylalanine, tyrosine, and tryptophan hydroxylation reactions. Defects in BH₄ synthesis result from guanosine triphosphate cyclohydrolase (GTPCH) deficiency or 6-pyruvoyl tetrahydrobiopterin synthase (PTPS) deficiency. Impaired recycling of BH₄ is caused by deficient dihydropteridine reductase (DHPR) or deficient pterin-4 acarbinolamine dehydratase (PCD). All of the HPAs caused by BH₄ deficiency are inherited in an autosomal recessive manner. They account for approximately 2% of individuals with HPA. BH₄ is also involved in catecholamine, serotonin, and nitric oxide biosynthesis (see www.bh4.org).

Scriver & Kaufman (2001) and Blau et al (2001) emphasize that all neonates with persistent hyperphenylalaninemia must be screened for the BH₄ deficiencies. The following tests are best performed in specialized centers. Prenatal diagnosis is possible for all forms of BH₄ deficiencies. The following screening tests are essential:

• Analysis of pterins in the urine
• Measurement of DHPR activity in blood from the Guthrie card. If screening test results are positive, the following confirmatory tests are recommended:
  • Loading test with BH₄
  • Analysis of folates and neurotransmitter metabolites in CSF
  • Enzyme activity measurements

The typical (severe) forms of GTPCH, PTPS, and DHPR deficiencies have the following variable, but common, symptoms: mental retardation, convulsions, disturbance of tone and posture, drowsiness, irritability, abnormal movements, recurrent hyperthermia without infections, hypersalivation, and swallowing difficulties. Microcephaly is common in PTPS and DHPR deficiencies. Plasma phenylalanine concentations can vary from slightly above normal (>120 µmol/L) to as high as 2500 µmol/L. Mild forms of BH₄ deficiency have no clinical signs. PCD deficiency, sometimes referred to as 'primapterinuria' is associated with benign transient hyperphenylalaninemia.

In principle, BH₄ deficiencies are treatable. Treatment requires the normalization of BH₄ availability and of blood Phe concentration and restoration of the BH₄-dependent hydroxylation of tyrosine and tryptophan. This is achieved by BH₄ supplementation along with dietary modification, neurotransmitter precursor replacement therapy, and supplements of folinic acid in DHPR deficiency. The treatment should be initiated early and probably continued for life [Blau et al 2001].

More information on the BH₄ deficiencies can be found on the Tetrahydrobiopterin Web site (www.bh4.org).

Management

Evaluations Following Initial Diagnosis

Infants with hyperphenylalaninemia are classified at the time of diagnosis based on blood phenylalanine concentration and phenylalanine tolerance [Kayaalp et al 1997 , Guldberg et al 1998]. Because the PAH genotype may not be a robust predictor of phenotype (see Genotype-Phenotype Correlations), the individual's diet should be tailored to calculate phenylalanine tolerance irrespective of genotype.

BH₄ loading tests determine which persons are BH₄-responsive and which are not in order to relax or discontinue restriction of dietary phenylalanine in those who are responsive. BH₄ responsiveness is determined based on response to a pharmacologic dose of BH₄ (10-20 mg/kg per day).

• The majority of BH₄ responders belong to the mild PKU and hyperphenyalaninemia group [Bernegger & Blau 2002 , Perez-Duenas et al 2004 , Mitchell et al 2005].
• A small number of BH₄ responders have classic disease [Perez-Duenas et al 2004 , Zurfluh et al 2006].

Note: Although the genotype/phenotype relationship for BH4 responsiveness is also fairly robust, discrepancies have been described; see Mutations (pdf).

Treatment of Manifestations

Classic PKU

• Restriction of dietary phenylalanine.   The generally accepted goal of treatment for the hyperphenylalaninemias is normalization of the concentrations of Phe and Tyr in the blood and thus prevention of the cognitive deficits that are attributable to this disorder [Burgard et al 1999]; Phe concentrations of 120-360 µmol/L (2-6 mg/dL) [Koch, Azen et al 1996] or 40-240 µmol/L (1-4 mg/dL) [Burgard et al 1999] are generally regarded as safe.

