Classification

A blood phenylalanine level above the normal range (30–110 μM) is referred to as hyperphenylalaninemia. Patients have been classified as having nonphenylketonuria hyperphenylalaninemia if their blood phenylalanine levels without dietary therapy are 360–600 μM. Classic phenylketonuria is characterized by untreated phenylalanine levels of more than 1000 μM [Scriver and Kaufman, 2001]. A range of reduced PAH-specific activity correlates broadly with the severity of the phenotype. When it has been measured directly (i.e., liver biopsy) or indirectly (i.e., l-[1-¹³C]phenylalanine breath test), residual liver PAH-specific activity is relatively high in milder hyperphenylalaninemic patients, whereas enzyme activity is zero to low in the more severe cases of classic phenylketonuria [Bartholome et al., 1975]. Measured PAH activity also correlates to some degree with tolerance for dietary protein [Güttler et al., 1996]. Patients with classic phenylketonuria can tolerate very little phenylalanine in the diet (<500 mg/day). The classification of phenylketonuria into subtypes based on blood phenylalanine levels is arbitrary, and some patients (i.e., mild, variant, or atypical phenylketonuria) fall between the two extreme biochemical phenotypes. Because environmental (phenylalanine intake) and genetic (modifier genes) factors alter the biochemical and neurodevelopmental phenotypes in this single-gene disorder, phenylketonuria in many ways behaves more like a complex trait than a monogenic disorder [Kayaalp et al., 1997].

Clinical Manifestations

Profound mental retardation is the most significant clinical finding in untreated or poorly treated phenylketonuria. Acute metabolic encephalopathy, a common feature of many inborn errors of metabolism, does not occur in phenylketonuria. Children with phenylketonuria appear normal at birth and have normal early development, even if untreated. Neurologic manifestations appear insidiously and include reduced rate of growth of head circumference, developmental delay, abnormalities in muscle tone, and hyperactive deep tendon reflexes. Affected children may have lighter pigmentation than other family members (i.e., reduced melanin synthesis) and a musty odor attributed to phenylacetic acid. Eczema and decreased bone mineral density may occur [Zeman et al., 1999]. Patients exposed to chronically elevated phenylalanine levels ultimately develop microcephaly, seizures (e.g., tonic-clonic, myoclonic, infantile spasms), tremors, athetosis, and spasticity, and they may be misdiagnosed as having cerebral palsy. Psychiatric and behavior problems, including autistic behavior and attention-deficit hyperactivity disorder, are common [Pietz et al., 1997; Smith and Knowles, 2000].

Brain magnetic resonance imaging (MRI) may detect dysmyelination, especially T2 enhancement in the periventricular white matter, a finding that is potentially reversible with the initiation of dietary therapy [Cleary et al., 1995]. Abnormal areas of white matter demonstrate restricted diffusion of water, possibly indicating increased myelin turnover [Phillips et al., 2001].

In the past, most untreated phenylketonuria patients were institutionalized, and many born before universal newborn screening remain so. An eloquent and inspiring description of the life of a child with untreated phenylketonuria is given in a short monograph by Pearl S. Buck [Buck, 1950].

As a rule, well-treated classic phenylketonuria patients have normal IQs. However, recent studies have found that children and adults with phenylketonuria may experience cognitive symptoms, such as problems in executive functioning, as well as disturbance in emotional and behavioral functioning despite early and continuous treatment [Enns et al., 2010]. Dietary control of the blood phenylalanine level appears to be the best predictor of ultimate IQ [Waisbren et al., 1987], but careful psychometric testing of well-treated individuals has detected instances and degrees of impairment in visual-motor skills, abstract reasoning, problem solving, specific aspects of executive control, attention, verbal memory, expressive naming, and verbal fluency [Fishler et al., 1987]. Such neuropsychologic impairments may be a consequence of mid-dorsolateral prefrontal cortex dysfunction caused by abnormal catecholamine levels [Huijbregts et al., 2002]. Abnormal EEG patterns, including general slowing and generalized paroxysmal activity with or without spikes, may be demonstrated for children with phenylketonuria, even if they are well treated [Pietz et al., 1988]. Emotional disorders (e.g., depression, anxiety, phobias) and hyperactive behavior are more frequently encountered in persons with classic phenylketonuria than in the general population [Smith and Knowles, 2000]. However, untreated mild hyperphenylalaninemic patients are not at risk for developing neuropsychologic impairment [Weglage et al., 1996].

Maternal Phenylketonuria Syndrome

Elevated maternal blood phenylalanine levels can cross the placenta and cause fetal birth defects, including microcephaly, dysmorphic features, and congenital heart defects. Children with the maternal phenylketonuria syndrome are typically heterozygous for the mutant PAH allele, and they are not affected with phenylketonuria. More than 90 percent of children born to women with untreated classic phenylketonuria have mental retardation; 70 percent have microcephaly, 40 percent have intrauterine growth retardation, and 12 percent have congenital heart disease [Lenke and Levy, 1980]. The risk to the fetus is greatest with increasing phenylalanine levels in maternal blood. Dietary control should ideally be achieved before 3 months prior to conception, and mothers with phenylketonuria should be monitored carefully by an experienced center throughout pregnancy [ACOG, 2009]. Optimal birth outcomes occur when blood phenylalanine levels between 120 and 360 μM are achieved by 8–10 weeks’ gestation [Widaman and Azen, 2003]. Mothers with phenylketonuria can safely breastfeed their children.

