Classic Phenylketonuria

Severe hyperphenylalaninemia (plasma phenylalanine levels >20 mg/dL), if untreated, invariably results in the development of signs and symptoms of classic PKU, except in rare unpredictable cases.

Milder Forms of Hyperphenylalaninemia, Non-PKU Hyperphenylalaninemias

In any screening program for PKU, a group of infants are identified in whom initial plasma concentrations of phenylalanine are above normal (2 mg/dL, 120 µmole/L) but <20 mg/dL (1,200 µmole/L). These infants do not excrete phenylketones. The term hyperphenylalaninemia implies lower plasma concentration of phenylalanine, but these patients may still require dietary therapy depending on their untreated plasma phenylalanine level. Attempts have been made to classify these patients in different subgroups depending on the degree of hyperphenylalaninemia, but such a practice has little clinical or therapeutic advantage. The possibility of deficiency of BH₄ should be investigated in all infants with the milder forms of hyperphenylalaninemia (see later).

Hyperphenylalaninemia due to Deficiency of the Cofactor BH₄

In 1-3% of infants with hyperphenylalaninemia, the defect resides in 1 of the enzymes necessary for production or recycling of the cofactor BH₄ (Fig. 79-2). If these infants are misdiagnosed as having PKU, they may deteriorate neurologically despite adequate control of plasma phenylalanine. BH₄ is synthesized from guanosine triphosphate (GTP) through several enzymatic reactions (see Fig. 79-1). In addition to acting as a cofactor for PAH, BH₄ is also a cofactor for tyrosine hydroxylase and tryptophan hydroxylase, which are involved in the biosynthesis of dopamine (see Fig. 79-2) and serotonin (see Fig 79-5), respectively. Therefore, patients with hyperphenylalaninemia due to BH₄ deficiency also manifest neurologic findings related to deficiencies of the neurotransmitters dopamine and serotonin. Four enzyme deficiencies leading to defective BH₄ formation cause hyperphenylalaninemia and deficiencies of dopamine and serotonin. These include autosomal recessive GTP cyclohydrolase deficiency, pterin-carbinolamine dehydratase (PCD) deficiency, dihydropteridine reductase (DHPR) deficiency, and 6-pyruvoyltetrahydropterin synthase (PTPS or 6-PTS) deficiency. More than half of the reported patients have had a deficiency of 6-pyruvoyltetrahydropterin synthase. Autosomal dominant form of GTP deficiency and sepiapterin reductase deficiency result in deficiencies of neurotransmitters without hyperphenylalaninemia (Chapter 79.11 and Fig. 79-1).

Figure 79-2 Other pathways involving tyrosine metabolism. PKU* indicates hyperphenylalanemia due to tetrahydrobiopterin (BH₄) deficiency (see Fig. 79-1). HVA, homovanillic acid; VMA, vanillymandelic acid. Enzymes: (1) tyrosine hydroxylase (TH), (2) aromatic L-amino acid decarboxylase (AADC), (3) dopamine hydroxylase, (4) phenylethanolamine-N-methyltransferase (PNMT), (5) catechol O-methyltransferase (COMT), (6) Monoamine oxidase (MAO).

Tyrosinemia Type I (Tyrosinosis, Hereditary Tyrosinemia, Hepatorenal Tyrosinemia)

This severe disease of the liver, kidney, and peripheral nerve, is caused by a deficiency of the enzyme FAH. Organ damage is believed to result from accumulation of metabolites of tyrosine degradation, especially succinylacetone.

Tyrosinemia Type II (Richner-Hanhart Syndrome, Oculocutaneous Tyrosinemia)

This rare autosomal recessive disorder is caused by deficiency of tyrosine aminotransferase, resulting in palmar and plantar hyperkeratosis, herpetiform corneal ulcers, and mental retardation (see Fig. 79-1). Ocular manifestations include excessive tearing, redness, pain, and photophobia and often occur before skin lesions. Corneal lesions are presumed to be due to tyrosine deposition. In contrast to herpetic ulcers, corneal lesions in tyrosinemia type II stain poorly with fluorescein and often are bilateral. Skin lesions, which may develop later in life, include painful, nonpruritic hyperkeratotic plaques on the soles, palms and fingertips. Mental retardation, which occurs in <50% of patients, is usually mild to moderate.

The principal laboratory finding in untreated patients is marked hypertyrosinemia (20-50 mg/dL; 1,100-2,750 µmole/L). Surprisingly, 4-hydroxyphenylpyruvic acid and its metabolites are also elevated in urine despite being downstream from the metabolic block (see Fig. 79-1). This is hypothesized to occur via the action of other transaminases in the presence of high tyrosine concentrations, producing 4HPP in cellular compartments like the mitochondrion in which it is not further degraded. In contrast to tyrosinemia type I, liver and kidney function are normal, as are serum concentrations of other amino acids and succinylacetone. Tyrosinemia type II is due to TAT gene mutations, causing deficiency of cytosolic tyrosine aminotransferase activity in liver.

Diagnosis of type II tyrosinemia is established by assay of plasma tyrosine concentration in patients with suggestive findings. Molecular diagnosis is possible. Assay of liver tyrosine aminotransferase activity is rarely indicated.

Treatment with a diet low in tyrosine and phenylalanine improves the biochemical abnormalities and can normalize the skin and eye. The claim that mental retardation may be prevented by early diet therapy is reasonable and is supported by some case reports. The TAT gene maps to chromosome 16q and several disease-causing mutations have been identified. About half of reported cases are of Italian descent.

Tyrosinemia Type III (Primary Deficiency of 4-HPPD)

Only a few cases have been reported; most were detected by amino acid chromatography performed for various neurologic findings. Age at presentation has been from 1 to 17 mo. Developmental delay, seizures, intermittent ataxia, and self-destructive behavior are reported. A causal link to 4HPP deficiency is not formally established. Liver or renal abnormalities are absent. Asymptomatic infants with 4-HPPD deficiency have been identified by neonatal screening for hypertyrosinemia.

The diagnosis is suspected in children with sustained moderate increases in plasma levels of tyrosine (typically 350-700 µmole/L on a normal diet) and the presence in urine of 4-hydroxyphenylpyruvic acid and its metabolites 4-hydroxyphenyllactic and 4-hydroxyphenylacetic acids. Diagnosis may be refined by demonstrating the presence of mutations in the gene for 4-HPPD on chromosome 12q, or rarely, by demonstrating a low activity of 4-HPPD enzyme in liver biopsy.

Given the possible association with neurologic abnormalities, dietary reduction of plasma tyrosine levels is prudent. It is also logical to attempt a trial of vitamin C, the cofactor for 4-HPPD. The condition is inherited as an autosomal recessive trait.

Transient Tyrosinemia of the Newborn

In a small number of newborn infants, plasma tyrosine may be as high as 60 mg/dL (3,300 µmole/L) during the 1st 2 weeks of life. Most affected infants are premature and are receiving high-protein diets. Transient tyrosinemia is felt to result from delayed maturation of 4-HPPD (see Fig. 79-1). Lethargy, poor feeding, and decreased motor activity are noted in some patients. Most are asymptomatic and are identified by a high blood phenylalanine or tyrosine level on screening. Laboratory findings include marked elevation of plasma tyrosine with a moderate increase in plasma phenylalanine. The finding of hypertyrosinemia differentiates this condition from PKU. 4-Hydroxyphenylpyruvic acid and its metabolites are present in the urine. Hypertyrosinemia usually resolves spontaneously in the 1st mo of life. It can be corrected promptly by reducing dietary protein to below 2 g/kg/24 hr and by administering vitamin C (200-400 mg/24 hr). Mild intellectual deficits have been reported in some infants that had this condition, but the causal relationship to hypertyrosinemia is not conclusively established.

Hawkinsinuria

This rare autosomal dominant condition is caused by a mutant 4-HPPD enzyme that catalyzes a partial reaction, releasing an intermediate compound used for diagnosis (see Fig. 79-1). This intermediate is either reduced to form 4-hydroxycyclohexylacetic acid (4-HCAA) or reacts with glutathione to form the unusual organic acid hawkinsin (2-L-cysteine-S-yl-1-4-dihydroxycyclohex-5-en-1-yl-acetic acid); secondary glutathione deficiency may occur.

Individuals with this disorder are symptomatic only during infancy. The symptoms usually appear after weaning from breast-feeding with the introduction of a high-protein diet. Severe metabolic acidosis, ketosis, failure to thrive, mild hepatomegaly, and an unusual odor (like that of a swimming pool) are described. Mental development is usually normal.

Affected children and adults excrete the organic acids 4-HCAA, 4-hydroxyphenylpyruvic acid and its metabolites (4-hydroxyphenyllactic and 4-hydroxyphenylacetic acids), 5-oxoproline (owing to secondary glutathione deficiency), and hawkinsin in their urine. Plasma tyrosine level is usually normal.

Treatment consists of a low-protein diet during infancy. Breast feeding is encouraged. A trial with large doses of vitamin C (up to 1,000 mg/24 hr) is also recommended. The same mutation, a substitution of threonine for the normal alanine codon at position 33 of the 4-HPPD gene, has been identified in unrelated patients with hawkinsinuria.

Alcaptonuria

This rare (with an incidence of ≈1/250,000) autosomal recessive disorder is due to a deficiency of homogentisic acid oxidase, which causes large amounts of homogentisic acid to accumulate in the body and then to be excreted in the urine (see Fig. 79-1).

Clinical manifestations of alcaptonuria consist of ochronosis and arthritis in adulthood. The only sign in children is a blackening of the urine on standing, caused by oxidation and polymerization of homogentisic acid. A history of grey- or black-stained diapers should suggest the diagnosis. This sign may never be noted, hence, diagnosis is often delayed until adulthood. Ochronosis, which is seen clinically as dark spots on the sclera or ear cartilage, results from the accumulation of the black polymer of homogentisic acid. Arthritis can be disabling with advancing age. It involves the large joints (spine, hip, and knee) and is usually more severe in males. Like rheumatoid arthritis, the arthritis has acute exacerbations, but the radiologic findings are typical of osteoarthritis, with characteristic narrowing of the joint spaces and calcification of the intervertebral discs. High incidences of heart disease (mitral and aortic valvulitis, calcification of the heart valves, and myocardial infarction) have been noted.

The diagnosis is confirmed by finding massive excretion of homogentisic acid on urine organic acid testing. Tyrosine levels are normal. The enzyme is expressed only in the liver and kidneys. The gene for alcaptonuria, HGD, maps to chromosome 3q. Several disease-causing mutations have been identified. Alcaptonuria is commonest in the Dominican Republic and Slovakia.

Treatment of the arthritis is symptomatic. Nitisinone efficiently reduces homogentisic acid production in alkaptonuria. If presymptomatic individuals are detected, treatment with nitisinone, combined with a phenylalanine- and tyrosine-restricted diet, seems reasonable, although no experience is available regarding long-term efficacy.

Tyrosine Hydroxylase Deficiency

See Chapter 79.11.

Albinism

Albinism is due to deficiency of melanin, the main pigment of the skin and eye (Table 79-1). Melanin is synthesized by melanocytes from tyrosine in a membrane-bound intracellular organelle, the melanosome. Melanocytes originate from the embryonic neural crest and migrate to the skin, eyes (choroid and iris), hair follicles, and inner ear. The melanin in the eye is confined to the retinal pigment epithelium, whereas in skin and hair follicles, it is secreted into the epidermis and hair shaft. Albinism can be caused by deficiencies of melanin synthesis, by some hereditary defects of melanosomes, or by disorders of melanocyte migration. Although albinism is a classical example of a biochemical genetic disease, neither the biosynthetic pathway of melanin nor many facets of melanocyte cell biology are completely elucidated (see Fig. 79-2). The end products are 2 pigments: pheomelanin, which is a yellow-red pigment; and eumelanin, a brown-black pigment.

Table 79-1 CLASSIFICATION OF ALBINISM

* This includes Amish, minimal pigment, yellow albinism, and platinum and temperature-sensitive variants.

^† Includes brown OCA.

^‡ Tyrosinase related protein 1.

Clinically, primary albinism can be generalized or localized. Primary generalized albinism can be either ocular or oculocutaneous. Some syndromes feature albinism in association with platelet, immunological, or neurological dysfunction.

In generalized oculocutaneous albinism, hypopigmentation can be either complete or partial. Individuals with complete albinism do not develop detectable skin pigmentation, either generalized (tanning) or localized (pigmented nevi).

The diagnosis of albinism is usually evident, but for some white children whose families are particularly light-skinned, normal variation may be a diagnostic consideration. Such normal fair-skinned children progressively develop pigmentation, the eye manifestations of albinism are absent, and other family members may have had a similar course. The clinical diagnosis of oculocutaneous albinism, as opposed to other types of cutaneous hypopigmentation, requires the presence of characteristic eye findings.

The ocular manifestations of albinism include hypopigmentation, including foveal hypoplasia with reduced visual acuity, refractive errors, nystagmus, alternating strabismus, and a red reflex (diffuse reddish hue of the iris produced during ophthalmoscopic or slit lamp examination of the eye). There is also an abnormality in routing of the optic fibers at the chiasm. Unlike normally pigmented individuals, in patients with albinism the majority of the nerve fibers from the temporal side of the retina cross to the contralateral hemisphere of the brain. This results in lack of biocular (stereoscopic) vision and of depth perception, and in repeated switching of vision from eye to eye, causing alternating strabismus. This abnormality also causes a characteristic pattern of visual evoked potentials. These findings are highly specific for albinism and can be used to formally establish the clinical diagnosis. Regular ophthalmological follow-up is recommended for patients with albinism. For instance, correction of refractive errors can maximize visual function. Normally the alternating strabismus does not result in amblyopia and does not require surgery.

Patients with albinism should be counseled to avoid ultraviolet radiation by wearing protective long-sleeved clothing and by using sunscreens with a sun protection factor (SPF) rating above 30. All forms of oculocutaneous albinism are autosomal recessive traits.

Melanin is also present in the cochlea. Albino individuals may be more susceptible to ototoxic agents such as gentamicin.

Many clinical forms of albinism have been identified. Some of the seemingly distinct clinical forms are caused by different mutations of the same gene. Several genes located on different chromosomes are shown to be involved in melanogenesis (see Table 79-1). Attempts to differentiate types of albinism based on the mode of inheritance, tyrosinase activity, or the extent of hypopigmentation have failed to yield a comprehensive classification. The following classification is based on the distribution of albinism in the body and the type of mutated gene.

Mutation detection is clinically available for most albinism genes (see Table 79-1). Molecular diagnosis is of little use therapeutically in isolated albinism but can be helpful for precise genetic counseling of families.

Homocystinuria (Homocystinemia)

Normally, most homocysteine, an intermediate compound of methionine degradation, is remethylated to methionine. This methionine-sparing reaction is catalyzed by the enzyme methionine synthase, which requires a metabolite of folic acid (5-methyltetrahydrofolate) as a methyl donor and a metabolite of vitamin B₁₂ (methylcobalamin) as a cofactor (see Fig. 79-3). Only 20-30% of total homocysteine (and its dimer homocystine) is in free form in the plasma of normal individuals. The rest is bound to proteins as mixed disulfides. Three major forms of homocystinemia and homocystinuria have been identified.

Homocystinuria Due to Defects in Methylcobalamin Formation

Methylcobalamin is the cofactor for the enzyme methionine synthase, which catalyzes remethylation of homocysteine to methionine. There are at least 5 distinct defects in the intracellular metabolism of cobalamin that may interfere with the formation of methylcobalamin. To better understand the metabolism of cobalamin, see methylmalonic acidemia (Fig. 79-4; Chapter 79.6 and Fig. 79-3). The 5 defects are designated as cblC, cblD (including cblD variant 1), cblE (methionine synthase reductase), cblG (methionine synthase), and cblF. Patients with cblC, cblD (not those with cblD variant 2), and cblF defects have methylmalonic acidemia in addition to homocystinuria because formation of both adenosylcobalamin and methylcobalamin is impaired (Chapter 79.6).

Figure 79-4 Pathways in the metabolism of the branched-chain amino acids, biotin, and vitamin B₁₂ (cobalamin). MMA, methylmalonic acidemia; HCU, homocystinuria; Cbl, cobalamin; OHCbl, hydroxycobalamin; cbl, defect in metabolism of cobalamin; cblDV₁, cblD variant 1; cblDV₂, cblD variant 2; TC, transcobalamin.

Patients with cblE and cblG defects are unable to form methylcobalamin and develop homocystinuria without methylmalonic acidemia (see Fig. 79-4); fewer than 40 patients are known with each of these diseases.

The clinical manifestations are similar in patients with all of these defects. Vomiting, poor feeding, lethargy, hypotonia, and developmental delay may occur in the 1st few months of life. One patient with the cblG defect was not symptomatic (except for mild developmental delay) until she was 21 yr old, however, when she developed difficulty in walking and numbness of the hands. Laboratory findings include megaloblastic anemia, homocystinuria, and hypomethioninemia. The presence of megaloblastic anemia differentiates these defects from homocystinuria due to methylenetetrahydrofolate reductase deficiency (see later). The presence of hypomethioninemia differentiates both of these conditions from cystathionine β-synthase deficiency (see earlier).

Diagnosis is established by complementation studies performed in cultured fibroblasts. Prenatal diagnosis has been accomplished by studies in amniotic cell cultures. All of these conditions are inherited as autosomal recessive traits. The gene for cblE (MTRR) is on chromosome 5p15.3-p15.2 and that for cblG (MTR) is on chromosome 1q43; several disease-causing mutations, including a common missense mutation (P1173L) in the MTR gene, have been described.

Treatment with vitamin B₁₂ in the form of hydroxycobalamin (1-2 mg/24 hr) is used to correct the clinical and biochemical findings. Results vary among both diseases and sibships.

Hypermethioninemia

Secondary hypermethioninemia occurs in liver disease, tyrosinemia type I, and classic homocystinuria. Hypermethioninemia has also been found in premature and some full-term infants receiving high-protein diets, in whom it may represent delayed maturation of the enzyme methionine adenosyltransferase. Lowering the protein intake usually resolves the abnormality. Primary hypermethioninemia caused by the deficiency of hepatic methionine adenosyltransferase (MAT I/III; MAT II, which is present in other tissues, is not affected; see Fig. 79-3) has been reported in approximately 60 patients. The majority of these patients have been diagnosed in the neonatal period through screening for homocystinuria. Affected individuals with residual enzyme activity remain asymptomatic throughout life despite persistent hypermethioninemia. Some complain of unusual odor to their breath (boiled cabbage). A few patients with complete enzyme deficiency have had neurologic abnormalities related to demyelination (mental retardation, dystonia, dyspraxia). Normal pregnancies producing normal offspring have been reported in mothers with methionine adenoslytransferase deficiency. The condition is inherited as an autosomal recessive trait. The gene for hepatic methionine adenosyltransferase is on chromosome 10q22 and several disease-causing mutations have been identified. A novel defect, glycine N-methyltransferase deficiency, also causes isolated hypermethioninemia.

Cystathioninemia (Cystathioninuria)

Secondary cystathioninuria occurs in patients with vitamin B₆ or B₁₂ deficiency, liver disease (particularly damage caused by galactosemia), thyrotoxicosis, hepatoblastoma, neuroblastoma, ganglioblastoma, or defects in remethylation of homocysteine.

Cystathionase deficiency results in massive cystathioninuria and mild to moderate cystathioninemia; cystathionine is not normally detectable in blood. Deficiency of this enzyme is inherited as an autosomal recessive trait and its prevalence is estimated to be about 1/14,000 live births. Affected subjects with a wide variety of clinical manifestations have been reported. Lack of a consistent clinical picture and the presence of cystathioninuria in a number of normal persons suggest that cystathionase deficiency may be of no clinical significance. A majority of reported cases are responsive to oral administration of large doses of vitamin B₆ (≥100 mg/24 hr). When cystathioninuria is discovered in a patient, vitamin B₆ treatment seems indicated, but its beneficial effect has not been established. The gene encoding for cystathionase is located on chromosome 16.

Maple Syrup Urine Disease (MSUD)

Decarboxylation of leucine, isoleucine, and valine is accomplished by a complex enzyme system (branched-chain α-ketoacid dehydrogenase) using thiamine pyrophosphate (vitamin B₁) as a coenzyme. This mitochondrial enzyme consists of 4 subunits: E_1α, E_1β, E₂, and E₃. The E₃ subunit is shared with 2 other dehydrogenases in the body, namely pyruvate dehydrogenase and α-ketoglutarate dehydrogenase. Deficiency of this enzyme system causes MSUD (see Fig. 79-4), named after the sweet odor of maple syrup found in body fluids, especially urine. Based on clinical findings and response to thiamine administration, 5 phenotypes of MSUD have been identified.

Isovaleric Acidemia

This rare condition is due to the deficiency of isovaleryl coenzyme A (CoA) dehydrogenase (see Fig. 79-5).

Clinical manifestations in the acute form include vomiting and severe acidosis in the 1st few days of life. Lethargy, convulsions, and coma may ensue, and death may occur if proper therapy is not initiated. The vomiting may be severe enough to suggest pyloric stenosis. The characteristic odor of “sweaty feet” may be present (see Fig. 79-6). Infants who survive this acute episode will go on to have the chronic intermittent form later on in life. A milder form of the disease (chronic intermittent form) also exists; in this, the 1st clinical manifestation (vomiting, lethargy, acidosis or coma) may not appear until the child is a few months or a few years old. In both forms, acute episodes of metabolic decompensations may occur during a catabolic state such as an infection. Sensitive methods for newborn screening have identified yet a milder and potentially asymptomatic phenotype of the condition; a few older siblings of these affected newborns were found to have identical genotype and biochemical abnormalities without any clinical manifestations.

Laboratory findings during the acute attacks include ketoacidosis, neutropenia, thrombocytopenia, and occasionally pancytopenia. Hypocalcemia, hyperglycemia, and moderate to severe hyperammonemia may be present in some patients. Increases in plasma ammonia may suggest a defect in the urea cycle. In urea cycle defects the infant is not acidotic (see Fig. 79-6).

Diagnosis is established by demonstrating marked elevations of isovaleric acid and its metabolites (isovalerylglycine, 3-hydroxyisovaleric acid) in body fluids, especially urine. The main compound in plasma is isovalerylcarnitine, which can be measured even in a few drops of dried blood on a filter paper. Measuring the enzyme in cultured skin fibroblasts confirms the diagnosis.

Treatment of the acute attack is aimed at hydration, reversal of the catabolic state (by providing adequate calories orally or intravenously), correction of metabolic acidosis (by infusing sodium bicarbonate), and removal of the excess isovaleric acid. Because isovalerylglycine has a high urinary clearance, administration of glycine (250 mg/kg/24 hr) is recommended to enhance formation of isovalerylglycine. L-carnitine (100 mg/kg/24 hr orally) also increases removal of isovaleric acid by forming isovalerylcarnitine, which is excreted in the urine. In patients with significant hyperammonemia (blood ammonia >200 µM), measures that reduce blood ammonia should be employed (Chapter 79.12). Exchange transfusion and peritoneal dialysis may be needed if the just described measures fail to induce significant clinical and biochemical improvement. After recovery from the acute attack, the patient should receive a low-protein diet (1.0-1.5 g/kg/24 hr) and should be given glycine and carnitine supplements. Pancreatitis (acute or recurrent forms) has been reported in survivors. Normal development can be achieved with early and proper treatment.

Prenatal diagnosis may be accomplished by measuring isovalerylglycine in amniotic fluid, by enzyme assay in cultured amniocytes, or by identification of the mutant gene. Successful pregnancy with favorable outcomes both for the mother and the infant has been reported. Mass screening of newborn infants is in use in the USA and other countries. Isovaleric acidemia is inherited as an autosomal recessive trait. The gene has been mapped to chromosome 15q14q15 and many disease-causing mutations have been identified. The prevalence of the condition is estimated from 1/62,500 (in parts of Germany) to 1/250,000 (in the USA).

Multiple Carboxylase Deficiencies (Defects in Utilization of Biotin)

Biotin is a water-soluble vitamin that is a cofactor for all 4 carboxylase enzymes in humans: pyruvate carboxylase, acetyl CoA carboxylase, propionyl CoA carboxylase, and 3-methylcrotonyl CoA carboxylase. The latter 2 are involved in the metabolic pathways of leucine, isoleucine, and valine (see Fig. 79-4).

Dietary biotin is bound to proteins; free biotin is generated in the intestine by the action of digestive enzymes, by intestinal bacteria, and perhaps by biotinidase. The latter enzyme, which is found in serum and most tissues in the body, is also essential for the recycling of biotin in the body by releasing it from the apoenzymes (carboxylases, see Fig. 79-4). Free biotin must form a covalent peptide bond with the apoprotein of the 4 carboxylases to activate them (holocarboxylase). This binding is catalyzed by holocarboxylase synthetase. Deficiencies in this enzyme or in biotinidase result in malfunction of all the carboxylases and in organic acidemia.

Isolated 3-Methylcrotonyl CoA Carboxylase Deficiency

This enzyme is 1 of 4 carboxylase enzymes in the body that require biotin as a cofactor (see Fig. 79-4). An isolated deficiency of this enzyme must be differentiated from disorders of biotin metabolism (multiple carboxylase deficiency), which cause diminished activity of all 4 carboxylases. 3-Methylcrotonyl CoA carboxylase is a heteromeric enzyme consisting of α (biotin containing) and β subunits.

Clinical manifestations are highly variable, ranging from fatal neonatal onset with acidosis, severe hypotonia, and seizures to completely asymptomatic individuals. In the severe form of the condition, the affected infant who has been seemingly normal develops an acute episode of vomiting, hypotonia, lethargy, and convulsions after a minor infection. Death may occur during the acute episode.

Laboratory findings during acute episodes include mild to moderate acidosis, ketosis, severe hypoglycemia, hyperammonemia, and elevated serum levels of liver transaminases. Large amounts of 3-hydroxyisovaleric acid and 3-methylcrotonylglycine are found in the urine. Urinary excretion of 3-methylcrotonic acid is not usually increased in this condition because the accumulated 3-methylcrotonyl CoA is converted to 3-hydroxyisovaleric acid. Severe secondary carnitine deficiency is common. The condition should be differentiated biochemically from multiple carboxylase deficiency (see above) in which lactic acid and metabolites of propionic acid are present in body fluids in addition to 3-hydroxyisovaleric acid. Diagnosis may be confirmed by measurement of the enzyme activity in cultured fibroblasts. Documentation of normal activities of other carboxylases is necessary for definitive diagnosis.

Aggressive treatment of acute episodes with hydration, intravenous infusion of glucose, and alkali is recommended. These patients are unresponsive to biotin therapy. Patients who in earlier reports were found to be biotin responsive were most probably suffering from multiple carboxylase deficiency due to biotinidase deficiency (see above). Long-term treatment includes a diet restricted in leucine in conjunction with the oral administration of L-carnitine (75-100 mg/kg/24 hr) and the prevention of catabolic states. Normal growth and development are expected in these patients.

The condition is inherited as an autosomal recessive trait. The gene for α subunit (MCC1) is located on chromosome 3q25-27 and that for the β subunit (MCC2) is mapped to chromosome 5q12-13. Mutation in either of these genes may result in the deficiency of the enzyme activity. Similar phenotype may be caused by different genotype. Several disease-causing mutations in either gene have been identified in different families. Newborn screening programs using tandem mass spectrometry have identified an unexpectedly high number of infants with 3-methylcrotonyl CoA carboxylase deficiency (1 : 50,000). Only a small number (<10%) of the affected infants become symptomatic; none of the symptoms reported so far could be clearly attributed to the enzyme deficiency. These findings have questioned the advisability of including this condition in the routine newborn screening programs because the psychological and financial burdens may outweigh the potential benefits.

3-Methylglutaconic Aciduria

At least 3 inherited conditions are known to be associated with excessive excretion of 3-methylglutaconic acid in the urine. Deficiency of the enzyme 3-methylglutaconyl CoA hydratase (see Fig. 79-4) has been documented in only 1 condition (type I). In the other 2 conditions, the enzyme activity is normal despite a modest 3-methylglutaconic aciduria.

β-Ketothiolase (3-Oxothiolase) Deficiency (Mitochondrial Acetoacetyl CoA Thiolase [T₂] Deficiency)

This reversible mitochondrial enzyme cleaves 2-methylacetoacetyl CoA (see Fig. 79-4) or acetoacetyl CoA in 1 direction and synthesizes these compounds in a reverse action (Fig. 79-7).

Figure 79-7 Formation (liver) and metabolism (peripheral tissues) of ketone bodies and cholesterol synthesis. Enzymes: (1) mitochondrial acetoacetyl CoA thiolase, (2) HMG-CoA synthase, (3) HMG-CoA lyase, (4) cytosolic acetoacetyl CoA thiolase, (5) HMG-CoA reductase, (6) mevalonic kinase, (7) succinyl CoA:3-ketoacid CoA transferase (SCOT).

Clinical manifestations are quite variable, ranging from an asymptomatic course in an adult to severe episodes of acidosis starting in the 1st yr of life. These children have intermittent episodes of unexplained ketosis and acidosis. These episodes usually occur after an intercurrent infection and respond quickly to intravenous fluids and bicarbonate therapy. Mild to moderate hyperammonemia may also be present during attacks. Both hypoglycemia and hyperglycemia have been reported in isolated cases. The child may be completely asymptomatic between episodes and may tolerate a normal protein diet well. Mental development is normal in most children. The episodes may be misdiagnosed as salicylate poisoning because of the similarity of clinical findings and the interference of elevated blood levels of acetoacetate with the colorimetric assay for salicylate.

Laboratory findings during the acute attack include acidosis, ketosis, and hyperammonemia. The urine contains large amounts of 2-methylacetoacetate and its decarboxylation product butanone, 2-methyl-3-hydroxybutyrate, and tiglylglycine. Lower concentrations of these urinary metabolites persist during the seemingly well periods. Mild hyperglycinemia may also be present. The clinical and biochemical findings should be differentiated from those seen with propionic and methylmalonic acidemias (see later). Diagnosis may be established by assay of the enzyme in leukocytes, cultured fibroblasts, or identification of the mutant gene.

Treatment of acute episodes includes hydration and infusion of bicarbonate to correct the acidosis; a 10% glucose solution with the appropriate electrolytes and intravenous lipids may be used to minimize the catabolic state. Restriction of protein intake (1-2 g/kg/24 hr) is recommended for long-term therapy. Oral L-carnitine (50-100 mg/kg/24 hr) is also recommended to prevent possible secondary carnitine deficiency. Long-term prognosis for achieving normal life seems very favorable. Three patients graduated from high school and 1 has attended college. All patients continued to have abnormal metabolites in body fluids. Successful pregnancy with normal outcomes for both mother and infant has been reported.

The pathogenesis of ketosis in this condition is not adequately explained because, in this enzyme deficiency, one expects impaired ketone formation (see Fig. 79-7). It is postulated that excess acetoacetyl CoA produced from other sources is used as a substrate for 3-hydroxy-3-methylglutaryl (HMG) CoA synthesis in the liver.

This condition is inherited as an autosomal recessive trait and may be more prevalent than has been appreciated. It is most prevalent in Tunisia. The gene (ACAT1) for this enzyme (T₂) is located on chromosome 11q22.3-q23.1.

Cytosolic Acetoacetyl CoA Thiolase (ACAT₂) Deficiency

This enzyme catalyzes the cytosolic production of acetoacetyl CoA from 2 moles of acetyl CoA (see Fig. 79-7). Cytosolic acetoacetyl CoA is the precursor of hepatic cholesterol synthesis. Cytosolic acetoacetyl CoA thiolase is a completely different enzyme from that of mitochondrial thiolase (see earlier and Fig. 79-4). Clinical manifestations in patients with this rare enzyme deficiency are similar to those in patients with mevalonic acidemia (see later). Severe progressive developmental delay, hypotonia, and choreoathetoid movements develop in the 1st few months of life. Laboratory findings are nonspecific; elevated levels of lactate, pyruvate, acetoacetate, and 3-hydroxybutyrate may be found in blood and urine. One patient had normal levels of acetoacetate and 3-hydroxybutyrate. Diagnosis can be established by demonstrating a deficiency in cytosolic thiolase activity in liver biopsy or in cultured fibroblasts or by DNA analysis. No effective treatment is available. The gene for this condition is mapped to chromosome 6q25.3-q26.

Mitochondrial 3-Hydroxy-3-Methylglutaryl (HMG) CoA Synthase Deficiency

This enzyme catalyzes synthesis of HMG-CoA from acetoacetyl CoA in the mitochondria. This is a critical step in ketone body synthesis in the liver (see Fig. 79-7). A few patients with deficiency of this enzyme have been reported. All patients have had similar presentations and outcomes. Signs and symptoms of acute hypoglycemia have occurred after an acute illness (gastroenteritis). Age at presentation has ranged from 18 mo to 6 yr. All children were asymptomatic before the episodes and remained normal after the recovery (except for mild hepatomegaly with fatty infiltration). None of the patients has had a 2nd episode, perhaps as a result of preventive measures to avoid prolonged fasting during ensuing intercurrent illnesses. Hepatomegaly was a consistent physical finding in all patients. Laboratory findings included hypoglycemia, acidosis with mild or no ketosis, elevation of liver function tests, and massive dicarboxylic aciduria. The clinical and laboratory findings may be confused with those of patients with defects in fatty acid metabolism (see Chapter 80.1). In contrast to the latter, blood concentrations of acylcarnitine conjugates are normal in patients with HMG-CoA synthase deficiency. Fasting of these patients has produced the abovementioned clinical and biochemical abnormalities.

Treatment consisted of provision of adequate calories and avoidance of prolonged periods of fasting. No dietary protein restriction was needed.

The condition is inherited as an autosomal recessive trait. The gene for this condition is located on chromosome 1p13-p12 and several disease-causing mutations have been identified. The condition should be considered in any child with fasting hypoglycemia and is perhaps more common than appreciated.

3-Hydroxy-3-Methylglutaric Aciduria

This condition is due to a deficiency of HMG-CoA lyase (see Fig. 79-4). This enzyme catalyzes the conversion of HMG-CoA to acetoacetate and is a rate-limiting enzyme for ketogenesis (see Fig. 79-7). Clinically, >60% of patients become symptomatic between 3 and 11 mo of age, whereas about 30% develop symptoms in the 1st few days of life. One child remained asymptomatic until 15 yr of age. Episodes of vomiting, severe hypoglycemia, hypotonia, acidosis with mild or no ketosis, and dehydration may rapidly lead to lethargy, ataxia, and coma. These episodes often occur during a catabolic state such as fasting or an intercurrent infection. Hepatomegaly is common. These manifestations may be mistaken for Reye syndrome or medium-chain acyl CoA dehydrogenase (MCAD) deficiency. Patients are usually clinically asymptomatic between the attacks; 1 patient died of acute cardiomyopathy at age 7 mo during a febrile illness. Development is usually normal, but mental retardation and seizure with abnormalities of white matter (shown by MRI) have been observed in patients with prolonged episodes of hypoglycemia.

Laboratory findings include hypoglycemia, moderate to severe hyperammonemia, and acidosis. There is mild or no ketosis (see Fig. 79-7). Urinary excretion of 3-hydroxy-3-methylglutaric acid and other proximal intermediate metabolites of leucine catabolism (3-methylglutaconic acid and 3-hydroxyisovaleric acid) is markedly increased causing the urine to smell like cat urine. These organic acids are excreted in the urine as carnitine conjugates, resulting in secondary carnitine deficiency. Glutaric and adipic acids may also be increased in urine during acute attacks. Diagnosis may be confirmed by enzyme assay in cultured fibroblasts, leukocytes, or liver specimens or by identification of the mutant gene. Prenatal diagnosis is possible by the assay of the enzyme in cultured amniocytes or a chorionic villi biopsy or by DNA analysis.

Treatment of acute episodes includes hydration, infusion of glucose to control hypoglycemia, provision of adequate calories, and administration of bicarbonate to correct acidosis. Hyperammonemia should be treated promptly (Chapter 79.12). Exchange transfusion and peritoneal dialysis may be required in patients with severe hyperammonemia. Restriction of protein and fat intake is recommended for long-term management. Oral administration of L-carnitine (50-100 mg/kg/24 hr) prevents secondary carnitine deficiency. Prolonged fasting should be avoided. One child died after routine immunization. The condition is inherited as an autosomal recessive trait. The gene for HMG-CoA lyase resides on chromosome 1pter-p33 and several disease-causing mutations have been identified in different families. The gene defect appears to be more common in the Arabic population, especially in Saudi Arabia.

Succinyl CoA:3-Ketoacid CoA Transferase (SCOT) Deficiency

This enzyme is necessary for the metabolism of ketone bodies (acetoacetate and 3-hydroxybutyrate) in peripheral tissue (see Fig. 79-7). A deficiency of this enzyme results in the underutilization and accumulation of ketone bodies and ketoacidosis. Only a few patients with SCOT deficiency have been reported to date; the condition may not be rare because many cases are undiagnosed.

The presentation is an acute episode of unexplained severe ketoacidosis in an infant who had been growing and developing normally. About half of the patients present in the 1st wk of life and all before 2 yr. The acute episode is often precipitated by an intercurrent infection or a catabolic state. Death may occur during these episodes. A chronic subclinical ketosis usually persists between the attacks. Development is usually normal.

Laboratory findings during the acute episode are nonspecific and include metabolic acidosis and ketonuria with high levels of acetoacetate and 3-hydroxybutyrate in blood and urine. No other organic acids are found in the blood or in the urine. Blood glucose levels are usually normal, but hypoglycemia has been reported in 2 newborn infants with severe ketoacidosis. Plasma amino acids are usually normal. Diagnosis can be established by demonstrating a deficiency of enzyme activity in cultured fibroblasts or by DNA analysis.

Treatment of acute episodes consists of hydration, correction of acidosis, and the provision of a diet adequate in calories. Long-term treatment with a high-carbohydrate diet and avoidance of catabolic states is recommended. This condition should be considered in any infant with unexplained bouts of ketoacidosis. The condition is inherited as an autosomal recessive trait. The gene for this enzyme is located on chromosome 5p13, and several disease-causing mutations have been found in different families.

Mevalonic Aciduria

Mevalonic acid, an intermediate metabolite of cholesterol synthesis, is converted to 5-phosphomevalonic acid by the action of the enzyme mevalonate kinase (MVK) (see Fig. 79-7). Based on clinical manifestations, 2 forms of this condition have been recognized.

Propionic Acidemia (Propionyl CoA Carboxylase Deficiency)

Propionic acid is an intermediate metabolite of isoleucine, valine, threonine, methionine, odd-chain fatty acids, and cholesterol catabolism. It is normally carboxylated to methylmalonic acid by the mitochondrial enzyme propionyl CoA carboxylase, which requires biotin as a cofactor (see Fig. 79-4). The enzyme is composed of 2 nonidentical subunits, α and β. Biotin is bound to the α subunit.

Clinical findings are nonspecific. In the severe form of the condition, patients develop symptoms in the 1st few days or weeks of life. Poor feeding, vomiting, hypotonia, lethargy, dehydration, and clinical signs of severe ketoacidosis progress rapidly to coma and death. Seizures occur in approximately 30% of affected infants. If an infant survives the 1st attack, similar episodes may occur during an intercurrent infection or constipation or after ingestion of a high-protein diet. Moderate to severe mental retardation and neurologic manifestations indicating extrapyramidal (dystonia, choreoathetosis, tremor), and pyramidal (paraplegia) dysfunction are common sequelae in the older survivors. These abnormalities, which usually occur after an episode of metabolic decompensation, have been shown by neuroimaging to be due to the destruction of basal ganglia, especially that of the globus pallidus. This phenomenon has been referred to in the literature as metabolic stroke. In the milder forms, the older infant may have mental retardation without acute attacks of ketosis. Some affected children may have episodes of unexplained severe ketoacidosis separated by periods of seemingly normal health. Mass screening of newborn infants has identified milder forms of the condition; a few of theses infants were completely asymptomatic at diagnosis. The severity of clinical manifestations may also be variable within a family; in 1 kindred, a brother was diagnosed at 5 yr of age whereas his 13 yr old sister, with the same level of enzyme deficiency, was asymptomatic.

Laboratory findings during the acute attack include severe metabolic acidosis with a large anion gap, ketosis, neutropenia, thrombocytopenia, and hypoglycemia. Moderate to severe hyperammonemia is common; plasma ammonia concentrations usually correlate with the severity of the disease. Measurement of plasma ammonia is especially helpful in planning therapeutic strategy during episodes of exacerbation in a patient whose diagnosis has been established. Pathogenesis of hyperammonemia is not well understood. Hyperglycinemia is a common finding. Elevations in plasma and urinary levels of glycine have also been observed in patients with methylmalonic acidemia. These disorders were collectively referred to as ketotic hyperglycinemia before the specific enzyme deficiencies were elucidated. Concentrations of propionic acid and methylcitric acid (presumably made by the condensation of propionyl CoA with oxaloacetic acid) are markedly elevated in the plasma and urine of infants with propionic acidemia. 3-Hydroxypropionic acid, propionylglycine, and other intermediate metabolites of isoleucine catabolism, such as tiglic acid, tiglylglycine, and 2-methyloacetoacetic acid, are also found in urine. Moderate elevations in blood levels of ammonia, glycine, and previously mentioned organic acids usually persist between the acute attacks. CT scan and MRI of the brain may reveal cerebral atrophy, demyelination, and abnormalities in the globus pallidus and basal ganglia as the evidence of a metabolic stroke in this condition (see earlier).

The diagnosis of propionic acidemia should be differentiated from multiple carboxylase deficiencies (see earlier and Fig. 79-6). Infants with the latter condition may have skin manifestations and excrete large amounts of lactic acid, 3-methylcrotonic acid, and 3-hydroxyisovaleric acid in addition to propionic acid. The presence of hyperammonemia may suggest a genetic defect in the urea cycle enzymes. Infants with defects in the urea cycle are usually not acidotic (see Fig. 79-1). Definitive diagnosis of propionic acidemia can be established by measuring the enzyme activity in leukocytes or cultured fibroblasts.

Treatment of acute attacks includes hydration, correction of acidosis, and amelioration of the catabolic state by provision of adequate calories through parenteral hyperalimentation. Minimal amounts of protein (0.25 g/kg/24 hr), preferably a protein deficient in propionate precursors, should be provided in the hyperalimentation fluid very early in the course of treatment. To curtail the possible production of propionic acid by intestinal bacteria, sterilization of the intestinal tract flora by antibiotics (oral neomycin, or metronidazole) should be promptly initiated. Constipation should also be treated. Patients with propionic acidemia may develop carnitine deficiency, presumably as a result of urinary loss of propionylcarnitine formed from the accumulated organic acid. Administration of L-carnitine (50-100 mg/kg/24 hr orally or 10 mg/kg/24 hr intravenously) normalizes fatty acid oxidation and improves acidosis. In patients with concomitant hyperammonemia, measures to reduce blood ammonia should be employed (Chapter 79.12). Very ill patients with severe acidosis and hyperammonemia require peritoneal dialysis or hemodialysis to remove ammonia and other toxic compounds efficiently. Although infants with true propionic acidemia are rarely responsive to biotin, this compound should be administered (10 mg/24 hr orally) to all infants during the 1st attack and until the diagnosis is established.

Long-term treatment consists of a low-protein diet (1.0-1.5 g/kg/24 hr) and administration of L-carnitine (50-100 mg/kg/24 hr orally). Synthetic proteins deficient in propionate precursors (isoleucine, valine, methionine, and threonine) may be used to increase the amount of dietary protein (to 1.5-2.0 g/kg/24 hr) while causing minimal change in propionate production. Excessive supplementation with these proteins may cause a deficiency of the essential amino acids. To avoid this problem, natural proteins should comprise most of the dietary protein (50-75%). Some patients may require chronic alkaline therapy to correct chronic acidosis. The concentration of ammonia in the blood usually normalizes between attacks, and chronic treatment of hyperammonemia is not usually needed. Catabolic states that may trigger acute attacks (infections, constipation) should be treated promptly and aggressively. Close monitoring of blood pH, amino acids, urinary content of propionate and its metabolites, and growth parameters is necessary to ensure the proper balance of the diet and the success of therapy.

Long-term prognosis is guarded. Death may occur during an acute attack. Normal psychomotor development is possible, especially in the mild forms identified through screening programs; most children identified clinically manifest some degree of permanent neurodevelopmental deficit such as tremor, dystonia, chorea, and pyramidal signs despite adequate therapy. These neurologic findings may be sequelae of a metabolic stroke occurring during an acute decompensation (see earlier).

Prenatal diagnosis is achieved by measuring the enzyme activity in cultured amniotic cells or in samples of uncultured chorionic villi, by measurement of methylcitrate in amniotic fluid, or by identification of the mutant gene.

The condition is inherited as an autosomal recessive trait and can be identified by mass screening of newborns. It is more prevalent in Saudi Arabia (1 : 2,000 to 1 :5 ,000). The gene for the α subunit (PCCA gene) is located on chromosome 13q32 and that of the β subunit (PCCB gene) is mapped to the chromosome 3q21-q22. Many mutations in either gene have been identified in different patients. Pregnancy with normal outcome has been reported in affected females.

Methylmalonic Acidemia

Methylmalonic acid, a structural isomer of succinic acid, is normally derived from propionic acid as part of the catabolic pathways of isoleucine, valine, threonine, methionine, cholesterol, and odd-chain fatty acids. Two enzymes are involved in the conversion of D-methylmalonic acid to succinic acid: methylmalonyl CoA racemase, which forms the L-isomer; and methylmalonyl CoA mutase, which converts the L-methylmalonic acid to succinic acid (see Fig. 79-4). The latter enzyme requires adenosylcobalamin, a metabolite of vitamin B₁₂, as a coenzyme. Deficiency of either the mutase or its coenzyme causes the accumulation of methylmalonic acid and its precursors in body fluids. A deficiency of the racemase can be associated with mild elevations of methylmalonic acid, but the clinical consequence of racemase deficiency is not known.

At least 2 forms of mutase apoenzyme deficiencies have been identified. These are designated mut⁰, meaning no detectable enzyme activity, and mut^–, indicating residual, although abnormal, mutase activity. The majority of reported patients with methylmalonic acidemia have a deficiency of the mutase apoenzyme (mut⁰ or mut⁻). These patients are not responsive to vitamin B₁₂ therapy. In the remaining patients with methylmalonic acidemia, the defect resides in the formation of adenosylcobalamin.

Combined Methylmalonic Aciduria and Homocystinuria (cblC, cblD, and cblF Defects)

Over 300 patients with methylmalonic acidemia and homocystinuria due to cblC have been reported. Indeed with the advent of expanded newborn screening, cblC may be as common as mutase deficiency. The cblD, and cblF defects are much rarer with less than a dozen patients known with each (see Figs. 79-3 and 79-4). Neurologic findings are prominent in patients with cblC and cblD defects. Most patients with the cblC defect present in the 1st yr of life because of failure to thrive, lethargy, poor feeding, mental retardation, and seizures. However, late-onset defects with sudden development of dementia and myelopathy have been reported, even with presentation is adulthood. Megaloblastic anemia was a common finding in patients with cblC defect. Mild to moderate increases in concentrations of methylmalonic acid and homocysteine were found in body fluids. Unlike patients with classic homocystinuria, plasma levels of methionine are low to normal in these defects. Neither hyperammonemia nor hyperglycinemia is present in these patients. The clinical findings in the cblF defect are quite variable; the 1st 2 patients had poor feeding, growth and developmental delay, and persistent stomatitis manifesting in the 1st 3 wk of life. One patient was not diagnosed until age 10 yr and had findings suggestive of rheumatoid arthritis, a pigmented skin abnormality, and encephalopathy. Vitamin B₁₂ malabsorption has been noted in patients with cblF defect.

Experience with treatment of patients with cblC, cblD, and cblF defects is limited. Large doses of hydroxycobalamin (OHCbl; 1-2 mg/24 hr) in conjunction with betaine (6-9 g/24 hr) seem to produce biochemical improvement with little clinical effect. Unexplained severe hemolytic anemia, hydrocephalus, and congestive heart failure have been major complications in patients with cblC defect. The cblC disorder is caused by mutations in the MMACHC gene on chromosome 1p34.1 and there are a number of common mutations and ones that are more common in specific ethnic groups. The cblD disorder is caused by mutations in the MMADHC gene on chromosome 2q23.2. Mutations resulting in the variant 1 (homocystinuria) affect the C-terminal domain of the gene product; those resulting in the variant 2 (methylmalonic aciduria) affect the N terminal. The classical form of cblD with both homocystinuria and methylmalonic aciduria had mutations resulting in decreased protein expression. The cblF disorder has been found to be due to mutations in the LMBRD1 gene on chromosome 6p13 coding for the lysosomal cobalamin transporter.

Patients with cblD variant 1, cblE and cblG defects do not have methylmalonic acidemia (Chapter 79.3).

Hypoglycinemia

Defects in the biosynthetic pathway of serine (Chapter 79.8) cause deficiency of glycine in addition to that of serine in body fluids, especially in the CSF. Isolated primary deficiency of glycine has not been reported.

Hyperglycinemia

Elevated levels of glycine in body fluids occur in propionic acidemia and methylmalonic acidemia that are collectively referred to as ketotic hyperglycinemia because episodes of severe acidosis and ketosis occur. The pathogenesis of hyperglycinemia in these disorders is not fully understood, but inhibition of the glycine cleavage enzyme system by the various organic acids has been shown to occur in some of the patients. The term nonketotic hyperglycinemia (NKH) is reserved for the clinical condition caused by the genetic deficiency of the glycine cleavage enzyme system (see Fig. 79-8). In this condition, hyperglycinemia is present without ketosis.

Nonketotic Hyperglycinemia, Glycine Encephalopathy

Four forms of this condition have been identified: neonatal, infantile, late onset, and transient.

Sarcosinemia

Increased concentrations of sarcosine (N-methylglycine) are observed in both blood and urine, but no consistent clinical picture has been attributed to this metabolic defect. This is a recessively inherited metabolic condition caused by a defect in sarcosine dehydrogenase, the enzyme that converts sarcosine to glycine (see Fig. 79-8). The gene for this enzyme is on chromosome 9q33-q34.

D-Glyceric Aciduria

D-Glyceric acid is an intermediate metabolite of serine and fructose metabolism (see Fig. 79-8). In this rare condition, clinical manifestations of severe encephalopathy (hypotonia, seizures, and mental and motor deficits) and the laboratory findings of hyperglycinemia and hyperglycinuria were suggestive of NKH. These patients excreted large quantities of D-glyceric acid. This compound is not normally detectable in urine. Enzyme studies indicated a deficiency of glycerate kinase in 1 patient and decreased activity of D-glyceric dehydrogenase in another.

No effective therapy is available. Restriction of fructose reduced the incidence of seizures in 1 patient. The gene for glycerate kinase is on chromosome 3p21.

Trimethylaminuria

Trimethylamine is normally produced in the intestine from the breakdown of dietary choline and trimethylamine oxide by bacteria. Egg yolk and liver are the main sources of choline, and fish is the major source of trimethylamine oxide. Trimethylamine is absorbed and oxidized in the liver by trimethylamine oxidase (flavin-containing monooxygenases) to trimethylamine oxide, which is odorless and excreted in the urine (see Fig. 79-8). Deficiency of this enzyme results in massive excretion of trimethylamine in urine. Several asymptomatic patients with trimethylaminuria have been reported; there is a foul body odor that resembles that of a rotten fish, which may have significant social and psychosocial ramifications. Restriction of fish, eggs, liver, and other sources of choline (such as nuts and grains) in the diet significantly reduce the odor. Treatment with short courses of oral metronidazole, neomycin or lactulose cause temporary reduction in the body odor. The gene for trimethylamine oxidase has been mapped to chromosome 1q23-q25.

Hyperoxaluria and Oxalosis

Normally, oxalic acid is derived mostly from oxidation of glyoxylic acid, and to a lesser degree, from oxidation of ascorbic acid (see Fig. 79-8). Glyoxylic acid is formed from the oxidation of glycolic acid and the deamination of glycine in the peroxisomes. The source of glycolic acid is poorly understood. Foods containing oxalic acid, such as spinach and rhubarb, are the main exogenous sources of this compound. Oxalic acid cannot be further metabolized in humans and is excreted in the urine as oxalates. Calcium oxalate is relatively insoluble in water and precipitates in tissues (kidneys and joints) if its concentration increases in the body.

Secondary hyperoxaluria has been observed in pyridoxine deficiency (cofactor for alanine-glyoxylate aminotransferase; see Fig. 79-8) after ingestion of ethylene glycol or high doses of vitamin C, after administration of the anesthetic agent methoxyflurane (which oxidizes directly to oxalic acid), and in patients with inflammatory bowel disease or extensive resection of the bowel (enteric hyperoxaluria). Acute, fatal hyperoxaluria may develop after ingestion of plants with a high oxalic acid content such as sorrel. Precipitation of calcium oxalate in tissues causes hypocalcemia, liver necrosis, renal failure, cardiac arrhythmia, and death. The lethal dose of oxalic acid is estimated to be between 5 and 30 g.

Primary hyperoxaluria is a rare genetic disorder in which large amounts of oxalates accumulate in the body. Two types of primary hyperoxaluria have been identified. The term oxalosis refers to deposition of calcium oxalate in parenchymal tissue.

3-Phosphoglycerate Dehydrogenase Deficiency

Deficiency of this enzyme causes deficiencies of serine and glycine in the body. Clinical manifestations, which are developed in the 1st few months of life, include microcephaly, severe psychomotor retardation, and intractable seizures. Other findings such as failure to thrive, spastic tetraplegia, nystagmus, cataracts, hypogonadism, and megaloblastic anemia may also be present.

Laboratory findings include low levels of serine and glycine in plasma and very low levels of serine and glycine in CSF. No abnormal organic acid is found in the urine. MRI of the brain shows significant attenuation of white matter and incomplete myelination.

Diagnosis can be confirmed by measurement of the enzyme activity in cultured fibroblasts and by DNA analysis.

Treatment with serine (200-600 mg/kg/24 hr, orally) alone or in conjunction with glycine (200-300 mg/kg/24 hr) normalizes the serine levels in the blood and CSF. This treatment produces significant improvement in all clinical findings except for the psychomotor retardation; seizure activity subsides within a few days of therapy and may be halted completely. Microcephaly improves in young affected infants. There is evidence to indicate that psychomotor retardation may be prevented if the treatment starts in the 1st few days of life.

The condition is inherited as an autosomal recessive trait. The gene for 3-phosphoglycerate dehydrogenase enzyme has been mapped to chromosome 1q12 and a few disease-producing mutations have been identified in different families. Prenatal diagnosis has been achieved by DNA analysis in a family with previously affected offspring; administration of serine to the mother corrected the microcephaly in the affected fetus as evidenced by ultrasound imaging. The favorable response of this condition to a simple treatment makes this diagnosis an important consideration in any child with microcephaly and neurologic defects such as psychomotor delay or a seizure disorder.

Phosphoserine Aminotransferase Deficiency

This enzyme catalyzes conversion of 3-phosphohydroxypyruvate to 3-phosphoserine (see Fig. 79-9). Deficiency of this enzyme has been reported in 2 siblings from an English family. Poor feeding, cyanotic episodes, and jerky movements developed shortly after birth in the 1st affected infant and progressed to intractable seizures by 9 wk of age. The infant was microcephalic. EEG was consistent with multifocal seizures. Neuroimaging showed generalized cerebral and cerebellar atrophies. Laboratory studies were all within normal limits except for mild decrease in plasma levels of serine and glycine with pronounced deficiencies of these 2 amino acids in the CSF. Treatment with serine (500 mg/kg/day) and glycine (200 mg/kg/day) was started at 11 wk of age but resulted in only marginal clinical improvement; the child died at 7 mo of age. The younger affected sibling, who was treated with serine and glycine within a few hours after birth, has remained asymptomatic at 3 yr of age.

The condition is inherited as an autosomal recessive trait and gene for the enzyme is mapped to chromosome 9q21.31. Based on this single report one can assume that this is a treatable genetic condition with favorable outcome if the treatment is initiated early in life. Measurements of serine and glycine in the CSF are critical for diagnosis since mild decreases of these amino acids in the plasma can be easily overlooked.

Hyperprolinemia

Two types of primary hyperprolinemia have been described.

Prolidase Deficiency

During collagen degradation, imidodipeptides (dipeptides containing proline such as glycylproline) are released and are normally cleaved by tissue prolidase. This enzyme requires manganese for its proper activity. Deficiency of prolidase, which is inherited as an autosomal recessive trait, results in the accumulation of imidodipeptides in body fluids.

The clinical manifestations of this rare condition and the age at onset are quite variable (19 mo to 19 yr) and include recurrent, painful skin ulcers, which are typically on hands and legs. Other skin lesions that may precede ulcers by several years may include scaly erythematous maculopapular rash, purpura, and telangiectasia. Most ulcers become infected. Healing of the ulcers may take 4 to 7 mo. Mild to severe mental and motor deficits and susceptibility to infections are also present in most patients (recurrent otitis media, sinusitis, respiratory infection, splenomegaly). Infection is the cause of death. Some patients may have some craniofacial abnormalities such as ptosis, ocular proptosis, and prominent cranial sutures. Asymptomatic cases have also been reported. Development of systemic lupus erythematosus (SLE) has been noted in affected children of 1 family; young patients with SLE should be screened for prolidase deficiency. High levels of urinary excretion of imidodipeptides are diagnostic. Enzyme assay may be performed in erythrocytes or cultured skin fibroblasts.

Oral supplementation with proline, ascorbic acid, and manganese and the topical use of proline and glycine result in an improvement in leg ulcers. These treatments have not been found to be consistently effective in all patients.

The gene for prolidase enzyme has been mapped to chromosome 19cen-q13.11 and several disease-causing mutations have been identified in different families.

Glutathione Synthetase Deficiency (See Fig. 79-11)

Three forms of this condition have been reported. In the severe form, which is due to generalized deficiency of the enzyme, severe acidosis and massive 5-oxoprolinuria are the rule. In the mild form, in which the enzyme deficiency causes glutathione deficiency only in erythrocytes, neither 5-oxoprolinuria nor acidosis has been observed. A moderate form has also been observed in which the hemolytic anemia is associated with variable degrees of acidosis and 5-oxoprolinuria. In all forms, patients have hemolytic anemia secondary to glutathione deficiency. All forms are rare; a total of 65 patients have been reported.

Glutathionemia (γ-Glutamyl Transpeptidase [GGT] Deficiency)

This enzyme is present in any cell that has secretory or absorptive functions. It is especially abundant in the kidneys, pancreas, intestines, and liver. The enzyme is also present in the bile. Measurement of this enzyme in the blood is commonly performed to evaluate liver and bile duct diseases.

Deficiency of this enzyme causes elevation in glutathione concentrations in body fluids, but the cellular levels remain normal. Only a few patients with enzyme deficiency have been reported; therefore, the scope of clinical manifestations has not yet been defined. Mild to moderate mental retardation and severe behavioral problems were observed in 3 patients. One of the 2 sisters with this condition had normal intelligence as an adult, however, and the other had Prader-Willi syndrome.

Laboratory findings include marked elevations in urinary concentrations of glutathione (up to 1 g/day), γ-glutamylcysteine, and cysteine. None of the reported patients has had generalized aminoaciduria, a finding that would have been expected to occur in this enzyme deficiency (see Fig. 79-11).

Diagnosis can be confirmed by measurement of the enzyme activity in leukocytes or cultured skin fibroblasts. No effective treatment is available.

The condition is inherited as an autosomal recessive trait. The enzyme GGT is a complex protein and is encoded by at least seven genes.

Genetic DIsorders of Metabolism of γ-Aminobutyric Acid (Chapter 79.11)

Genetic Causes of Hyperammonemia

In addition to genetic defects of the urea cycle enzymes, a marked increase in plasma level of ammonia is observed in other genetic disorders of metabolism (Table 79-3); the pathogenesis for hyperammonemia in some of these conditions is not fully understood.

Clinical Manifestations of Hyperammonemia

In the neonatal period, symptoms and signs are mostly related to brain dysfunction and are similar regardless of the cause of the hyperammonemia. The affected infant is normal at birth but becomes symptomatic within a few days of protein feeding. Refusal to eat, vomiting, tachypnea, and lethargy can quickly progress to a deep coma. Convulsions are common. Physical examination may reveal hepatomegaly in addition to the neurologic signs of deep coma. Hyperammonemia can trigger increased intracranial pressure that may be manifested with bulging fontanel and dilated pupils.

In infants and older children acute hyperammonemia is manifested by vomiting and neurologic abnormalities such as ataxia, mental confusion, agitation, irritability, and combativeness. These manifestations may alternate with periods of lethargy and somnolence that may progress to coma.

Routine laboratory studies show no specific findings when hyperammonemia is due to defects of the urea cycle enzymes. Blood urea nitrogen is usually low in these patients; serum pH is usually normal or mildly elevated. There may be mild increases in serum transaminases (ALT, AST) since ammonia can cause swelling of hepatic mitochondria. In infants with organic acidemias, hyperammonemia is commonly associated with severe acidosis as well as ketonuria. Newborn infants with hyperammonemia are often misdiagnosed as having sepsis; they may succumb without a correct diagnosis. Neuroimaging may reveal cerebral edema. Autopsy is usually unremarkable. It is imperative to measure plasma ammonia levels in any ill infant whose clinical manifestations cannot be explained by an obvious infection.

Diagnosis

The main criterion for diagnosis is hyperammonemia. Each clinical laboratory should establish its own normal values for blood ammonia; some variation is common. In the older child and adult, the normal limit is typically <35 µmole/L. Blood concentrations in healthy newborn infants often are higher (as high as 100 µmole/L in a full-term baby and 150 µmole/L in a premature infant). An approach to the differential diagnosis of hyperammonemia in the newborn infant is illustrated in Figure 79-13. Plasma amino acids reveal abnormalities that may help the diagnosis. In patients with deficiencies of either CPS, OTC, or N-acetylglutamate (NAG) synthetase frequent findings are elevations in plasma glutamine and alanine with concurrent decrements in citrulline and arginine. These disorders cannot be differentiated from one another by the plasma amino acid levels alone. A marked increase in urinary orotic acid in patients with OTC deficiency differentiates this defect from CPS deficiency. Differentiation between the CPS deficiency and the NAG synthetase deficiency may require an assay of the respective enzymes. Clinical improvement occurring after oral administration of carbamylglutamate, however, may suggest NAG synthetase deficiency. Patients with deficiencies of AS, AL, or arginase have marked increases in the plasma levels of citrulline, argininosuccinic acid, or arginine, respectively. Indeed, the combination of hyperammonemia and marked hypercitrullinemia or argininosuccinic acidemia is virtually pathognomonic for these disorders.

Figure 79-13 Clinical approach to a newborn infant with symptomatic hyperammonemia. CPS, carbamyl phosphate synthetase; HHH syndrome, hyperammonemia-hyperornithinemia-homocitrullinemia; NAG, N-acetylglutamate; OTC, ornithine transcarbamylase.

Treatment of Acute Hyperammonemia

The outcome for patients with hyperammonemic episodes depends mainly on the severity and duration of the hyperammonemia. Serious neurologic sequelae are highly likely in newborn infants with severe elevations in blood ammonia (>300 µmole/L) for more than 12 hr. Thus, hyperammonemia should be treated promptly and vigorously. The goal of therapy is to lower the concentration of ammonia in the body. This is accomplished in two ways: (1) by removal of ammonia from the body in a form other than urea, and (2) by provision of adequate calories and essential amino acids to minimize the endogenous protein degradation and favor protein synthesis (Table 79-4). Fluid, electrolytes, glucose (5-15%), and lipids (1-2 g/kg/24 hr) should be infused intravenously together with a minimal amount of protein (0.25 g/kg/24 hr), preferably in the form of essential amino acids. As soon as the clinical condition of the patient allows, oral feeding with a low-protein formula (0.5-1.0 g/kg/24 hr) through a nasogastric tube should be started.

An important advance in treatment of hyperammonemia has been the advent of acylation therapy in which exogenously administered organic acids form acyl adducts with endogenous nonessential amino acids. These adducts are nontoxic compounds with high renal clearances. The main organic acids used for this purpose are sodium salts of benzoic acid and phenylacetic acid. Benzoate forms hippuric acid with endogenous glycine in the liver (see Fig. 79-12). Each mole of benzoate removes 1 mole of ammonia as glycine. Phenylacetate conjugates with glutamine to form phenylacetylglutamine, which is readily excreted in the urine. One mole of phenylacetate removes 2 moles of ammonia as glutamine from the body (see Fig. 79-12).

Arginine administration is effective in the treatment of hyperammonemia that is due to most defects of the urea cycle because it supplies the urea cycle with ornithine and NAG (see Fig. 79-12). In patients with citrullinemia, 1 mole of arginine reacts with 1 mole of ammonia (as carbamyl phosphate) to form citrulline. In patients with argininosuccinic acidemia, 2 moles of ammonia (as carbamyl phosphate and aspartate) react with arginine to form argininosuccinic acid. Citrulline and argininosuccinic acid are far less toxic and more readily excreted by the kidneys than ammonia. In patients with CPS or OTC deficiency, arginine administration is indicated because arginine becomes an essential amino acid in these disorders. Patients with OTC deficiency benefit from supplementation with citrulline (200 mg/kg/24 hr), which reacts with 1 mole of ammonia (as aspartic acid) to form arginine. Administration of arginine or citrulline is contraindicated in patients with arginase deficiency, a rare condition in which the presenting clinical picture is one of spastic diplegia rather than hyperammonemia (see later). Furthermore, arginine therapy is of no benefit if hyperammonemia is secondary to an organic acidemia. However, in a newborn infant with a 1st attack of hyperammonemia, arginine should be used until the diagnosis is established.

Benzoate, phenylacetate, and arginine may be administered together for maximal therapeutic effect. A priming dose of these compounds is followed by continuous infusion until recovery from the acute state occurs (see Table 79-4). Both benzoate and phenylacetate are usually supplied as concentrated solutions and should be properly diluted (1-2% solution) for intravenous use. The recommended therapeutic doses of both compounds deliver a substantial amount of sodium to the patient that should be calculated as part of the daily sodium requirement. A commercial preparation of sodium benzoate plus sodium phenylacetate is available for intravenous use (Ammonul; www.ammonul.com). Benzoate and phenylacetate should be used with caution in newborn infants with hyperbilirubinemia because they may displace bilirubin from albumin. However, despite this theoretical risk, no documented case of kernicterus (Chapter 96.4) has yet been reported in neonates with hyperammonemia who have received such therapies.

If the foregoing therapies fail to produce any appreciable change in the blood ammonia level within a few hours, peritoneal dialysis, or preferably, hemodialysis should be used. Exchange transfusion has little effect on reducing total body ammonia. It should be used only if dialysis cannot be employed promptly or when the patient is a newborn infant with hyperbilirubinemia. Hemodialysis is the most effective measure, but if it is unavailable or technically unfeasible, peritoneal dialysis can decrease the plasma ammonia level within hours. When hyperammonemia is due to an organic acidemia, dialysis effectively removes both the offending organic acid and ammonia from the body.

Oral administration of neomycin limits growth of intestinal bacteria that can produce ammonia. Oral lactulose acidifies the intestinal lumen, thereby reducing the diffusion of ammonia across the intestinal epithelium. Neither compound has been used extensively to treat acute hyperammonemia in the newborn infants. There may be considerable lag between the normalization of ammonia and an improvement in the neurologic status of the patient. Several days may be needed before the infant becomes fully alert.

Carbamyl Phosphate Synthetase (CPS) and N-Acetylglutamate (NAG) Synthetase Deficiencies (See Fig. 79-12)

Deficiencies of these 2 enzymes produce similar clinical and biochemical manifestations. There is a wide variation in severity of symptoms and in the age of presentation. In near complete enzymatic deficiency, symptoms appear during the 1st few days or even hours of life with signs and symptoms of hyperammonemia (refusal to eat, vomiting, lethargy, convulsion, and coma). Increased intracranial pressure is a frequent finding. Late forms (as late as 32 yr of age) may present as an acute bout of hyperammonemia (lethargy, headache, seizures, psychosis) in a seemingly normal individual. Coma and death may occur during these episodes (a previously asymptomatic 26 yr old female died from hyperammonemia during childbirth). Diagnostic confusion with migraine is frequent. Intermediate forms with mental retardation and chronic subclinical hyperammonemia interspersed with bouts of acute hyperammonemia have also been observed.

Laboratory findings include hyperammonemia. The plasma aminogram commonly shows a marked increase of glutamine and alanine with a relatively low levels of citrulline and arginine. Urinary orotic acid is usually low or may be absent (see Fig. 79-13).

Treatment of acute hyperammonemic attacks and the long-term therapy of the condition is outlined earlier (see Table 79-4). Patients with NAG synthetase deficiency benefit from oral administration of carbamylglutamate. It is therefore important to differentiate between CPS and NAG synthetase deficiencies by assay of the enzyme activities in liver biopsy specimens. Deficiency of NAG synthetase is rare in North America.

CPS deficiency is inherited as an autosomal recessive trait; the enzyme is normally present in the liver and intestine. The gene is on chromosome 2q35. Several disease-causing mutations have been found in different families. The prevalence of the condition is not known.

Ornithine Transcarbamylase (OTC) Deficiency (See Fig. 79-12)

In this X-linked partially dominant disorder, the hemizygote males are more severely affected than heterozygote females. The heterozygous females may have a mild form of the disease, but the majority (75%) is asymptomatic, although subtle neurologic defects may be present in women without a frank history of hyperammonemia. This is the most common form of all the urea cycle disorders, comprising about 40% of all cases.

Clinical manifestations in male newborn infants are usually those of severe hyperammonemia (see earlier) occurring in the 1st few days of life. Milder forms of the condition are commonly seen in heterozygous females and in some affected males. Mild forms characteristically have episodic manifestations, which may occur at any age (usually after infancy). Episodes of hyperammonemia (manifested by vomiting and neurologic abnormalities such as ataxia, mental confusion, agitation, and combativeness) are separated by periods of wellness. These episodes usually occur after ingestion of a high-protein diet or as a result of a catabolic state such as infection. Hyperammonemic coma, cerebral edema, and death may occur during 1 of these attacks. Mental development may proceed normally. Mild to moderate mental retardation, however, is common. Gallstones have been seen in the survivors; the mechanism remains unclear.

The major laboratory finding during the acute attack is hyperammonemia accompanied by marked elevations of plasma concentrations of glutamine and alanine with low levels of citrulline and arginine. Blood level of urea (BUN) is usually low. A marked increase in the urinary excretion of orotic acid differentiates this condition from CPS deficiency (see Fig. 79-13). Orotates may precipitate in urine as pink gravel. In the mild form, these laboratory abnormalities may revert to normal between attacks. This form should be differentiated from all the episodic conditions of childhood. In particular, patients with lysinuric protein intolerance (Chapter 79.13) may demonstrate some features of OTC deficiency, but the former can be differentiated by increased urinary excretion of lysine, ornithine, and arginine and elevated blood concentrations of citrulline.

The diagnosis may be confirmed by performing an assay of enzyme activity that is normally present only in the liver or by mutational analysis of the gene. Several commercial laboratories now offer sequencing of the OTC gene, although as many as 20% of affected patients demonstrate a normal sequence, perhaps because the mutation involves an intron or a leader sequence. Prenatal diagnosis has been achieved by means of fetal liver biopsy or by analysis of DNA in amniocytes or in chorionic villi samples. An oral protein load, which increases plasma ammonia and urinary orotic acid levels, may identify asymptomatic heterozygous female carriers. A marked increase in urinary excretion of orotidine after an allopurinol loading test also detects obligate female carriers. The importance of a detailed family history should be emphasized. A history of migraine or protein aversion is common in maternal female relatives of the proband. Indeed, careful scrutiny of the family history may reveal a pattern of unexplained deaths in male newborns in the maternal lineage.

Treatment of acute hyperammonemic attacks and the long-term therapy of the condition were outlined earlier. Citrulline is used in place of arginine in patients with OTC deficiency. Liver transplantation is a successful and definite treatment that has been utilized even in infants.

The gene for OTC is on Xp21.1. Many disease-causing mutations (>300) have been identified in different patients. The degree of enzyme deficiency determines severity of the phenotype in most cases. Mothers of affected infants are expected to be carriers of the mutant gene unless a de novo mutation has occurred. A mother who gave birth to 2 affected male offspring was found to have normal genotype, suggesting gonadal mosaicism in the mother.

Argininosuccinate Synthetase (AS) Deficiency (Citrullinemia) (See Fig. 79-12)

Two clinically and genetically distinct forms of citrullinemia have been identified. The classic form (type I) is due to the deficiency of the AS enzyme. The adult form (type II) is due to deficiency of a mitochondrial transport protein named citrin.

Argininosuccinate Lyase (AL) Deficiency (Argininosuccinic Aciduria) (See Fig. 79-12)

The severity of the clinical and biochemical manifestations varies considerably. In the neonatal form, signs and symptoms of severe hyperammonemia (see earlier) develop in the 1st few days of life and mortality is usually high. Survivors manifest a subacute or late form that is characterized by mental retardation, failure to thrive and hepatomegaly. Abnormalities of the hair characterized by dryness and brittleness are of special diagnostic value (trichorrhexis nodosa). Gallstones have been seen in some of the survivors. Acute attacks of severe hyperammonemia commonly occur during a catabolic state.

Laboratory findings include hyperammonemia, moderate elevations in liver enzymes, nonspecific increases in plasma levels of glutamine and alanine, moderate increase in plasma levels of citrulline (less than that seen in citrullinemia), and marked increase in plasma levels of argininosuccinic acid (see Fig. 79-13). In most amino acid analyzers, argininosuccinic acid appears as series of anhydrides within the isoleucine or methionine region, which may cause confusion in the diagnosis. Argininosuccinic acid can also be found in large amounts in urine and spinal fluid. The levels in the spinal fluid are usually higher than those in plasma. The enzyme is normally present in erythrocytes, the liver, and cultured fibroblasts. Prenatal diagnosis is possible by measurement of the enzyme activity in cultured amniotic cells or by identification of the mutant gene. Argininosuccinic acid is also elevated in the amniotic fluid of affected fetuses.

Treatment of acute hyperammonemic attacks and the long-term therapy of the condition were outlined earlier. Mental retardation, persistent hepatomegaly with mild increases in liver enzymes, and bleeding tendencies due to abnormal clotting factors are common sequelae. This deficiency is inherited as an autosomal recessive trait with a prevalence of ≈1/70,000 live births. The gene is located on chromosome 7cen-q11.2

Arginase Deficiency (Hyperargininemia) (See Fig. 79-12)

This defect is inherited as an autosomal recessive trait. There are 2 genetically distinct arginases in humans. One is cytosolic (A1) and is expressed in the liver and erythrocytes, and the other (A2) is found in the renal and brain mitochondria. The gene for cytosolic enzyme, which is the 1 deficient in patients with arginase deficiency, is mapped to chromosome 6q23. The role of the mitochondrial enzyme is not well understood; its activity increases in patients with argininemia but has no protective effect. Several disease-causing mutations have been identified in different families.

The clinical manifestations of this rare condition are quite different from those of other urea cycle enzyme defects. The onset is insidious; the infant usually remains asymptomatic in the 1st few months or, sometimes, years of life. A progressive spastic diplegia with scissoring of the lower extremities, choreoathetotic movements, and loss of developmental milestones in a previously normal infant may suggest a degenerative disease of the CNS. Two children were treated for several years as cerebral palsy before the diagnosis of arginase deficiency was confirmed. Mental retardation is progressive; seizures are common, but episodes of severe hyperammonemia are not usually seen in this disorder. Hepatomegaly may be present. The acute neonatal form with intractable seizures, cerebral edema, and death has also been reported.

Laboratory findings include marked elevations of arginine in plasma and CSF (see Fig. 79-13). Urinary orotic acid is moderately increased. Plasma ammonia levels may be normal or mildly elevated. Urinary excretions of arginine, lysine, cystine, and ornithine are usually increased, but normal levels have also been noted. Therefore, determination of amino acids in plasma is a critical step in the diagnosis of argininemia. The guanidino compounds (α-keto-guanidinovaleric acid, argininic acid) are markedly increased in urine. The diagnosis is confirmed by assaying arginase activity in erythrocytes.

Treatment consists of a low-protein diet devoid of arginine. Administration of a synthetic protein made of essential amino acids usually results in a dramatic decrease in plasma arginine concentration and an improvement in neurologic abnormalities. The composition of the diet and the daily intake of protein should be monitored by frequent plasma amino acid determinations. Sodium benzoate (250-375 mg/kg/24 hr) is also effective in controlling hyperammonemia, when present; lowering of plasma arginine levels has been noted with this treatment. Mental retardation is a common sequela of the condition. One patient developed type 1 diabetes at age 9 yr while his argininemia was under good control.

Transient Hyperammonemia of the Newborn

Blood concentration of ammonia in some healthy full-term infants may be as high as 100 µmole/L, or 2 to 3 times higher than that of the older child or adult. In premature infants the upper limit of normal for blood ammonia may be as high as 150 µmole/L. Blood levels approximate the adult normal values after a few weeks of life. These infants are asymptomatic, and follow-up studies up to 18 mo of age have not revealed any significant neurologic deficits.

Severe transient hyperammonemia has been observed in newborn infants. The majority of affected infants is premature and has mild respiratory distress syndrome. Hyperammonemic coma may develop within 2-3 days of life, and the infant may succumb to the disease if treatment is not started immediately. Laboratory studies reveal marked hyperammonemia (plasma ammonia as high as 4,000 µmole/L) with moderate increases in plasma levels of glutamine and alanine. Plasma concentrations of urea cycle intermediate amino acids are usually normal. The cause of the disorder is unknown. Urea cycle enzyme activities are normal. Treatment of hyperammonemia should be initiated promptly and continued vigorously (see above). Recovery without sequelae is common, and hyperammonemia does not recur even with a normal protein diet.

Ornithine

Ornithine is 1 of the intermediate metabolites of the urea cycle that is not incorporated into natural proteins. Rather, it is generated in the cytosol from arginine and must be transported into the mitochondria where it is used as a substrate for the enzyme OTC to form citrulline. Excess ornithine is catabolized by 2 enzymes, ornithine 5-aminotransferase, which is a mitochondrial enzyme and converts ornithine to a proline precursor, and ornithine decarboxylase, which resides in the cytosol and converts ornithine to putrescine (see Fig. 79-12). Two genetic disorders result in hyperornithinemia: gyrate atrophy of the retina and hyperammonemia-hyperornithinemia-homocitrullinemia syndrome.

Hyperammonemia-Hyperornithinemia-Homocitrullinemia (HHH) Syndrome

In this rare autosomal recessively inherited disorder, the defect is in the transport system of ornithine from the cytosol into the mitochondria, resulting in accumulation of ornithine in the cytosol and its deficiency in the mitochondria. The former causes hyperornithinemia and the latter results in disruption of the urea cycle and hyperammonemia (see Fig. 79-12). Homocitrulline is presumably formed from the reaction of mitochondrial carbamyl phosphate with lysine, which occurs because of the intramitochondrial deficiency of ornithine. Clinical manifestations of hyperammonemia may develop shortly after birth or may be delayed until adulthood. Acute episodes of hyperammonemia manifest as refusal to feed, vomiting, and lethargy; coma may occur during infancy. Progressive neurologic signs, such as lower limb weakness, increased deep tendon reflexes, spasticity, clonus, seizures, and varying degrees of psychomotor retardation may develop if the condition remains undiagnosed. No clinical ocular findings have been observed in these patients.

Laboratory findings reveal marked increases in plasma levels of ornithine and homocitrulline in addition to hyperammonemia. Restriction of protein intake improves hyperammonemia. Ornithine supplementation may produce clinical improvement in some patients. The gene for this disorder (SLC25A15) is on chromosome 13q14.

Pyridoxine (Vitamin B₆)-Dependent Epilepsy

Pyridoxal 5′-phosphate, the active form of pyridoxine, is the cofactor for many enzymes including those involved in the metabolism of neurotransmitters. Intracellular deficiency of pyridoxal 5′-phosphate in the brain may result in a seizure disorder that is responsive to high doses of pyridoxine. This pyridoxine-dependent epilepsy is seen in the following genetic metabolic conditions:

Glutaric Aciduria Type I

Glutaric acid is an intermediate in the degradation of lysine (see Fig. 79-14), hydroxylysine, and tryptophan. Glutaric aciduria type I, a disorder caused by a deficiency of glutaryl CoA dehydrogenase, should be differentiated from glutaric aciduria type II, a distinct clinical and biochemical disorder caused by defects in the electron transport system (Chapter 80.1).

Lysinuric Protein Intolerance (Familial Protein Intolerance)

This rare autosomal recessive disorder is due to a defect in the transport of the cationic amino acids lysine, ornithine, and arginine in both kidneys and intestine. Unlike patients with cystinuria, urinary excretion of cystine is not increased in these patients. About half of the reported cases have been from Finland, where the prevalence has been estimated to be 1/60,000.

Clinical manifestations consist of refusal to feed, nausea, aversion to protein, vomiting, and mild diarrhea, which may result in failure to thrive, wasting, and hypotonia. Breast-fed infants usually remain asymptomatic until shortly after weaning. This may be due to the low protein content of breast milk. Episodes of hyperammonemia may occur after ingestion of a high-protein diet. Mild to moderate hepatosplenomegaly, osteoporosis, sparse brittle hair, thin extremities with moderate centripetal adiposity, and growth retardation are common physical findings in patients whose condition has remained undiagnosed. Mental development is usually normal, but moderate mental retardation has been observed in 20% of patients. Interstitial pneumonitis manifesting with fever, fatigue, cough, and dyspnea occur as an acute episode or as a chronic progressive process. Some patients have remained undiagnosed until the appearance of pulmonary manifestations. Radiographic evidence of pulmonary fibrosis has been observed in up to 65% of patients without clinical manifestations of pulmonary involvement. Acute pulmonary proteinosis with renal involvement resembling glomerulonephritis has occurred in older patients and may cause death.

Laboratory findings may reveal hyperammonemia and an elevated concentration of urinary orotic acid, which develop only after protein feeding. Fasting blood ammonia and urinary orotic acid excretion are usually normal. Plasma concentrations of lysine, arginine, and ornithine are usually mildly decreased, but urinary levels of these amino acids, especially lysine, are greatly increased. The mechanism producing hyperammonemia is not clear. All enzymes of the urea cycle are normal. Hyperammonemia may be related to a disturbance of the urea cycle secondary to a deficiency of arginine and ornithine. However, in patients with cystinuria who also have defects in the transport of lysine, arginine, and ornithine in both intestine and kidneys, hyperammonemia is not observed. Plasma concentrations of alanine, glutamine, serine, glycine, proline, and citrulline are usually increased. These abnormalities may be secondary to hyperammonemia and are not specific to this disorder.

Mild anemia and increased serum levels of ferritin, lactic dehydrogenase (LDH), and thyroxine-binding globulin have also been observed in these patients. This condition should be differentiated from hyperammonemia due to urea cycle defects (Chapter 79.11), especially in heterozygous females with OTC deficiency. Increased urinary excretion of lysine, ornithine, and arginine and elevated blood levels of citrulline are not seen in patients with OTC deficiency.

The transport defect in this condition resides in the basolateral (antiluminal) membrane of enterocytes and renal tubular epithelia. This explains the observation that cationic amino acids are unable to cross these cells even when administered as dipeptides. Lysine in the form of dipeptide crosses the luminal membrane of the enterocytes but hydrolyzes to free lysine molecules in the cytoplasm. Free lysine, unable to cross the basolateral membrane of the cells, diffuses back into the lumen.

Treatment with a low-protein diet (1.0-1.5 g/kg/24 hr) supplemented with citrulline (3-8 g/day) has produced biochemical and clinical improvements. Episodes of hyperammonemia should be treated promptly (Chapter 79.12). Supplementation with lysine is not useful because it is poorly absorbed and tends to produce diarrhea and abdominal pain. Treatment with high doses of prednisone and bronchoalveolar lavage has been effective in the management of acute pulmonary complications.

The gene for lysinuric protein intolerance (SLC7A7) is mapped to chromosome 14q11.2 and several disease-causing mutations have been identified in different families. Pregnancies in affected mothers have been complicated by anemia, thrombocytopenia, toxemia, and bleeding, but offspring have been normal.

Canavan Disease

Canavan disease, an autosomal recessive disorder characterized by spongy degeneration of the white matter of the brain, leads to a severe form of leukodystrophy. It is more prevalent in individuals of Ashkenazi Jewish descent than in other ethnic groups.

Diagnosis

In a typical patient with Canavan disease, CT scan and MRI reveal diffuse white matter degeneration, primarily in the cerebral hemispheres, with less involvement of the cerebellum and brainstem (Fig. 79-15). Repeated evaluations may be required. MRS performed at the time MRI is done can show the high peak of N-acetylaspartic acid, suggesting Canavan disease. The differential diagnosis of Canavan disease should include Alexander disease, which is another leukodystrophy associated with macrocephaly. Progression is usually slower in Alexander disease; hypotonia is not as pronounced as it is in Canavan disease. Brain biopsies of patients with Canavan disease show spongy degeneration of the myelin fibers, astrocytic swelling, and elongated mitochondria. Definitive diagnosis can be established by finding elevated amounts of N-acetylaspartic acid in the urine or blood. A deficiency of aspartoacylase can be found in cultured skin fibroblasts. The biochemical method is the preferred choice for diagnosis. N-acetylaspartic acid is found only in trace amounts (24 ± 16 µmol/mmol creatinine) in the urine of unaffected individuals, whereas in patients with Canavan disease its concentration is in the range of 1,440 ± 873 µmol/mmol creatinine. High levels of N-acetylaspartic acid can also be detected in plasma, CSF, and brain tissue. The activity of aspartoacylase in the fibroblasts of obligate carriers is less than or equal to half of the activity found in normal individuals.

Figure 79-15 Axial T weighted MRI of a 2 yr old patient with Canavan disease. Extensive thickening of the white matter is seen.

The gene for aspartoacylase has been cloned, and mutations leading to Canavan disease have been identified. There are 2 mutations predominant in the Ashkenazi Jewish population. The 1st is an amino acid substitution (E285A) in which glutamic acid is substituted for alanine. This mutation is the most frequent and encompasses 83% of 100 mutant alleles examined in Ashkenazi Jewish patients. The second common mutation is a change from tyrosine to a nonsense mutation, leading to a stop in the coding sequence (Y231X). This mutation accounts for 13% of the 100 mutant alleles. In the non-Jewish population, more diverse mutations have been observed, and the 2 mutations common in Jewish people are rare. A different mutation (A305E), substitution of alanine for glutamic acid, accounts for 40% of 62 mutant alleles in non-Jewish patients. There have been more than 50 mutations described in the non-Jewish population. With Canavan disease, it is important to obtain a molecular diagnosis because this will lead to accurate counseling and prenatal guidance for the family. If the mutations are not known, prenatal diagnosis relies on the level of N-acetylaspartic acid in the amniotic fluid. In Ashkenazi Jewish patients, the carrier frequency can be as high as 1 : 36, which is close to that of Tay-Sachs disease. Carrier screening for Canavan disease is now in practice for Jewish individuals.