  A diet restricted in Phe should be initiated as soon as possible after birth and continued at least into adolescence [Pietz et al 1998], perhaps for life [Medical Research Council Working Party on PKU 1993 , NIH Consensus Development Panel 2000].

  It is clear that if the diet is not followed closely, especially during childhood, and if plasma Phe concentration is allowed to rise frequently above the recommended concentration, some impairment is inevitable.

  The restricted phenylalanine diet is adapted to individual tolerance for phenylalanine and includes appropriate protein and energy for age.

  Plasma concentrations of Phe within the effective treatment range and normal nutritional status cannot be achieved by a low-protein diet alone, but rather require the use of a Phe-free medical formula.

  The diet must be carefully monitored so that growth and nutritional status are unaffected and deficiency of phenylalanine or tyrosine is not created. The diet must be adjusted for growth, illness, activity, etc. Adequate calcium and vitamin D intake as assessed by the metabolic dietician is an important component of care.

  Treatment for affected individuals of all ages can be difficult and is enhanced with the teaching and support of an experienced healthcare team consisting of physicians, nutritionists, genetic counselors, social workers, nurses, and psychologists.

• Supplementation with BH₄.  An ever-growing body of evidence indicates that many individuals with primary phenylalanine hydroxylase deficiency are responsive to the 6R-BH₄ stereoisomer in pharmacologic doses (20 mg/kg daily in divided oral doses) [Kure et al 1999 , Bernegger & Blau 2002 , Matalon et al 2004]. The majority of individuals with mild or moderate PKU may be responsive to BH₄ while up to 10% of individuals with classic PKU can show a response [Bernegger & Blau 2002 , Perez-Duenas et al 2004]. Most responders can be detected with a standard 24-hour load, but some slow responders escape detection unless a more prolonged protocol is used [Shintaku et al 2004 , Fiege et al 2005 , Mitchell et al 2005].

  Individuality in BH₄ pharmacokinetics implies the need for patient-specific dosage schedules [Blau & Erlandsen 2004]. Long-term treatment in a small number of BH₄-responsive persons has documented the maintenance of the phenylalanine lowering effect and the absence of major side effects [Trefz et al 2005]. In the majority of individuals, the BH₄ response is likely a result of correction of PAH mutant kinetic effects and/or a chaperone-like effect of BH₄ [Erlandsen et al 2004].

  Whatever the mechanism of the therapeutic effect, the 6R-BH₄ enhances in vivo phenylalanine hydroxylation and the corresponding oxidative flux and lowers plasma phenylalanine concentration with improved tolerance of dietary phenylalanine [Muntau et al 2002].

• Treatment in infancy.  A Phe-restricted diet and a Phe-free medical formula must be started as soon as possible after birth under the direction of a nutritionist. Breastfeeding is encouraged along with Phe-free formula [NIH Consensus Development Panel 2000]. Intake of tyrosine and total amino acids must be monitored. Children under age two years should maintain a total amino acid intake of at least 3 g/kg/day including 25 mg tyrosine/kg/day. The consumption of Phe-free formula should be spread out evenly over the 24 hours of the day to minimize fluctuations in blood amino acid concentrations [Medical Research Council Working Party on PKU 1993]. Care must be taken to avoid long periods of low blood Phe concentration, which is also harmful to brain development. Blood Phe concentration should be monitored weekly or biweekly to evaluate control [Burgard et al 1999].

• Treatment in childhood.  Children older than age two years should maintain a total amino acid consumption of 2 g/kg/day including 25 mg tyrosine/kg/day. Monitoring on a biweekly basis is recommended until age seven years and on a monthly basis thereafter.