Diagnosis

The Guthrie bacterial inhibition assay was a technical breakthrough, allowing newborn screening of large populations. The growth inhibition of Bacillus subtilis by β-2-thienylalanine is prevented by phenylalanine, phenylpyruvic acid, and phenyllactic acid, and this forms the basis of the Guthrie test. Fluorometric assays or tandem mass spectrometry is used in screening and monitoring [Chace et al., 1993]. False-positive results may be seen in neonates with low birth weight or liver disease, or in infants on parenteral alimentation. False-negative results may occur if the newborn screen is performed too early (especially less than 12 hours after birth) [Hanley et al., 1997]. Confirmation of the diagnosis is made by analysis of blood phenylalanine and tyrosine concentrations by means of high-performance liquid chromatography, fluorescent methods, or tandem mass spectrometry. Without the introduction of a phenylalanine-restricted diet, maximal elevation in plasma phenylalanine is typically reached within or soon after the first week of life in patients with classic phenylketonuria. Urine phenylpyruvic acid causes the appearance of a deep green color when ferric chloride is added. This ferric chloride test is sometimes performed as part of a metabolic screening panel for the evaluation of patients suspected of having an inborn error of metabolism, but it should not be used to confirm a diagnosis of phenylketonuria because of a lack of sensitivity and specificity. All patients with confirmed hyperphenylalaninemia must have urine pterins analyzed for defects in tetrahydrobiopterin metabolism. Phenylketonuria may be suspected in a child or adult and should reasonably be included in the differential diagnosis of a given patient of any age presenting with neurodevelopmental delay of unknown origin. In such settings, diagnostic testing for phenylketonuria (i.e., serum phenylalanine levels) must be done, even if there is a history or a record of a normal newborn screen.

Genetics

Phenylketonuria is an autosomal-recessive disorder with an incidence of 1 case per 10,000 people in the general white population of northern European ancestry [Eisensmith and Woo, 1994]. Phenylketonuria is more common in Turkey, Scotland, and Czechoslovakia, and among Arabic populations and Yemenite Jews (1 case per 2500–5000 persons). It is relatively uncommon in Japan and Finland, and among African Americans (1 case per 100,000–200,000 persons) [Scriver and Kaufman, 2001]. Nonphenylketonuria hyperphenylalaninemia has an overall incidence of 1 case per 50,000 persons.

The PAH gene on chromosome 12q24.1 spans 90 kb and contains 13 exons [Kwok et al., 1985]. Almost 500 mutations have been reported throughout all exons and flanking sequences. A detailed account of PAH mutant alleles and other DNA variations is maintained at the PAH Mutation Analysis Consortium website (http://www.pahdb.mcgill.ca/) [Waters and Scriver, 1998]. Most DNA alterations are missense mutations, although splice, nonsense, and frameshift mutations and large deletions and insertions have been identified. Most patients are compound heterozygotes, carrying a different mutant allele on each chromosome. Prevalences of specific mutant alleles differ from population to population [Eisensmith and Woo, 1994]. For example, R408W, IVS12nt1, and IVS10nt11 are severe mutations that account for about 50 percent of mutant alleles in Europeans, but they are rare in Asians. Conversely, mutant alleles R243Q, R413P, and Y204C are common in Asians, but they rare in Europeans [Eisensmith and Woo, 1994].

Pathogenesis

Although PAH is a hepatic enzyme, the major effect of its deficiency is brain dysfunction. Elevated phenylalanine appears to be the cause of neurotoxicity. However, the precise cause of the mental retardation observed in untreated phenylketonuria is not understood. Defective brain myelination may be related to decreased biosynthesis of myelin proteins, because brain protein synthesis is inhibited by excessive phenylalanine [Huether et al., 1982]. CNS effects may be ascribable to more global amino acid imbalances; elevated phenylalanine may affect the CNS concentrations of neutral amino acids by competitive inhibition of a shared amino acid transporter, with relative brain deprivation of tyrosine, tryptophan, and branched-chain amino acids [Huttenlocher, 2000]. Decreased brain tyrosine and tryptophan may lead to decreased neurotransmitter synthesis. The cause of abnormal brain myelination is also unclear. In a phenylketonuria mouse model (i.e., enu2 mouse), there is evidence that oligodendrocytes overexpress glial fibrillary acid protein and become nonmyelinating [Dyer et al., 1996]. Increased myelin turnover has also been observed in the enu2 mouse [Hommes and Moss, 1992]. In phenylketonuria patients studied by positron emission tomography (PET), brain protein synthesis appears to be impaired, which could also affect the myelination process [Paans et al., 1996]. Brain pathology in untreated classic phenylketonuria includes abnormalities in width of the cortical plate, cell density and organization, dendritic arborization and number of synaptic spines, and abnormal myelination [Bauman and Kemper, 1982].

Genotype-Phenotype Correlations

Mutations in the PAH gene cause phenylketonuria and nonphenylketonuria hyperphenylalaninemia. However, the final biochemical phenotype (i.e., blood phenylalanine level and dietary phenylalanine tolerance) and clinical phenotype (i.e., IQ) depend on the severity of the mutations, and are influenced by patient adherence to a strict diet, the effects of modifying genetic factors, and other environmental factors. Potential modifier genes may encode proteins mediating interindividual rates of protein synthesis (and phenylalanine use) or basal metabolism; synthesis and degradation of the PAH enzyme protein; gastrointestinal absorption of phenylalanine; hepatic uptake of circulating phenylalanine; metabolism of the tetrahydrobiopterin co-factor; and rate of phenylalanine transport across the blood–brain barrier [Dipple and McCabe, 2000; Treacy et al., 1997]. Because patients – including sibling patients – with identical mutations can have divergent neurodevelopmental progress, mutation identification may not predict the severity of the disease with certainty in a given patient [DiSilvestre et al., 1991; Enns et al., 1999b].

Genes encoding proteins responsible for transport of amino acids across the blood–brain barrier are especially attractive candidates for modifying factors in phenylketonuria. In a study of two siblings with identical genotypes but widely different IQ, in vivo nuclear MR spectroscopy documented lower peak brain phenylalanine levels and more rapid decreases in brain phenylalanine concentration in the less severely affected sibling after a phenylalanine load [Weglage et al., 1998]. Subsequent studies have confirmed the wide interindividual variation of brain to blood phenylalanine concentrations in classic phenylketonuria patients with divergent cognitive phenotypes [Moller et al., 2003].