• Treatment in adolescence and adulthood.  Recommendations for treatment of adolescents and adults vary [Lee 2004]. In general, support for 'diet therapy for life' is increasing [Brenton et al 1996 , NIH Consensus Development Panel 2000]. Some recommendations are more liberal than others and indicate that relaxation (not elimination) of the strict diet in adolescence does not affect non-executive functions when plasma Phe concentration remains below 1200 µmol/L [Griffiths 2000]. However, other studies indicate that if the diet is relaxed after the age of 12 years, IQ can remain stable but other functions deteriorate. Adults who have abandoned the Phe-restricted diet tend to have a reduced attention span, slow information-processing abilities, and slow motor reaction time. They also develop changes in the frequency distribution of brain electrical activity [Pietz et al 1998], increased muscle tone and tremor, lowered bone mineral content, and mental health disorders. Controversy remains over the plasma Phe concentration to be achieved for individuals older than age 12 years. The general consensus is that the closer the Phe concentration is to the recommended normal value, the better the individual's general state of well-being.

Non-PKU HPA.   Individuals with non-PKU HPA who have plasma Phe concentrations consistently below 600 µmol/L (10 mg/dL) are not at higher risk of developing intellectual, neurologic, and neuropsychological impairment than are individuals without PAH deficiency. While some specialists debate the advisability of non-treatment, others believe that dietary treatment is unnecessary for the individuals in this class [Weglage et al 1996 , Burgard et al 1999 , Weglage et al 2001].

Care should be taken so that girls in this group receive proper counseling about the teratogenic effects of elevated maternal plasma Phe concentration (i.e., 'maternal HPA/PKU') when they reach childbearing age [Weglage et al 2001].

Pregnant women with PAH deficiency.   Women with PAH deficiency who have been properly treated throughout childhood and adolescence have normal physical and intellectual development. However, if the woman has high plasma Phe concentrations, her intrauterine environment will be hostile to a developing fetus as phenylalanine is a potent teratogen [Jardim et al 1996]. It is strongly recommended that women with PAH deficiency use reliable methods of contraception to prevent unplanned pregnancies.

Women with PAH deficiency who are off diet and are planning a pregnancy should start a Phe-restricted diet prior to conception and should maintain plasma Phe concentrations between 120 and 360 µmol/L (2-6 mg/dL), ideally over several months, before attempting conception [Koch, Levy et al 1996]. After conception, they should be offered continuous nutritional guidance and weekly or biweekly measurement of plasma Phe concentration because dietary Phe and protein requirements change considerably during pregnancy. It is important that pregnant women with PAH deficiency have an adequate energy intake with the proper proportion of protein, fat, and carbohydrates. They should try to attain normal weight gain patterns to provide the most favorable conditions for fetal growth [Michals et al 1996 , Koch et al 2000].

The abnormalities that result from exposure of a fetus to high maternal plasma Phe concentration are the result of 'maternal HPA/PKU' [Lenke & Levy 1980]. The likelihood that the fetus will have congenital heart disease, intrauterine and postnatal growth retardation, microcephaly, and mental retardation depends upon the severity of the maternal HPA and the effectiveness of the mother's dietary management. Although studies have shown that women with non-PKU HPA who have plasma Phe concentrations lower than 400 µmol/L (7 mg/dL) when untreated [Levy, Waisbren et al 1996 ; Jardim et al 1996] can give birth to offspring who seem to be normal [Levy, Waisbren et al 1996], the Maternal PKU Collaborative Study reports that even at maternal plasma Phe concentrations of 120-360 µmol/L (2-6 mg/dL), 6% of infants are born with microcephaly and 4% with postnatal growth retardation [Rouse et al 1997]. If maternal plasma Phe concentrations are greater than 900 µmol/L (15 mg/dL), the risk is 85% for microcephaly, 51% for postnatal growth retardation, and 26% for intrauterine growth retardation [Rouse et al 1997]. The risk for these abnormalities is both dose dependent [Levy & Ghavami 1996] and time dependent [Rouse et al 1997]. Thus, optimal plasma Phe concentrations must be strictly maintained throughout pregnancy to reduce the risk of each individual abnormality; continuing studies corroborate this position [Rouse & Azen 2004].