Despite these considerations, trends can be identified in whole populations. In general, individuals with classic phenylketonuria and poor dietary control have mental retardation, although exceptions exist. If patients with severe mutations are started on strict dietary therapy in the neonatal period and maintained on such treatment throughout life, cognition will be normal. Using in vitro expression analysis in cultured cells transfected with mutant cDNAs, specific mutant alleles (i.e., genotype) can be categorized as severe or mild; such categorization correlates with biochemical or clinical severity (i.e., phenotype) in most patients in relatively homogenous populations. In a study of German and Dutch subjects, the predicted level of PAH activity correlated strongly with neonatal pretreatment levels of blood phenylalanine and dietary phenylalanine tolerance in both populations [Okano et al., 1991]. In relatively homogeneous German, Swedish, and southeastern U.S. populations, similar genotype-phenotype correlations were observed [Eisensmith et al., 1996; Kayaalp et al., 1997; Trefz et al., 1993]. However, when populations with high ethnic diversity are studied in this way, a clear genotype-phenotype correlation may not be apparent [Enns et al., 1999b; Treacy et al., 1997], and the genotype-phenotype correlation is not straightforward in some patients. Although phenylketonuria is a single-gene mendelian disorder, the observed clinical spectrum is more in keeping with a complex multifactorial trait [Scriver and Waters, 1999].

Genetic Counseling

After the diagnosis of phenylketonuria and once the newborn screening process has confirmed the diagnosis, a family is likely to be first seen by the medical care team. The initial emphasis is on the institution of nutritional therapy, the support of the family, and the conveying of information on the broadly favorable prognosis. Genetic counseling in phenylketonuria cases is a continuing process. Understanding on the part of the parents and patients of the nature of the disorder, the genetics of the disorder, and the complexities of the phenotype and prognosis occurs over time during the course of multiple clinic visits. Mutation analysis, with characterization of both parental alleles, is possible, and it may facilitate detection of carrier status in other family members and subsequent prenatal diagnosis. Genotyping may eventually prove valuable in predicting a patient’s phenotype, helping to optimize therapy, and aiding determination of long-term prognosis. However, because phenylketonuria behaves in many ways like a complex trait, care is needed when interpreting genotypic data.

In the instance of a child with milder hyperphenylalaninemia or atypical phenylketonuria, recurrence risk counseling must consider the possibility that such mild hyperphenylalaninemia may not be the only outcome in a subsequent homozygous affected child. If one parent is a compound heterozygote for a mild and a severe mutation, but the other parent is a heterozygote for a severe mutation, their child may be born with classic phenylketonuria. Accordingly, after ascertainment of a child with milder hyperphenylalaninemia or atypical phenylketonuria, measuring the blood phenylalanine levels of the parents and characterizing both parents’ mutations are important studies to obtain.

Patients who are diagnosed in the neonatal period and who adhere to the phenylalanine-restricted diet have normal intelligence. However, learning problems can occur in well-treated patients and include problems in basic spelling, reading, and mathematical calculation skills. Patients may also be more prone to depression, anxiety, phobic tendencies, and isolation from their peers [Smith and Knowles, 2000; Welsh et al., 1990]. Such potential adverse and unpredictable manifestations should be brought to the attention of parents and carefully explained, with care and support, during the on-going genetic counseling process.

Treatment

Medical nutrition therapy consists of using modified low-phenylalanine and low-protein products, supplemented by a small amount of natural protein to provide required amounts of phenylalanine. After an initial positive newborn screen, a confirmatory determination of blood phenylalanine level must be obtained without delay. Dietary restriction of phenylalanine is begun only after a diagnosis of phenylketonuria has been established or if the initial phenylalanine level is highly elevated in a term infant not being supplemented with total parenteral nutrition as anticipatory management with the advice and collaboration of a metabolic center skilled in the management of phenylketonuria patients. Total elimination of phenylalanine from the diet is not done for longer than 1–2 days, because phenylalanine deficiency leads to tissue catabolism and rebound elevation of blood phenylalanine levels. It is important to screen for the presence of disorders affecting tetrahydrobiopterin metabolism if confirmatory testing corroborates persistent hyperphenylalaninemia. If the low-phenylalanine diet is initiated in the neonatal period (between 7 and 14 days) and maintained throughout life, the underlying biochemical toxicity is ameliorated, and mental retardation is prevented. In the neonatal period, breastfeeding is possible and should be encouraged in any mother who desires to do so. Breast milk contains a lower protein (and lower phenylalanine) concentration than commercial formulas, and it can be used in conjunction with the special phenylketonuria formulas required to provide the infant with appropriate calories, nutrients, and protein for sustained, normal growth.

Significant restriction of dietary phenylalanine is required for treatment, but the exact level of daily phenylalanine intake varies from patient to patient, and varies with age in an individual patient. Because phenylalanine is an essential amino acid, detrimental effects on growth and development may occur if restriction of phenylalanine intake is too severe and the blood level drops to below normal. Although there is no worldwide consensus about optimal plasma phenylalanine levels, most clinics in the United States strive to maintain levels between 120 and 360 μM in children younger than 12 years and between 120 and 600 μM in individuals older than 12 years [Phenylketonuria, 2000]. Phenylalanine levels in unaffected individuals are usually below 120 μM. In general, phenylketonuria patients who harbor severe mutations require a greater limitation of phenylalanine intake to maintain acceptable blood phenylalanine levels. However, individual variations of phenylalanine tolerance may occur, even in patients with identical genotypes. Blood phenylalanine levels therefore are monitored frequently, especially in the first year of life, and the diet is adjusted with care for each individual patient. The regimen must be initiated and overseen by experts in phenylketonuria at a specialized center, and referral of the patient to such a specialized center is mandatory. An expert, coordinated team approach is clearly the most effective way of managing phenylketonuria; stricter management improves developmental outcome [Camfield et al., 2004].

In earlier therapeutic protocols, phenylalanine restriction was continued only through the first few years of life, theoretically corresponding to the age at which brain myelination is complete. As developmental data accumulated, it became evident that treatment throughout childhood and adolescence was the best course to preserve IQ [Smith et al., 1991]. In later studies, it has been found that characteristic periventricular T2 white matter signal abnormalities on conventional MRI, restricted white matter diffusion in diffusion-weighted imaging, and electrophysiologic testing abnormalities referable to the CNS are observed in adults who are on unrestricted phenylalanine intake or poorly compliant with dietary therapy [Phillips et al., 2001]. There is evidence that MRI changes in cases of phenylketonuria are at least partially reversible if patients return to a low-phenylalanine diet [Cleary et al., 1995]. Accordingly, it is reasonable to continue therapy into adulthood, and most centers recommend lifelong treatment. Reassessment of adult phenylalanine tolerance may be necessary as body mass changes with age [MacLeod et al., 2009].

A variety of medical food products is available as special formulas for the treatment of phenylketonuria. These special formulas are low in phenylalanine or do not contain any phenylalanine, and they typically contain supplemental tyrosine and a balanced mixture of the additional amino acids, carbohydrates, essential fatty acids, vitamins, and minerals, including zinc, selenium, and molybdenum. The metabolic medical foods provide a variable amount of calories (up to 70 percent of daily requirement) in the form of starch (e.g., dextrose, cornstarch, dextromaltose, Polycose) and fat (i.e., corn or other oils), and they constitute a major source of nutrition for the lifetime of the patient. The special formula or medical food, containing no phenylalanine, is continued even after solid food is introduced. The special formula or medical food is ingested together with regular food during the same meal, providing the phenylalanine in food plus the amino acids, vitamins, and nutrients in the special formula in a complementary, beneficial manner. The medical and regular foods therefore should be given in a calculated proportion together in intervals throughout the day. Overall, the targeted total amino acid intake for children younger than 2 years is approximately 3 g/kg/day, and it is about 2 g/kg/day for older children [Cockburn and Clark, 1996]. If the medical food is ingested in a single sitting, the supply of amino acids may induce hyperinsulinism and hypoglycemia.

Medical food products continue to be modified to increase palatability and optimize the treatment of phenylketonuria. Amino acid powders and gels with added carbohydrate and with or without fats, vitamins, and minerals are examples of commonly used protein substitutes. Newer protein substitutes include amino acid tablets and capsules, which do not contain carbohydrate, vitamins, or minerals. Special amino acid bars and a protein that is almost phenylalanine-free (glycomacropeptide) are also available [van Spronsen and Enns, 2010]. Phenylketonuria dietary research has focused on making the medical food products more palatable. Although low-phenylalanine flour, pastas, cookies, and nutrition bars are available, the phenylketonuria diet remains very bland, and poor dietary compliance can be a major problem, especially after childhood. Early efforts to make a more palatable amino acid mixture have met with preliminary success, with some patients preferring the new products to traditional medical foods.

It is important to monitor complete blood cell counts and serum vitamin B₁₂ levels periodically, because clinical and subclinical B₁₂ deficiency has been reported in adolescents and adults with classic phenylketonuria, even in those poorly compliant with the restricted diet [Hanley et al., 1996].

In contrast to the strict dietary control required in the treatment of classic phenylketonuria, patients with nonphenylketonuria hyperphenylalaninemia (i.e., untreated blood phenylalanine levels of 360–600 μM) are not necessarily placed on the special diet as long as their phenylalanine levels are in treatment range; many of these patients are able to maintain acceptable blood phenylalanine levels with protein restriction alone. These patients have normal intelligence, and they do not have the psychologic findings or head MRI changes that have been documented in classic phenylketonuria [Weglage et al., 1996]. Dietary therapy may be recommended in some instances for pregnant women with nonphenylketonuria hyperphenylalaninemia to minimize the risk of maternal phenylketonuria syndrome.

Additional and Novel Therapies

A complementary therapeutic approach has received U.S. Food and Drug Administration approval: administration of dietary supplementation of large, neutral amino acids. Large, neutral amino acids compete with phenylalanine for transport across the blood–brain barrier by the L-type amino acid carrier and consequently decrease the level of phenylalanine in the CNS [Matalon et al., 2003; van Spronsen and Enns, 2010]. In a study of six adult subjects with classic phenylketonuria, large, neutral amino acid supplementation resulted in increased blood concentrations of tyrosine and tryptophan (the respective precursors for dopamine and serotonin) and decreased brain phenylalanine concentration, as measured by ¹H-MR spectroscopy, toward the carrier range. All patients reported improvements in well-being and energy levels [Koch et al., 2003].

Deficiencies of carnitine and long-chain polyunsaturated fatty acids (i.e., arachidonic and docosahexaenoic acids) may contribute to CNS toxicity in uncontrolled phenylketonuria. Dietary supplementation of these fatty acids and of carnitine may benefit phenylketonuria patients who have low plasma levels of these essential metabolites [Infante and Huszagh, 2001]. Supplementation with omega-3, long-chain, polyunsaturated fatty acids resulted in improvement in visual-evoked potential latencies in 36 children with early-treated phenylketonuria [Beblo et al., 2001].

A novel therapeutic approach uses the nonmammalian enzyme phenylalanine lyase [van Spronsen and Enns, 2010]. This enzyme converts phenylalanine to trans-cinnamic acid, a harmless compound, and it has been found to reduce hyperphenylalaninemia in phenylketonuria rat and mouse models [Bourget and Chang, 1986; Sarkissian et al., 1999]. Enteral phenylalanine lyase therapy has the theoretic potential to increase dietary phenylalanine tolerance substantially, but significant practical hurdles need to be overcome; phenylalanine lyase is destroyed by gastric acidic pH and intestinal proteolysis. Alternative approaches being considered include the use of polyethylene glycol derivatization to produce protected forms of PAH for potential enzyme replacement therapy [Gamez et al., 2004; Kang et al., 2010].

Oral administration of tetrahydrobiopterin, the naturally occurring co-factor for the PAH reaction, reduces serum phenylalanine concentrations, especially in patients with mild hyperphenylalaninemia [Muntau et al., 2002]. However, response to tetrahydrobiopterin has also been documented in patients with classic or variant phenylketonuria [Matalon et al., 2004; van Spronsen and Enns, 2010]. These patients have mutations in the PAH gene, not in one of the genes encoding enzymes involved in tetrahydrobiopterin biosynthesis (see the section on biopterin disorders below). It has been suggested that the PAH mutations in such patients affect the structure of domains that are involved in the binding of tetrahydrobiopterin to the PAH enzyme. Tetrahydrobiopterin also may act as a chemical chaperone, preventing the PAH enzyme from misfolding or protecting PAH from inactivation [Pey et al., 2004]. If these observations and hypotheses are borne out, it may be possible to define by mutation analysis a subset of patients who would predictably benefit from co-factor supplementation. Tetrahydrobiopterin may prove useful in the treatment of maternal phenylketonuria [Trefz and Blau, 2003]. These, and other, novel therapies are under close investigation, especially given recent findings of suboptimal outcomes in phenylketonuria patients who have been continuously treated from the neonatal period [Enns et al., 2010].

Clinical Manifestations

Most patients with GTPCH, DHPR, and PTPS deficiencies have severe forms of disease, although mild forms of DHPR and PTPS deficiencies exist, and some forms of PTPS deficiency may be transient [Blau et al., 2001]. PCD deficiency is usually not associated with significant abnormalities other than transient tone abnormalities. Untreated patients with typical severe disorders of tetrahydrobiopterin synthesis or recycling usually develop neurologic manifestations by 4 months, although symptoms can appear in the neonatal period. Clinical manifestations include microcephaly, progressive neurologic deterioration, movement disorders, delayed motor development, seizures, tone disturbances, oculogyric spasms, swallowing difficulties, hypersalivation, and hyperthermia [Blau et al., 1996, 2001; Dhondt, 1993]. Diurnal fluctuation of dystonia may occur. The clinical course is similar in severe untreated tetrahydrobiopterin deficiency, regardless of the enzymatic defect. Head MRI findings have only rarely been reported [Pietz et al., 1996]. However, in DHPR deficiency, brain abnormalities such as diffuse demyelination, atrophy, spongy vacuolation of brainstem long tracts, and basal ganglia calcification may occur. Abnormal vascular proliferation in the cortex, white matter, and basal ganglia may also be detected [Gudinchet et al., 1992; Schmidt et al., 1988].

Diagnosis

Patients with tetrahydrobiopterin defects are often identified by mandatory newborn screening programs because of hyperphenylalaninemia. All children with persistent hyperphenylalaninemia must be screened for aberrations in the levels of pterin metabolites (i.e., neopterin and biopterin). Patients with GTPCH deficiency have decreased urinary excretion of neopterin and biopterin. In PTPS deficiency, neopterin is increased and biopterin decreased, resulting in a greatly elevated neopterin to biopterin ratio (normally, the ratio is about 1:1). The neopterin to biopterin ratio in PCD deficiency is also increased but not to the same extent as in PTPS deficiency. In PCD deficiency, the characteristic feature is the presence of primapterin (7-biopterin) in the urine [Ayling et al., 2000]. In DHPR deficiency, the percentage of biopterin is elevated (>80 percent in most cases), but urine screening may miss it in some patients [Dhondt, 1984]. The measurement of DHPR activity in neonatal dried blood spots using a spectrophotometric assay is an effective method for diagnosis of DHPR deficiency. Urine pterin analysis and DHPR activity screening should be performed early in the management of a new patient with persistent hyperphenylalaninemia, or these disorders may be missed. Mutation analysis has yet to identify clear genotype-phenotype correlations [Blau et al., 1996].

Treatment

The goals of therapy are to decrease the level of phenylalanine to an acceptable range (120–360 μM) and correct the neurotransmitter deficiencies with exogenous supplementation. The diet is similar to that used to treat classic phenylketonuria, but patients tend to have a higher phenylalanine tolerance (300–700 mg/day) [Blau et al., 2001]. Tetrahydrobiopterin supplementation (2–20 mg/kg/day) is also used to help control blood phenylalanine levels. Lower tetrahydrobiopterin doses (2–5 mg/kg/day) may be effective in GTPCH and PTPS deficiencies, whereas higher doses (up to 20 mg/kg/day) may be required in DHPR deficiency. l-DOPA and 5-hydroxytryptophan are administered in a dose of 1–10 mg/kg/day. Carbidopa, an inhibitor of peripheral aromatic amino acid decarboxylase, decreases the conversion rates of l-DOPA to dopamine and 5-hydroxytryptophan to serotonin, allowing for the use of lower doses of these compounds; these therapeutic adjuncts may be especially helpful in severe forms of tetrahydrobiopterin deficiency. The optimal dose of each medication must be determined for each patient. Mild forms of disease may respond to tetrahydrobiopterin supplementation alone. Measuring levels of cerebrospinal fluid neurotransmitter metabolites (i.e., homovanillic acid and 5-hydroxyindolacetic acid) is useful in monitoring the efficacy of treatment [Blau et al., 2001; Shintaku, 2002].

Side effects of therapy include choreoathetosis, dystonia, and on-off phenomena, which are also features of the underlying disorders [Tanaka et al., 1989]. Tachycardia, diarrhea, and anorexia are associated with 5-hydroxytryptophan administration [Dhondt, 1993]. l-Deprenyl, a monoamine oxidase inhibitor, has been useful in decreasing catabolism of l-DOPA and 5-hydroxytryptophan, allowing lower dosing [Schuler et al., 1995; Spada et al., 1995, 1996]. A low concentration of cerebrospinal fluid folate is typical in DHPR deficiency, and it is treated by folinic acid supplementation (10–20 mg/day) [Shintaku, 2002]. Trimethoprim-sulfamethoxazole and methotrexate are DHPR inhibitors, and they may cause serious side effects in patients with tetrahydrobiopterin deficiency [Millot et al., 1995; Woody and Brewster, 1990]. Neurologic function may improve with therapy, but the overall prognosis for these disorders is largely unknown.

Pathophysiology

Hepatorenal tyrosinemia is caused by a deficiency of fumarylacetoacetate hydrolase, a distal enzymatic step in the processing of the amino acid tyrosine (Figure 32-2). Some investigations suggest that metabolites of tyrosine accumulating proximal to the blocked reaction step are toxic to liver and kidney, acting as alkylating agents or by disruption of sulfhydryl metabolism [Russo et al., 2001]. One of the accumulating metabolites, succinylacetone, has been implicated in the peripheral neuropathy of tyrosinemia [Sassa and Kappas, 1983; Sassa et al., 1983].

Fig. 32-2 The tyrosine metabolic pathway.

The pathway involves several enzymes: tyrosine aminotransferase (1); p-OH-phenylpyruvic acid dioxygenase (2); homogentisic acid oxidase (3); maleylacetoacetic acid isomerase (4); and fumarylacetoacetic acid hydrolase (5).

(From Wilcox WR, Cederbaum SD. Amino acid metabolism. In: Rimoin D, Connor J, Pyeritz R, Korf B, eds. Principles and practice of medical genetics, 4th edn. Philadelphia: Churchill Livingstone, 2002:2411.)

Clinical Manifestations

In states and countries that include tyrosinemia in newborn screening panels, infants are detected within the first weeks of life. Onset of disease manifestations may be sudden and may occur in the first month of life; a more gradual clinical course may also be seen. Children often manifest failure to thrive, and vomiting, diarrhea, and hepatosplenomegaly are common.

Traditional classifications of acute and chronic disease have been replaced by assessment of disease status in target organs (e.g., peripheral nerve, liver, and kidney). Liver disease can include acute decompensations and cirrhosis. There is a high incidence of progression to hepatocellular carcinoma, likely caused by accumulation of mutagenic metabolites. Renal dysfunction ranges from mild tubular dysfunction to frank renal failure. Vitamin D-resistant renal rickets is a common feature.

Neurologic involvement can include paresthesias, opisthotonic-like posture, bruxism and tongue biting, and in some cases, motor paralysis leading to respiratory failure and death [Mitchell et al., 1990]. Neurologic crises occur in up to 42 percent of individuals with tyrosinemia [Kvittingen, 1991]. These crises are biphasic, with an active period of pain, autonomic dysfunction, and sometimes paralysis lasting 1–7 days, followed by a period of recuperation. Succinylacetone blocks the heme biosynthetic pathway, and the neurologic crises – a major source of morbidity – therefore have a physiologic basis similar to those in porphyria [Russo et al., 2001].

Laboratory Tests

Plasma amino acid analysis will reveal moderate elevations of tyrosine, often together with methionine and phenylalanine, reflecting generalized liver damage. The presence of succinylacetone in the urine is pathognomonic for tyrosinemia type I. Other findings include diminished serum phosphorus and elevated urinary excretion of phosphate, due to reduced tubular phosphorus reabsorption. Glucosuria and hyperaminoaciduria result from renal tubular impairment. Liver dysfunction leads to hyperbilirubinemia and hypoproteinemia. Patients usually have leukopenia, anemia, and thrombocytopenia.

Management

2-[2-Nitro-4-trifluoromethylbenzoyl]-1,3-cyclohexanedione (NTBC) is an inhibitor of 4-hydroxyphenylpyruvate dioxygenase (4-HPD), a proximal step in tyrosine catabolism. Inhibition thereby prevents the synthesis of succinylacetone and related metabolites, which accumulate because of the enzymatic block at fumarylacetoacetate hydrolase, a distal step in the pathway. Treatment with this compound is effective within hours and dramatically reduces the risk of neurologic and hepatic crises [Holme and Lindstedt, 2000; McKiernan, 2006]. Dietary restriction of phenylalanine and tyrosine is used in combination with NTBC. Liver transplantation is curative for hepatic and nervous system disease, and it is used in those who are refractory to nonsurgical treatment.

Acute management of neurologic crises includes analgesia, glucose (which inhibits ALA synthetase), and symptomatic treatment of hypertension. Repletion of sodium, potassium, and phosphate is necessary. The use of barbiturates and other medications that aggravate porphyria should be avoided before stabilization on NTBC [Kang and Gerald, 1970].

Clinical Manifestations

Classic maple syrup urine disease

In the classic form, the clinical phenotype is one of severe neonatal encephalopathy, unless presymptomatic therapy is initiated because of abnormal newborn screening, prenatal diagnosis, or positive family history. Untreated neonates typically develop symptoms by the end of the first week of life. Feeding difficulties, alternating hypertonia and hypotonia, opisthotonic posturing, abnormal movements (“fencing” or “bicycling”), and seizures commonly occur. The characteristic urine smell develops on day 5–7 of life [Strauss and Morton, 2003a]. Unless an underlying inborn error of metabolism is suspected, affected children may be misdiagnosed as having sepsis and progress to coma and death. Ketosis is often found, and hypoglycemia may occur, but severe metabolic acidosis tends not to occur. Plasma amino acid analysis reveals elevated levels of branched-chain amino acids and the diagnostic presence of alloisoleucine in plasma [Schadewaldt et al., 1999]. Urine organic acid analysis demonstrates excretion of branched-chain α-ketoacids. Hyponatremia and cerebral edema are frequent sequelae during acute metabolic decompensation [Morton et al., 2002]. Other complications include pseudotumor cerebri, pancreatitis, and eye abnormalities [Burke et al., 1991; Kahler et al., 1994]. Ocular findings in untreated or late-diagnosed patients include optic atrophy, gray optic papilla, nystagmus, ophthalmoplegia, strabismus, and cortical blindness [Burke et al., 1991]. Children who survive the initial metabolic crisis typically have significant neurodevelopmental delays and spasticity [Chuang and Shih, 2001]. Although motor, visual, and learning deficits may occur, rapid identification of affected infants and careful institution of appropriate therapy can result in normal development [Kaplan et al., 1991; Morton et al., 2002].

Neuroimaging studies (Figure 32-3) are typically abnormal in patients with untreated classic maple syrup urine disease who are in crisis. Computed tomographic (CT) scans appear normal in the first few days of life, but they reveal progression to marked generalized cerebral edema if the patient remains untreated [Brismar et al., 1990]. An unusual pattern of edema may occur, characterized by involvement of the cerebellar deep white matter, posterior brainstem, cerebral peduncles, posterior limb of the internal capsule, and posterior aspect of the centrum semiovale. Edema tends to subside in the second month of life [Brismar et al., 1990]. Patients with classic maple syrup urine disease in metabolic crisis with associated hyponatremia demonstrate a prominently increased T2 signal on brain MRI in the brainstem reticular formation, dentate nucleus, red nucleus, globus pallidus, hypothalamus, septal nuclei, and amygdala [Morton et al., 2002]. One report observed that brain MRI abnormalities were absent or only slight in sick patients with maple syrup urine disease in the absence of hyponatremia [Morton et al., 2002]. Cranial ultrasonography of neonates in acute metabolic crisis reveals symmetrically increased echogenicity of the periventricular white matter, basal ganglia, and thalami [Fariello et al., 1996]. Chronic changes, including hypomyelination of the cerebral hemispheres, cerebellum, and basal ganglia and cerebral atrophy, may supervene in poorly controlled patients. CT- and MRI-defined abnormalities and the clinical phenotype may improve after implementation of appropriate dietary therapy [Taccone et al., 1992]. Diffusion-weighted imaging and spectroscopy have also documented abnormalities during the acute phase of disease [Cavalleri et al., 2002]. Markedly restricted proton diffusion, suggestive of cytotoxic or intramyelinic sheath edema, was demonstrated in the brainstem, basal ganglia, thalami, cerebellar and periventricular white matter, and cerebral cortex in six patients with maple syrup urine disease. MR spectroscopy demonstrated abnormal elevations of branched-chain amino acids, branched-chain α-ketoacids, and lactate in the four patients. All of these changes were reversed after the institution of appropriate nutritional and antibiotic therapy to treat intercurrent illness [Jan et al., 2003].

Fig. 32-3 Maple syrup urine disease.

A, Axial view, T2-weighted image shows edema in the internal capsules, lateral thalami, and globus pallidi. B, Axial view, calculated apparent diffusion coefficient image at the same level shows hypointensity, indicated by reduced water diffusion, in the affected areas. C, Proton MR spectroscopy (echo time of 26 msec) shows a large peak at 0.9 ppm, believed to represent resonances of methyl protons from branched-chain amino acids and branched-chain α-ketoacids that accumulate as a result of defective oxidative decarboxylation of leucine, isoleucine, and valine.

(Courtesy of Dr. A James Barkovich, University of California, San Francisco, CA.)

A characteristic comblike EEG pattern may be demonstrated for some patients with classic maple syrup urine disease between the second and third weeks of life [Tharp, 1992]. This unusual rhythm pattern resolves with the institution of dietary therapy [Tharp, 1992].

Laboratory Tests

Maple syrup urine disease can be detected easily and accurately by tandem mass spectrometry analysis of the newborn blood spot [Chace et al., 1995]. Tandem mass spectrometry used in newborn screening is effective in identifying maple syrup urine disease, and is performed in all states in the U.S. (see http://genes-r-us.uthscsa.edu/nbsdisorders.htm) and many countries worldwide. Urine screening tests for the presence of α-ketoacids (i.e., ferric chloride and 2,4-dinitrophenylhydrazine [DNPH]) may be positive, but are nonspecific and insensitive. Plasma amino acid analysis demonstrates elevations of leucine, isoleucine, and valine (5- to 10-fold greater than normal) [Strauss and Morton, 2003a], as well as the pathognomonic finding of elevated alloisoleucine [Schadewaldt et al., 1999]. Levels of branched-chain amino acids are greatly elevated in urine and cerebrospinal fluid [Chuang and Shih, 2001]. The branched-chain α-ketoacids 2-oxoisocaproic acid, 2-oxo-3-methylvaleric acid, and 2-oxoisovaleric acid, derived from the branched-chain amino acids leucine, isoleucine, and valine, respectively, are found to be elevated on urine organic acid analysis during metabolic crises. Branched-chain amino acids levels and excretion of branched-chain α-ketoacids may be normal between episodes of decompensation in the intermittent form of disease.

The branched-chain α-ketoacid dehydrogenase complex consists of three catalytic components – a thiamine pyrophosphate-dependent carboxylase (E1) with an α₂β₂ structure, a transacylase (E2), and a dehydrogenase (E3) – as well as two regulatory enzymes (a kinase and a phosphatase) [Chuang and Shih, 2001]. Deficient activity of this complex leads to the accumulation of leucine, isoleucine, and valine and their corresponding α-ketoacids. The decarboxylation activity can be measured in leukocytes, lymphoblasts, or fibroblasts, and it is loosely related to the clinical phenotype: 0–2 percent of normal activity in classic maple syrup urine disease, 3–30 percent activity in intermediate, 5–20 percent in intermittent, 2–40 percent in thiamine-responsive, and 0–25 percent in E3 deficiency [Chuang and Shih, 2001; Scriver et al., 1971]. Because significant overlap exists between measured enzyme activity and clinical phenotype, enzymatic activity cannot be used to predict the clinical course with certainty.

Genetics

Maple syrup urine disease is a pan-ethnic, autosomal-recessive condition that can be caused by mutations in any of the components of the mitochondrial branched-chain α-ketoacid dehydrogenase complex. In a study of 63 individuals, E1β subunit mutations were most common (38 percent), followed by E1α (33 percent), and E2 (19 percent) mutations [Nellis and Danner, 2001]. Branched-chain α-ketoacid dehydrogenase phosphatase or kinase mutations are also predicted to cause maple syrup urine disease, but such abnormalities have not yet been detected. The overall incidence is approximately 1 case per 150,000 people in the general population, but maple syrup urine disease is more common in Old Order Mennonites in southeastern Pennsylvania (1 in 176 births) [Danner and Doering, 1998]. A novel founder mutation in the E1β subunit has been reported in the Ashkenazi Jewish population [Edelmann et al., 2001]. In general, increased residual branched-chain α-ketoacid dehydrogenase activity should convey some advantage, but there is a wide overlap between measured enzymatic activities and clinical outcome. Given the complexity of the molecular genetics, the potential for modifier gene and environmental interactions, and the multiple clinical phenotypes associated with maple syrup urine disease, a lack of definitive genotype-phenotype relationships is not surprising.

Treatment

Chronic care of the child with maple syrup urine disease includes regular visits to an integrated metabolic clinic for medical and nutritional assessment. Adequate calories (100–120 kcal/kg/day) and protein (2–3 g/kg/day) are needed for growth. Chronic valine or isoleucine deficiency may cause an exfoliative dermatitis, and supplementation of these amino acids is often needed [Koch et al., 1993]. Thiamine supplementation is administered to patients with thiamine-responsive forms of maple syrup urine disease. Because patients on restricted diets are at risk for micronutrient and essential fatty acid deficiencies, patients should be periodically monitored for such deficits and supplementation given as needed.

Because significant metabolic intoxication may occur rapidly, even in patients with apparently well-controlled disease, it is crucial to have carefully considered home and hospital emergency protocols in place for each child [Morton et al., 2002; Strauss and Morton, 2003a]. Acute metabolic decompensation (e.g., fasting or illness severe enough to cause catabolism) is a medical emergency that requires prompt intervention. Initial intervention is aimed at correcting dehydration, starting high-dose intravenous thiamine, and providing adequate calories (approximately 120–140 kcal/kg/day) to prevent further protein catabolism and higher rise in plasma leucine levels. To this end, high-dextrose intravenous fluids (to provide approximately 10 mg/kg/min) and intralipid are often administered. Branched-chain amino acid-free parenteral nutrition or enteral formula, delivered by continuous nasogastric drip, can also be used [Nyhan et al., 1998; Parini et al., 1993]. The rate of decrease of leucine is slowed in the face of valine and isoleucine levels inadequate to stimulate protein synthesis. Acute valine and isoleucine deficiency can be avoided by careful supplementation of these amino acids [Parini et al., 1993]. Leucine is reintroduced to the diet after therapeutic levels are achieved [Morton et al., 2002].

In a study of 36 maple syrup urine disease patients, plasma leucine levels fell to less than 400 μM 2–4 days after the initiation of therapy with enteral and parenteral nutrition. Initial leucine levels ranged from 233 to 778 μM in a group diagnosed on the first day of life (n = 18) and 1489 to 3359 μM in a group diagnosed between days 3 and 16 (n = 18). Over an 11-year period, neurologic examinations, gross motor development, and speech were normal in 34 of 36 children [Morton et al., 2002]. Enteral nutrition was also found to be beneficial when instituted within the first 20 days of life. Four patients receiving nasogastric drip feeding as the only treatment of neonatal classic maple syrup urine disease had normal development when 3–5 years old [Parini et al., 1993]. Hemodialysis and continuous venovenous extracorporeal removal therapies result in more rapid fall in plasma levels of branched-chain amino acids, but these modalities typically have been described in single case reports or small series with relatively short follow-up, and it is difficult to ascertain the long-term outcome of such intervention [Gouyon et al., 1996; Puliyanda et al., 2002]. Nevertheless, normal development has been reported for 8 of 12 children after continuous venovenous extracorporeal removal therapy [Jouvet et al., 2001]. Branched-chain amino acids levels often rebound after initial dialysis in cases of severe metabolic imbalance characterized by extremely high leucine levels, and dialysis may need to be repeated in such cases. Peritoneal dialysis is no longer routinely used; there is a tendency for leucine levels to plateau between 1000 and 1500 μM after 24 hours, limiting the utility of this therapeutic modality [Gortner et al., 1989]. Levels of branched-chain amino acids and branched-chain α-ketoacids tend to plateau with exchange transfusion therapy [Nyhan et al., 1998; Wendel et al., 1982].

Although enteral and intravenous therapy may be sufficient to manage many patients with maple syrup urine disease in acute crisis, various dialysis methods are commonly used and should be considered, especially when clearance of branched-chain amino acids by nutritional support is not effective or when other considerations, such as life-threatening cerebral edema, renal imbalance, or cardiovascular abnormalities, exist [Jouvet et al., 2001; Nyhan et al., 1998]. Liver transplantation has been performed rarely for maple syrup urine disease. Three patients who underwent successful transplantation were able to resume normal diets and were no longer at risk for metabolic decompensation [Wendel et al., 1999]. More recently, domino hepatic transplantion for maple syrup urine disease was successfully performed [Barshop and Khanna, 2005].

Because hyponatremia and subsequent brain edema are serious and relatively common complications, it is important to monitor serum sodium and serum and urine osmolalities closely and to replace urinary losses with saline [Morton et al., 2002]. Critical brain swelling and abnormal brainstem function may develop with only a moderate reduction in serum sodium level (by only 8–10 mEq/L) [Morton et al., 2002]. Low-dose diuretics may also be used to prevent water retention [Strauss and Morton, 2003a]. Mannitol is reserved for life-threatening episodes of increased intracranial pressure [Morton et al., 2002].