Unfortunately, many pregnancies are unplanned. Women with HPA who conceive while off the Phe-restricted diet and who manage to bring their plasma Phe concentrations into the recommended range (120 to 360 µmol/L) as early as possible after conception and no later than eight weeks of pregnancy can be encouraged by the existing data [Koch, Levy et al 1996]. Though the risks of congenital abnormalities, especially for congenital heart disease, are greater than if plasma Phe concentration is controlled preconceptually, the possibility for a normal child still exists. If plasma Phe concentrations are not controlled until the second or third trimester, the risks are the same as if the plasma Phe concentration were never brought under control [Koch et al 2000].

Proper prenatal care should include serial ultrasonography to: (1) identify nonviable pregnancies in the first trimester; (2) monitor fetal growth; and (3) identify congenital abnormalities (such as congenital heart disease) that are relatively common in babies whose mothers have PAH deficiency. This information can be useful in anticipating the postnatal needs of the infant [Levy, Lobbregt et al 1996].

Waisbren et al (1998) contend that suboptimal home environments can have as much of an adverse effect on offspring as a delay in control of maternal plasma Phe concentration. Of concern, the limited intellectual abilities, the reduced social resources, and the emotional difficulties of many women with HPA may complicate adherence to the diet, prenatal care, and the care of their infant [Koch et al 2000]. Support for women with HPA/PKU starting before conception and continuing after delivery is essential for optimal outcome. A successful pilot project involving mothers of children with PKU serving as resources for these women resulted in better dietary control during pregnancy and better outcome for the children [Waisbren et al 2000].

Too little published data exist on children born to mothers with PKU. One interesting observation of this cohort of children is a decline in the scores achieved on developmental tests at age four years from those achieved on similar tests taken at age two years [Waisbren et al 2000]. Longitudinal studies of these children are necessary to interpret the significance of these findings.

Prevention of Primary Manifestations

See Treatment of Manifestations .

Surveillance

Plasma Phe and Tyr concentrations in individuals with classic PKU must be monitored regularly [Medical Research Council Working Party on PKU 1993 , NIH Consensus Development Panel 2000].

Currently, there are no recommendations for surveillance for osteopenia, but baseline bone scans may be of value for long-term follow-up.

Agents/Circumstances to Avoid

Aspartame, an artificial sweetener in widespread use, contains phenylalanine. Persons with PKU should either avoid products containing aspartame or calculate intake of Phe and adapt diet components accordingly.

Testing of Relatives at Risk

Newborn sibs of an individual with PKU who have not been tested prenatally should have blood concentration of phenylalanine measured shortly after birth, in addition to their newborn screen. This will allow earlier detection than by newborn screening alone and thus, treatment as soon as possible after birth.

See Genetic Counseling for issues related to testing of at-risk relatives for genetic counseling purposes.

Therapies Under Investigation

Although the treatment of PKU with phenylalanine-restricted diets has been hugely successful, the poor palatability of the diet results in poor compliance in adolescence and adulthood. A number of attempts are ongoing to find other treatment modalities for PKU.

Large neutral amino acids (LNAA) transporters.  At the blood-brain barrier, phenylalanine shares a transporter with other large neutral amino acids (LNAA). Some individuals exclude excess phenylalanine, more or less efficiently, because they show evidence of variation in the high-capacity/high k_m component of phenylalanine transport across the blood-brain barrier [Weglage et al 2002]. LNAA supplementation has reduced brain phenylalanine concentration despite consistently high serum concentrations of phenylalanine by competition at this transporter [Pietz et al 1999 , Moats et al 2003]. In non-compliant adults, this may help to protect the brain from acute toxic effects of phenylalanine.

Significant improvement in ability to concentrate and decreased self-injurious behavior were seen with trial of LNAA supplements in a small number of untreated adults with PKU [Kalkanoglu et al 2005].

A similar transporter for LNAA also exists in the intestine and supplementation with a different formulation of LNAA reduced the blood Phe concentration by up to 50% in a small number of individuals with PKU [Matalon et al 2006]. These supplements will not replace the phenyalanine-restricted diet but may help relax dietary restriction of treated individuals or may aid in management of adults who are not treated. Clinical trials are needed before conclusions on the effectiveness of these treatments can be made.

BH₄ supplementation.  BH₄ may have important implications for treatment of maternal PKU [Trefz et al 2003]. By increasing the phenylalanine tolerance, BH₄ supplementation may allow for a more complete diet, thus minimizing risk of nutritional deficiencies associated with a low-protein diet. In one case report, pregnancies were successfully managed with BH₄ supplementation; however, more data are needed to prove the safety and efficacy of this alternative treatment [Koch et al 2005].

Under investigation is the administration of the enzyme phenylalanine ammonia lyase (PAL) by oral routes to degrade Phe to trans-cinnamic acid and ammonia [Sarkissian et al 1999 , Gamez et al 2004]. The oral route is complicated by proteolytic degradation, while injected PAL is complicated by increased immunogenicity [Gamez et al 2005 , Sarkissian & Gamez 2005]. Research continues in this area to find modifications to the PAL enzyme that will decrease immunogenicity and prolong activity.

Somatic gene therapy is also being explored in animal models and holds some promise for possible future treatment [Eisensmith et al 1999 , NIH Consensus Development Panel 2000 , Ding et al 2004 , Ding et al 2006].

Search ClinicalTrials.gov for access to information on clinical studies for a wide range of diseases and conditions.

Other

Genetics clinics are a source of information for individuals and families regarding the natural history, treatment, mode of inheritance, and genetic risks to other family members as well as information about available consumer-oriented resources. See the GeneTests Clinic Directory.

Support groups have been established for individuals and families to provide information, support, and contact with other affected individuals. The Resources section may include disease-specific and/or umbrella support organizations.

Genetic Counseling

Genetic counseling is the process of providing individuals and families with information on the nature, inheritance, and implications of genetic disorders to help them make informed medical and personal decisions. The following section deals with genetic risk assessment and the use of family history and genetic testing to clarify genetic status for family members. This section is not meant to address all personal, cultural, or ethical issues that individuals may face or to substitute for consultation with a genetics professional. To find a genetics or prenatal diagnosis clinic, see the GeneTests Clinic Directory. —ED.

Mode of Inheritance

Phenylalanine hydroxylase (PAH) deficiency is inherited in an autosomal recessive manner.

Risk to Family Members

Parents of a proband

• Unaffected parents of a child with PAH deficiency are obligate heterozygotes and therefore carry one mutant allele.
• Heterozygotes (carriers) are asymptomatic and do not have hyperphenylalaninemia.

Sibs of a proband

• At conception, each sib of a proband has a 25% chance of being affected (homozygous), a 50% chance of being an asymptomatic carrier of a disease-causing allele (heterozygous), and a 25% chance of having two normal alleles.
• Once an at-risk sib is known to be unaffected, the risk of his/her being a carrier is 2/3.

Offspring of a proband

• Because PAH deficiency is treatable, affected individuals who have the benefit of effective treatment can live physically and intellectually normal lives and have children of their own.
• Children born of one parent with PAH deficiency and one parent with two normal alleles are obligate heterozygotes.
• If one parent is affected and the other parent is a carrier, the offspring have a 50% chance of being heterozygous and a 50% chance of being affected.
• If the father is the affected parent, no additional risks exist. If the mother is the affected parent, maternal HPA/PKU becomes a critical issue (see Management: Pregnant women with PAH deficiency).

Carrier Detection

• Carrier testing for at-risk family members is available on a clinical basis once the mutations have been identified in the proband.
• Reproductive partners of carriers can now access such testing on a limited basis.
• Three methods can be used: