103.9

Proline

Oleg A. Shchelochkov, Charles P. Venditti

Keywords

• proline
• hydroxyproline
• hyperprolinemia
• hyperprolinemia type 1
• proline oxidase
• PRODH
• hyperprolinemia type 2
• aldehyde dehydrogenase 4
• ALDH4A1
• pyrroline-5-carboxylate
• P5C
• prolidase
• prolidase deficiency
• PEPD
• De novo proline synthesis
• pyrroline-5-carboxylate synthase
• P5C synthase
• ALDH18A1
• pyrroline-5-carboxylate reductase
• PYCR1
• de Barsy syndrome

Proline is a nonessential amino acid synthesized endogenously from glutamic acid, ornithine, and arginine (see Fig. 103.9 ). Proline and hydroxyproline are found in high concentrations in collagen. Normally, neither of these amino acids is found in large quantities in urine. Excretion of proline and hydroxyproline as iminopeptides (dipeptides and tripeptides containing proline or hydroxyproline) is increased in disorders of accelerated collagen turnover, such as rickets or hyperparathyroidism. Proline is also found in synapses, where it can interact with glycine and glutamate receptors (see Chapter 103.11 ). The catabolic pathway of proline and hydroxyproline produces glyoxylic acid, which can be further metabolized to glycine or oxalic acid (see Fig. 103.8 ).

Accumulation of proline in tissues is associated with disorders of hyperprolinemia type 1 and hyperprolinemia type 2. Reduced de novo synthesis of proline causes syndromes manifesting with cutis laxa (see Fig. 678.8 ) with progeroid features or spastic paraplegia . Two types of primary hyperprolinemia have been described.

Hyperprolinemia Type I

This rare autosomal recessive condition is caused by deficiency of proline oxidase (proline dehydrogenase; see Fig. 103.9 ). Most patients with hyperprolinemia type 1 appear asymptomatic, although some may present with intellectual disability, seizures, and behavioral problems. Hyperprolinemia may also be a risk factor for autism spectrum disorders and schizophrenia. The nature of such wide phenotypic range in this biochemical condition has not been elucidated. The gene encoding proline oxidase (PRODH) is mapped to 22q11.2 and is located within the critical region for the velocardiofacial syndrome . Laboratory studies reveal high concentrations of proline in plasma, urine, and CSF. Increased urinary excretion of hydroxyproline and glycine is also present, which could be related to saturation of the shared tubular reabsorption mechanism due to massive prolinuria.

No effective treatment has yet emerged. Restriction of dietary proline causes modest improvement in plasma proline with no proven clinical benefit.

Hyperprolinemia Type II

This is a rare autosomal recessive condition caused by the deficiency of Δ¹ -pyrroline-5-carboxylate dehydrogenase (aldehyde dehydrogenase 4; see Fig. 103.9 ). Intellectual disability and seizures (usually precipitated by an intercurrent infection) have been reported in affected children, but asymptomatic patients have also been described. The cause for such disparate clinical outcomes is incompletely understood. The gene encoding P5C dehydrogenase (ALDH4A1) is mapped to chromosome 1p36.13.

Laboratory studies reveal increased concentrations of proline and Δ¹ -pyrroline-5-carboxylic acid (P5C) in blood, urine, and CSF. The presence of P5C differentiates this condition from hyperprolinemia type I. Increased level of P5C in body fluids, especially in the CNS, appears to antagonize vitamin B₆ and lead to vitamin B₆ dependency (see Chapter 103.14 ). Vitamin B₆ dependency may be the main cause of seizures and neurologic findings in this condition and may explain the variability in clinical manifestations in different patients. Treatment with high doses of vitamin B₆ is recommended.

Prolidase Deficiency

During collagen degradation, imidodipeptides are formed and are normally cleaved by tissue prolidase. Deficiency of prolidase, which is inherited as an autosomal recessive trait, results in the accumulation of imidodipeptides in body fluids. Age at onset varies from 6 mo to the 3rd decade of life.

The clinical manifestations of this rare condition also vary and include recurrent, severe, and painful skin ulcers, which are typically on hands and legs. Other skin lesions that may precede ulcers by several years may include a scaly erythematous maculopapular rash, purpura, and telangiectasia. Most ulcers become infected. Healing of the ulcers may take months. Other findings include developmental delays, intellectual disability, organomegaly, anemia, thrombocytopenia, and immune dysfunction resulting in increased susceptibility to infections (recurrent otitis media, sinusitis, respiratory infection, splenomegaly). Some patients may have craniofacial abnormalities such as ptosis, ocular proptosis, hypertelorism, small beaked nose, and prominent cranial sutures. Asymptomatic cases have also been reported. Increased incidence of systemic lupus erythematosus has been noted in children. High levels of urinary excretion of imidodipeptides are diagnostic. The gene for prolidase (PEPD) has been mapped to chromosome 19q13.11. The diagnosis can be confirmed using DNA analysis. Enzyme assay may be performed in erythrocytes or cultured skin fibroblasts.

Treatment of prolidase deficiency is supportive. Infectious complications can be fatal and warrant close and proactive antibiotic management. Oral supplementation with proline, ascorbic acid, and manganese and topical proline and glycine have not been found to be consistently effective in all patients.

Disorders of De Novo Proline Synthesis

De novo synthesis of proline and ornithine from glutamate appears to be critical in the normal biology of connective tissue and to maintain urea cycle in a repleted state. Correspondingly, clinical manifestations of these disorders encompass connective tissue abnormalities, nervous system abnormalities, and variable biochemical abnormalities reflecting urea cycle dysfunction. This section summarizes clinical and laboratory findings associated with the deficient function of Δ¹ -pyrroline-5-carboxylate (P5C) synthase (see Fig. 103.9 ) encoded by ALDH18A1 (mapped to 10q24.1) and PSC reductase encoded by PYCR1 (mapped to 17q25.3).

Deficient activity of P5C synthase has been associated with several phenotypes, including de Barsy syndrome , characterized by cataracts, growth retardation, intellectual disability, a prematurely aged appearance (progeroid features), and cutis laxa. Some patients may show pyramidal signs. Skin biopsy may reveal decreased size of elastic fibers and collagen abnormalities. Brain imaging studies show cortical atrophy, ventriculomegaly, and reduced creatine. Laboratory findings include reduced levels of proline, ornithine, citrulline, and arginine as well as mild fasting hyperammonemia. Patients may show only intermittent abnormalities of plasma amino acids, likely related to the time of blood sampling in relation to the last meal. Interestingly, both autosomal recessive and autosomal dominant forms of inheritance have been described. The diagnosis can be suspected in a patient presenting with cutis laxa, developmental delay, mild hyperammonemia, and amino acid abnormalities. The diagnosis can be confirmed using molecular DNA analysis or using the glutamine loading test on skin fibroblasts. Treatment is supportive, although supplementation with citrulline or arginine to address hyperammonemia and cerebral creatine depletion have been proposed.

Deleterious mutations in PYCR1 result in the abnormal function of the mitochondrial Δ¹ -pyrroline-5-carboxylate reductase, which catalyzes the last step in the synthesis of proline from P5C. The most consistent finding in patients carrying proven pathogenic variants in PYCR1 include triangular facies, cutis laxa (de Barsy–like syndrome ), joint hypermobility, wrinkled skin, gerodermia osteodysplastica, and progeroid features. Skin biopsy reveals reduction of the elastic fibers and infiltration with inflammatory cells. Some patients may have epilepsy, developmental delays, intellectual disability, cataracts, osteopenia, and failure to thrive. However, many of the affected families are consanguineous, thus confounding the phenotype. Of note, plasma amino acid analysis typically reveals no specific abnormalities. The diagnosis depends on the recognition of the skin findings and can be confirmed using molecular DNA analysis. Available pedigrees of families affected by PYCR1 -related disorder supports the autosomal recessive mode of inheritance.

Bibliography

Coutelier M, Goizet C, Durr A, et al. Alteration of ornithine metabolism leads to dominant and recessive hereditary spastic paraplegia. Brain . 2015;138(Pt 8):2191–2205.

Dimopoulou A, Fischer B, Gardeitchik T, et al. Genotype-phenotype spectrum of PYCR1-related autosomal recessive cutis laxa. Mol Genet Metab . 2013;110(3):352–361.

Ferreira C, Wang H. Prolidase Deficiency. 2015 Jun 25. Adam MP, Ardinger HH, Pagon RA, et al. GeneReviews [Internet] . University of Washington, Seattle: Seattle (WA); 1993-2017 [Available from] https://www.ncbi.nlm.nih.gov/books/NBK299584/ .

Kurien BT, D'Sousa A, Bruner BF, et al. Prolidase deficiency breaks tolerance to lupus-associated antigens. Int J Rheum Dis . 2013;16:674–680.

Martinelli D, Häberle J, Rubio V, et al. Understanding pyrroline-5-carboxylate synthetase deficiency: clinical, molecular, functional, and expression studies, structure-based analysis, and novel therapy with arginine. J Inherit Metab Dis . 2012;35(5):761–776.

Mitsubuchi H, Nakamura K, Matsumoto S, et al. Inborn errors of proline metabolism. J Nutr . 2008;138:2016S–2020S.

Raux G, Bunsel E, Hecketsweiler B, et al. Involvement of hyperprolinemia in cognitive and psychiatric features of the 22q11 deletion syndrome. Hum Mol Genet . 2007;16:83–91.

103.10

Glutamic Acid

Oleg A. Shchelochkov, Charles P. Venditti

Keywords

• glutamic acid
• glutamate
• γ-glutamylcysteinylglycine
• glutathione
• γ-glutamyl cycle
• glutathione synthetase
• GSSD
• 5-oxoprolinase
• OPLAH
• glutamate-cysteine ligase
• γ-glutamylcysteine synthetase
• GCLC
• γ-glutamyl transpeptidase
• GGT
• glutathionemia
• hemolytic anemia
• 5-oxoproline
• 5-oxoprolinemia
• high–anion gap metabolic acidosis
• 5-oxoprolinuria
• secondary 5-oxoprolinuria

Glutamic acid and its amide derivative glutamine have a wide range of functions in the body. Glutamate plays numerous biologic roles, functioning as a neurotransmitter, an intermediate compound in many fundamental biochemical reactions, and a precursor of an inhibitory neurotransmitter γ-aminobutyric acid (GABA) (see Chapter 103.11 ). Another major product of glutamate is glutathione (γ-glutamylcysteinylglycine). This ubiquitous tripeptide, with its function as the major antioxidant in the body, is synthesized and degraded through a complex cycle called the γ-glutamyl cycle (Fig. 103.11 ). Because of its free sulfhydryl (–SH) group and its abundance in the cell, glutathione protects other sulfhydryl-containing compounds (e.g., enzymes, coenzyme A) from oxidation. It is also involved in the detoxification of peroxides, including hydrogen peroxide, and in keeping the intracellular milieu in a reduced state. In addition, glutathione participates in amino acid transport across the cell membrane through the γ-glutamyl cycle.

Fig. 103.11 The γ-glutamyl cycle and related pathways. Defects of the glutathione (GSH) synthesis and degradation are noted. Enzymes: (1) γ-Glutamyl transpeptidase (GGT), (2) γ-glutamyl cyclotransferase, (3) 5-oxoprolinase, (4) γ-glutamyl-cysteine synthetase, (5) glutathione synthetase, (6) glutamate decarboxylase, (7) γ-aminobutyric acid (GABA) transaminase, (8) succinate-semialdehyde dehydrogenase, (9) glutamine synthetase, (10) dipeptidase.

One of the biochemical manifestations of γ-glutamyl cycle deficiency is increased urinary excretion of 5-oxoproline, which could be the result of both genetic and non-genetic causes. 5-Oxoprolinemia should be routinely considered in the differential diagnosis of high–anion gap metabolic acidosis (HAGMA). Two metabolic disorders can present with massive 5-oxoprolinuria: glutathione synthetase deficiency and 5-oxoprolinase deficiency (Fig. 103.11 ). However, a more common clinical scenario is a transient and mild urinary elevation of 5-oxoproline in urine that can be seen in a variety of metabolic and acquired conditions, such as exposure to acetaminophen and some hydrolyzed-protein formulas, severe burns, Stevens-Johnson syndrome, homocystinuria, urea cycle defects, and tyrosinemia type I.

Glutathione Synthetase Deficiency

Three forms of this rare condition have been reported. In the mild form , enzyme deficiency causes glutathione deficiency only in erythrocytes. These patients present with hemolytic anemia without chronic metabolic acidosis and demonstrate high residual activity of glutathione synthetase on enzymatic testing. A moderate form has also been observed in which the hemolytic anemia is associated with variable degrees of metabolic acidosis and 5-oxoprolinuria. Its severe form is distinguished by presence of hemolytic anemia accompanied by severe acidosis, massive 5-oxoprolinuria, and neurologic manifestations.

Glutathione Synthetase Deficiency, Severe and Moderate Forms

Affected newborn infants with this rare condition usually develop acute symptoms of metabolic acidosis, jaundice, and mild to moderate hemolytic anemia in the 1st few days of life. Chronic acidosis continues after recovery. Similar episodes of life-threatening acidosis may occur during an infection (e.g., gastroenteritis) or after a surgical procedure. Progressive neurologic damage develops with age, manifested by intellectual disability, spastic tetraparesis, ataxia, tremor, dysarthria, and seizures. Susceptibility to infections, presumably because of granulocyte dysfunction, is observed in some patients. Patients with the moderate form of glutathione synthetase deficiency have milder acidosis and less 5-oxoprolinuria than is seen in the severe form, with no neurologic manifestations.

Laboratory findings include metabolic acidosis, mild to moderate degrees of hemolytic anemia, and 5-oxoprolinuria. High concentrations of 5-oxoproline are also found in blood. The urinary and blood levels of 5-oxoproline is less pronounced in patients with moderate form of the condition. The glutathione content of erythrocytes is markedly decreased. Increased synthesis of 5-oxoproline in this disorder is thought to be the result of the conversion of γ-glutamylcysteine to 5-oxoproline by the enzyme γ-glutamyl cyclotransferase (see Fig. 103.11 ). γ-Glutamylcysteine production increases greatly because the normal inhibitory effect of glutathione on the γ-glutamylcysteine synthetase enzyme is removed.

Treatment of acute attack includes hydration, correction of acidosis (by infusion of sodium bicarbonate), and measures to correct anemia and hyperbilirubinemia. Chronic administration of alkali is usually needed indefinitely. Supplementation with vitamin C, vitamin E, and selenium is recommended. Drugs and oxidants known to cause hemolysis and stressful catabolic states should be avoided. Oral administration of glutathione analogs has been tried with variable success.

Prenatal diagnosis can be achieved by the measurement of 5-oxoproline in amniotic fluid, by enzyme analysis in cultured amniocytes or chronic villus samples, or by DNA analysis. Successful pregnancy in an affected female (moderate form) has been reported, with favorable outcomes for both mother and infant.

Glutathione Synthetase Deficiency, Mild Form

The mild form has been reported in only a few patients. Mild to moderate hemolytic anemia has been the only clinical finding. Splenomegaly has been reported in some patients. Cognitive development is normal. Chronic metabolic acidosis typically is not seen. Some patients can have increased concentrations of 5-oxoproline in the urine. Pathogenic variants in the gene for this enzyme (GSSD) appear to decrease the half-life of the enzyme, which causes an increased rate of protein turnover without affecting its catalytic function. The expedited rate of enzyme turnover caused by these pathogenic variants is of little or no consequence for tissues with protein synthetic capability. However, inability of mature erythrocytes to synthesize protein results in glutathione deficiency in the erythrocytes. Treatment is that of hemolytic anemia and avoidance of drugs and oxidants that can trigger the hemolytic process.

All forms of glutathione synthetase deficiency are inherited as an autosomal recessive trait. GSSD is located on chromosome 20q11.22. Diagnosis can be confirmed by DNA analysis or enzyme activity in erythrocytes or skin fibroblasts.

5-Oxoprolinase Deficiency

More than 20 patients with 5-oxoprolinuria (4-10 g/day) caused by 5-oxoprolinase (see Fig. 103.11 ) deficiency have been described. No specific clinical picture has yet emerged; completely asymptomatic affected individuals have also been identified. It is therefore not clear whether 5-oxoprolinase deficiency is of any clinical consequence. No treatment is currently recommended. The gene for the enzyme (OPLAH) is on chromosome 8q24.3.

γ-Glutamylcysteine Synthetase Deficiency (Glutamate-Cysteine Ligase Deficiency)

Only a few patients with this enzyme deficiency have been reported. The most consistent clinical manifestation has been mild chronic hemolytic anemia. Acute attacks of hemolysis have occurred after exposure to sulfonamides. Peripheral neuropathy and progressive spinocerebellar degeneration have been noted in 2 siblings in adulthood. Laboratory findings of chronic hemolytic anemia were present in all patients. Generalized aminoaciduria is also present because the γ-glutamyl cycle is involved in amino acid transport in cells (see Fig. 103.11 ). Treatment should focus on the management of hemolytic anemia and avoidance of drugs and oxidants that may trigger the hemolytic process. The condition is inherited as an autosomal recessive trait; the gene (GCLC) is mapped to chromosome 6p12.1.

γ-Glutamyl Transpeptidase Deficiency (Glutathionemia)

γ-Glutamyl transpeptidase (GGT) 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 GGT in the blood is frequently performed to evaluate liver and bile duct diseases.

GGT deficiency causes elevation in glutathione concentrations in body fluids, but the cellular levels remain normal (see Fig. 103.11 ). Because only a few patients with GGT deficiency have been reported, the scope of clinical manifestations has not yet been defined. Mild to moderate intellectual disability and severe behavioral problems were observed in 3 patients. However, 1 of 2 sisters with this condition had normal intelligence as an adult, 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. 103.11 ).

Diagnosis can be confirmed by measurement of the enzyme activity in leukocytes or cultured skin fibroblasts. No effective treatment has been proposed. The condition is inherited as an apparent autosomal recessive trait. γ-Glutamyl transpeptidases represent a large family of enzymes encoded by at least 7 genes.

Genetic Disorders of Metabolism of γ-Aminobutyric Acid

See Chapter 103.11 .

Bibliography

Almusafri F, Elamin HE, Khalaf TE, et al. Clinical and molecular characterization of 6 children with glutamate-cysteine ligase deficiency causing hemolytic anemia. Blood Cells Mol Dis . 2017;65:73–77.

Ristoff E, Mayatepek E, Larsson A. Long-term clinical outcome in patients with glutathione synthetase deficiency. J Pediatr . 2001;139(1):79–84.

Sass JO, Gemperle-Britschgi C, Tarailo-Graovac M, et al. Unravelling 5-oxoprolinuria (pyroglutamic aciduria) due to bi-allelic OPLAH mutations: 20 new mutations in 14 families. Mol Genet Metab . 2016;119(1–2):44–49.

103.11

Genetic Disorders of Neurotransmitters

Oleg A. Shchelochkov, Charles P. Venditti

Keywords

• neurotransmitters
• tyrosine hydroxylase deficiency
• autosomal recessive Segawa syndrome
• aromatic L -amino acid decarboxylase deficiency
• AADC
• GTP cyclohydrolase 1 deficiency
• GCH1
• autosomal dominant Segawa syndrome
• BH₄ deficiency
• hyperphenylalaninemia
• sepiapterin reductase deficiency
• SPR
• dopamine β-hydroxylase deficiency
• DBH
• monoamine oxidase deficiency
• monoamine oxidase A deficiency
• MAOA
• MAOB
• γ-aminobutyric acid transaminase deficiency
• ABAT
• succinic semialdehyde dehydrogenase deficiency
• γ-hydroxybutyric aciduria
• SSADH
• ALDH5A1
• dopamine transporter protein deficiency
• SLC6A3
• dopamine-serotonin vesicular transporter disease
• vesicular monoamine transporter deficiency
• SLC18A2
• vesicular monoamine transporter 2
• VMAT2
• hyperprolinemia

Neurotransmitters are chemical substances released from the axonal end of excited neurons at the synaptic junctions; they mediate initiation and amplification or inhibition of neural impulses. A number of amino acids and their metabolites comprise the bulk of neurotransmitters. Pathogenic variants in genes responsible for the synthesis, transport, or degradation of these substances may cause conditions that manifest neurologic and/or psychiatric abnormalities (Table 103.3 ). Previously, children affected by disorders of neurotransmitters have been given syndromic diagnoses such as cerebral palsy, epilepsy, parkinsonism, dystonia, or autism. Diagnosis, in most cases, requires specialized laboratory studies of the cerebrospinal fluid (CSF), because some of the neurotransmitters generated in the central nervous system (CNS), dopamine and serotonin, do not cross the blood-brain barrier, and their abnormal concentrations are not detected in the serum or urine. A growing number of these conditions are being identified; diseases once thought to be rare are now diagnosed with increasing frequency.

Table 103.3

Genetic Disorders of Neurotransmitters in Children

TRANSMITTER	SYNTHESIS DEFECTS	DEGRADATION DEFECTS
MONOAMINES
Dopamine	TH deficiency	MAO deficiency
Serotonin and dopamine	AADC deficiency	MAO deficiency
	BH4 deficiency With and without hyperphenylalaninemia
Norepinephrine	DβH deficiency	MAO deficiency
GABA	?	GABA transaminase deficiency GHB aciduria
Histamine	HDC deficiency	?
TRANSPORTER PROTEINS
Dopamine transporter	DAT deficiency	?
Vesicular monoamine transporter	VMAT2 deficiency	?
AMINO ACIDS
Proline	?	Hyperprolinemia
Serine	3-PGD, PSAT, PSPH deficiencies	?
Glycine	3-PDG, PSAT deficiencies	NKH

AADC, Aromatic L -amino acid decarboxylase; BH₄ , tetrahydrobiopterin; DAT, dopamine transporter; DβH, dopamine β-hydroxylase; GABA, γ-aminobutyric acid; GHB, γ-hydroxybutyric acid; HDC, histidine decarboxylase; hyperphe, hyperphenylalaninemia; MAO, monoamine oxidase; NKH, nonketotic hyperglycinemia; 3-PGD, 3-phosphoglycerate dehydrogenase; PSAT, phosphoserine aminotransferase; PSPH, 3-Phosphoserine Phosphatase Deficiency; TH, tyrosine hydroxylase; VMAT2, vesicular monoamine transporter 2.

Tyrosine Hydroxylase Deficiency (Infantile Parkinsonism, Autosomal Recessive Dopa-Responsive Dystonia, Autosomal Recessive Segawa Syndrome)

Tyrosine hydroxylase catalyzes the formation of L -dopa from tyrosine. Deficiency of this enzyme results in deficiencies of dopamine and norepinephrine (see Fig. 103.2 ). The differential diagnosis includes a wide range of inherited dystonias, including autosomal dominant dystonia caused by GTP cyclohydrolase 1 deficiency.

Clinical manifestations range from mild to very severe. In general, 2 phenotypes have been recognized. In the mild form (dopa-responsive dystonia, or type A ), symptoms of unilateral limb dystonia causing gait incoordination and postural tremor occur in childhood and worsen with age if the condition remains untreated. Diurnal variation of symptoms (worse at the end of the day) may be present. Cognitive development is usually normal.

In the severe form of tyrosine hydroxylase deficiency (infantile parkinsonism, infantile encephalopathy, or type B ), the clinical manifestations occur at birth or shortly thereafter and include microcephaly, developmental delay, involuntary movements of the limbs with spasticity, dystonia, ptosis, expressionless face, oculogyric crises (upward eye-rolling movements), and autonomic dysfunction (temperature instability, excessive sweating, hypoglycemia, salivation, tremor, gastrointestinal reflux, constipation). Brisk reflexes, myoclonus, athetosis, and distal chorea may be present. The patient with the severe form usually shows incomplete response to treatment with L -dopa and is prone to developing L -dopa–induced dyskinesia as a side effect.

Laboratory findings include reduced levels of dopamine and its metabolite homovanillic acid (HVA) and normal concentrations of tetrahydrobiopterin (BH₄ ), neopterin, and 5-hydroxyindoleacetic acid (5-HIAA, a metabolite of serotonin) in the CSF. Serum prolactin levels are usually elevated. These findings are not diagnostic of the condition; diagnosis should be established by molecular gene analysis.

Treatment with L -dopa/carbidopa results in significant clinical improvement in most patients, but the severe forms are invariably associated with L -dopa–induced dyskinesias. To minimize the side effects of therapy, the treatment should be started with a low dose and increased very slowly, if needed. Other therapeutic interventions include anticholinergics, serotonergic agents, and monoamine oxidase (MAO) B inhibitors, including amantadine, biperiden, and selegiline. Bilateral subthalamic nucleus deep brain stimulation has shown clinical efficacy in one case. Tyrosine hydroxylase deficiency is inherited as an autosomal-recessive trait. Molecular testing for pathogenic variants in the TH gene is available clinically.

Aromatic l-Amino Acid Decarboxylase Deficiency

Aromatic L -amino acid decarboxylase (AADC) is a vitamin B₆ –dependent enzyme that catalyzes the decarboxylation of both 5-hydroxytryptophan to form serotonin (see Fig. 103.5 ) and L -dopa to generate dopamine, (see Fig. 103.2 ). Clinical manifestations are related to reduced availability of dopamine and serotonin. Poor feeding, lethargy, hypotension, hypothermia, oculogyric crises, and ptosis have been observed in affected neonates. Clinical findings in infants and older children include developmental delay, truncal hypotonia with hypertonia of limbs, oculogyric crises, extrapyramidal movements (choreoathetosis, dystonia, myoclonus), and autonomic abnormalities (sweating, salivation, irritability, temperature instability, hypotension). Symptoms may have a diurnal variation, becoming worse by the end of the day.

Laboratory findings include decreased concentrations of dopamine and serotonin and their metabolites (HVA, 5-HIAA, norepinephrine, vanillylmandelic acid [VMA]) and increased levels of 5-hydroxytryptophan, L -dopa, and its metabolite (3-O -methyldopa) in body fluids, especially in CSF. Elevated serum concentrations of prolactin (a result of dopamine deficiency) have also been observed. MRI of the brain reveals cerebral atrophy with degenerative changes in the white matter. A urine screening program, focused on 3-O -methyl-dopa and VMA, has demonstrated diagnostic promise in high-disease prevalence populations.

Treatment with neurotransmitter precursors has produced limited clinical improvement. Dopamine and serotonin have no therapeutic value because of their inability to cross the blood-brain barrier. Dopamine agonists (L -dopa/carbidopa, bromocriptine), MAO inhibitors (tranylcypromine), serotonergic agents and high doses of pyridoxine, a cofactor for AADC enzyme, have been tried. Pyridoxine supplementation in patients harboring the p.S250F variant in AADC may be beneficial. The recent demonstration of CNS-directed gene therapy with an adeno-associated viral vector has shown benefit in some patients. Preimplantation genetic diagnosis after in vitro fertilization has been achieved in the high-prevalence Taiwanese population. The gene encoding AADC (DDC ) is on chromosome 7p12.1. The condition is inherited as an autosomal recessive trait.

Tetrahydrobiopterin Deficiency

See Chapter 103.1 .

BH₄ is the cofactor for phenylalanine hydroxylase (see Fig. 103.1 ), tyrosine hydroxylase (see Fig. 103.2 ), tryptophan hydroxylase (see Fig. 103.5 ), and nitric oxide synthase. It is synthesized from GTP in many tissues (see Fig. 103.1 ). Deficiencies of enzymes involved in the biosynthesis of BH₄ result in inadequate production of this cofactor, which causes deficiencies of monoamine neurotransmitters with or without concomitant hyperphenylalaninemia.

Tetrahydrobiopterin Deficiency With Hyperphenylalaninemia

See Chapter 103.1 .

Tetrahydrobiopterin Deficiency Without Hyperphenylalaninemia

GTP Cyclohydrolase 1 Deficiency (Hereditary Progressive Dystonia, Autosomal Dominant Dopa-Responsive Dystonia, Autosomal Dominant Segawa Syndrome)

This form of dystonia, caused by guanosine triphosphate (GTP) cyclohydrolase 1 deficiency, is inherited as an autosomal dominant trait and is more common in females than males (4 : 1 ratio) (see Chapter 615.4 ). Clinical manifestations usually start in early childhood with tremor and dystonia of the lower limbs (toe gait ), which may spread to all extremities within a few years. Torticollis, dystonia of the arms, and poor coordination may precede dystonia of the lower limbs. Early development is generally normal. Symptoms have an impressive diurnal variation, becoming worse by the end of the day and improving with sleep. Autonomic instability is not uncommon. Parkinsonism may also be present or develop with advancing age. Late presentation in adult life has also been reported, associated with action dystonia (“writer's cramp”), torticollis, or generalized rigid hypertonia with tremor but without postural dystonia. Additionally, limited data on adults suggest symptoms related to serotonin deficiency (sleep disturbance, cognitive impairment, impulsivity).

Laboratory findings show reduced levels of BH₄ and neopterin in the CSF without hyperphenylalaninemia (see Chapter 103.1 ). Dopamine and its metabolite (HVA) may also be reduced in CSF. The serotonergic pathway is less affected by this enzyme deficiency; thus concentrations of serotonin and its metabolites are usually normal. Plasma phenylalanine is normal, but an oral phenylalanine loading test (100 mg/kg) produces an abnormally high plasma phenylalanine level with an elevated phenylalanine/tyrosine ratio. The ratio, obtained at the 2-3 hr after the load, in combination with urine neopterin level, has optimal diagnostic specificity and sensitivity. The existence of asymptomatic carriers indicates that other factors or genes may play a role in pathogenesis. The asymptomatic carrier may be identified by the phenylalanine loading test. Diagnosis may be confirmed by reduced levels of BH₄ and neopterin in CSF, measurement of the enzyme activity, and molecular genetic analysis (see Chapter 103.1 ). Clinically, the condition should be differentiated from other causes of dystonias and childhood parkinsonism, especially tyrosine hydroxylase, sepiapterin reductase, and aromatic amino acid decarboxylase deficiencies.

Treatment with L -dopa/carbidopa usually produces dramatic clinical improvement. Oral administration of BH₄ is also effective but is rarely used. The gene for GTP cyclohydroxylase 1 (GCH1 ) is located on chromosome 14q22.2.

Sepiapterin Reductase Deficiency

Sepiapterin reductase is involved in conversion of 6-pyruvoyl-tetrahydropterin to BH₄ . It also participates in the salvage pathway of BH₄ synthesis (see Fig. 103.1 ). Sepiapterin reductase deficiency results in accumulation of 6-lactoyl-tetrahydropterin, which can be converted to sepiapterin nonenzymatically. The majority of sepiapterin is metabolized to BH₄ through the salvage pathway in peripheral tissues (see Fig. 103.1 ), but because of the low activity of dihydrofolate reductase in brain, the amount of BH₄ remains insufficient for proper synthesis of dopamine and serotonin. This explains the absence of hyperphenylalaninemia and the often-delayed diagnosis.

Clinical manifestations usually appear within a few months of life. Cardinal manifestations include paroxysmal stiffening, oculogyric crises, and hypotonia. Additional findings include motor and language delays, weakness, limb hypertonia, dystonia, hyperreflexia, and early-onset parkinsonism. The symptoms usually have a diurnal variation. Misdiagnosis as cerebral palsy is common and a wide variability of symptoms have been reported. Diagnosis is established by measurement of CSF neurotransmitters and pterin metabolites, which reveal decreased dopamine, HVA, norepinephrine, and 5-HIAA and marked elevations of sepiapterin and dihydrobiopterin. The serum concentration of prolactin may be elevated. The phenylalanine loading test may have diagnostic utility. Diagnosis may be confirmed by molecular genetic analysis or enzyme assay in fibroblasts. Treatment with slowly increasing doses of L-dopa/carbidopa and 5-hydroxytryptophan usually produces dramatic clinical improvement. The condition is inherited as an autosomal recessive trait; the gene SPR encoding sepiapterin reductase is located on chromosome 2p13.2.

Dopamine β-Hydroxylase Deficiency

Dopamine β-hydroxylase catalyzes the conversion of dopamine to norepinephrine (see Fig. 103.2 ). The deficiency of this enzyme results in reduced or absent synthesis of norepinephrine, leading to dysregulation of the sympathetic function. Infants and children may present with difficulty opening eyes, ptosis, hypotension, hypothermia, hypoglycemia, and nasal stuffiness. Adult patients may present with profound deficits of autonomic regulation, resulting in severe orthostatic hypotension, and sexual dysfunction in males. Presyncopal symptomatology includes dizziness, blurred vision, dyspnea, nuchal discomfort, and chest pain; olfactory function remains relatively intact. The diagnosis can be aided by performing autonomic function testing (measurement of the sinus arrhythmia ratio, blood pressure studies during controlled hyperventilation, Valsalva maneuver, cold pressor, handgrip exercise). Laboratory findings include decreased or absent norepinephrine and epinephrine and their metabolites, with elevated levels of dopamine and its metabolite (HVA), in plasma, CSF, and urine. Elevated plasma dopamine may be pathognomonic for this disease. MRI of the brain shows decreased brain volume, consistent with the neurotrophic role of norepinephrine. Treatment with L -dihydroxyphenylserine, which is converted to norepinephrine directly in vivo by the action of AADC, leads to significant improvement in orthostatic hypotension and normalizes noradrenaline and its metabolites. The condition is inherited as an autosomal recessive trait; the gene (DBH) encoding dopamine β-hydroxylase resides on chromosome 9q34.2.

Monoamine Oxidase a Deficiency

Human genome encodes 2 monoamine oxidase (MAO) isoenzymes: MAO A and MAO B. Both enzymes catalyze oxidative deamination of most biogenic amines in the body, including serotonin (see Fig. 103.5 ), norepinephrine, epinephrine, and dopamine (see Fig. 103.2 ). The genes for both isoenzymes are on the X chromosome (Xp11.3), residing in close proximity. A deletion of both genes can also encompass a neighboring gene, NDP, resulting in a contiguous deletion syndrome, which can present as an atypical Norrie disease (see Chapter 640 ). Male patients with MAO A deficiency manifest borderline intellectual deficiency and impaired impulse control. The consequences of the isolated MAO B deficiency are incompletely understood. Combined MAO A and B deficiency causes severe intellectual disability and behavioral problems and can be associated with pronounced laboratory abnormalities (e.g., 4-6–fold serotonin elevation in physiologic fluids, elevated O -methylated amine metabolites, and reduced deamination products [VMA, HVA]). Dietary intervention (low tyramine, phenylethylamine, and L -dopa/dopamine intake) did not improve patients' blood serotonin levels. Inheritance of MAO deficiency is X-linked. Treatment of MAO A deficiency is supportive.

Disorders of γ-Aminobutyric Acid (GABA) Metabolism

GABA is the main inhibitory neurotransmitter synthesized in the synapses through decarboxylation of glutamic acid by glutamate decarboxylase (GAD). The same pathway is responsible for production of GABA in other organs, especially the kidneys and the β-cells of the pancreas. GAD enzyme requires pyridoxine (vitamin B₆ ) as cofactor. Two GAD enzymes, GAD1 (GAD₆₇ ) and GAD2 (GAD₆₅ ) have been identified. GAD1 is the main enzyme in the brain, and GAD2 is the major enzyme in the β-cells. Antibodies against GAD₆₅ and GAD₆₇ have been implicated in the development of type 1 diabetes and stiff-person syndrome , respectively. GABA is catabolized to succinic acid by 2 enzymes, GABA transaminase and succinic semialdehyde dehydrogenase (SSADH) (see Fig. 103.11 ).

γ-Aminobutyric Acid Transaminase Deficiency

Clinical manifestations in the 2 index infant siblings included severe psychomotor retardation, hypotonia, hyperreflexia, lethargy, refractory seizures, and increased linear growth likely related to GABA-mediated increased secretion of growth hormone. Increased concentrations of GABA and β-alanine were found in CSF (see Fig. 103.11 ). Evidence of leukodystrophy was noted in the postmortem examination of the brain. A 3rd patient showed severe psychomotor retardation, recurrent episodic lethargy, and intractable seizures with comparable CSF metabolite abnormalities to those of the index probands. GABA transaminase deficiency is demonstrated in brain and lymphocytes. Treatment is symptomatic. Intervention with vitamin B₆ , the cofactor for the enzyme, was without therapeutic benefit. The gene (ABAT), maps to chromosome 16p13.2; the condition is inherited as an autosomal recessive trait.

Succinic Semialdehyde Dehydrogenase Deficiency (γ-Hydroxybutyric Aciduria)

Clinical manifestations of SSADH deficiency usually begin in infancy with developmental delays with a disproportionate deficit in expressive language, hypotonia, and ataxia; seizures occur in approximately 50% of patients (see Fig. 103.11 ). Many patients also carry the diagnosis of autism spectrum disorder . Neuropsychiatric comorbidity (especially oppositional defiance, obsession-compulsion, and hyperactivity) can be disabling, particularly in adolescents and adults. Abnormal EEG findings include background slowing and generalized spike-wave paroxysms, with variable lateralization in hemispheric onset and voltage predominance. Photosensitivity and electrographic status epilepticus of sleep have been reported in combination with difficulties in sleep maintenance and excessive daytime somnolence. MRI of the brain shows an increased T2-weighted hyperintensity involving the globus pallidi, cerebellar dentate nuclei, and subthalamic nuclei, usually in a bilaterally symmetric distribution.

The biochemical hallmark, γ-hydroxybutyric acid (GHB), is elevated in physiologic fluids (CSF, plasma, urine) in all patients. Increased concentrations of GABA are also found in CSF. Heightened diagnostic suspicion evolves through documentation of elevated urinary GHB, and confirmation is achieved by molecular genetic testing.

Treatment remains elusive; vigabatrin (GABA-transaminase inhibitor) has been employed empirically, with mixed outcomes, and there is concern with its use as it further elevates CNS GABA in an already hyper-GABAergic disorder. Additionally, vigabatrin can cause constriction of the visual field and long-term use is contraindicated.

The gene for SSADH (ALDH5A1) is located on chromosome 6p22, and inheritance follows an autosomal-recessive pattern. Prenatal diagnosis has been achieved by measurement of GHB in the amniotic fluid, assay of the enzyme activity in the amniocytes, chorionic villus sampling, or DNA analysis.

Defects in Neurotransmitter Transporter Proteins

More than 20 different proteins are involved in transporting different neurotransmitters across the neuronal membranes. The main function of most of these transporters is to remove the excess neurotransmitters from the synaptic junction back into the presynaptic neurons (reuptake). This recycling process not only regulates the precise effect of neurotransmitters at the synaptic junction, but also resupplies the presynaptic neurons with neurotransmitters for future use. A few transporter proteins are involved in shuttling neurotransmitters from the neuronal cytoplasm across the membrane of synaptic vesicles for storage (vesicular transporters). On neuronal stimulation, these vesicles release a bolus of neurotransmitters through exocytosis. As expected, pathogenic variants in transporter proteins interfere with the proper reuptake and storage of neurotransmitters and may result in clinical manifestations similar to those seen in deficiencies of neurotransmitters metabolism. Several conditions caused by pathogenic variants of neurotransmitter protein transporters have been described, including dopamine transporter protein deficiency and dopamine-serotonin vesicular transporter disease.

Dopamine Transporter Protein Deficiency

This transporter protein is involved in reuptake of dopamine by the presynaptic neurons, and its deficiency causes depletion of dopamine and thus a dopamine deficiency state. Dopamine transporter protein (DAT) is encoded by SLC6A3 gene on chromosome 5p15.33. Pathogenic variants of this gene has been reported in 13 children. These children presented with symptoms of infantile parkinsonism-dystonia syndrome . Irritability and feeding difficulties started shortly after birth and progressed to hypotonia, lack of head control, parkinsonism, dystonia, and global developmental delay by early infancy. Brain MRI usually shows no abnormalities.

CSF examination revealed elevation of HVA and normal level of 5-HIAAs. The urinary level of HVA and serum concentration of prolactin were increased. Diagnosis was established by demonstrating the loss-of-function mutation in the SLC6A3 gene. No effective treatment has been identified; L -dopa/carbidopa did not result in improvements in clinical or biochemical parameters.

Dopamine-Serotonin Vesicular Transporter Disease (Vesicular Monoamine Transporter Deficiency)

This autosomal recessive condition, described in 8 children from a consanguineous Saudi Arabian family, is caused by a pathogenic variant in the SLC18A2 gene. This gene encodes the vesicular monoamine transporter 2 (VMAT2), which is involved in transporting dopamine and serotonin from the cytoplasm into the synaptic storage vesicles located in the axonal terminals of the presynaptic neurons. Most affected children presented in the 1st yr of life with symptoms consistent with deficiencies of dopamine (hypotonia progressing into dystonia, parkinsonism, oculogyric crises), serotonin (sleep and psychiatric disturbances), and norepinephrine-epinephrine (excessive sweating, tremors, temperature instability, postural hypotension, ptosis). Neurocognitive delays become apparent in the 1st yr of life. No diurnal variation of the symptoms was noted. Brain imaging studies were within normal limits. Changes in the levels of CNS neurotransmitters and their metabolites have been inconsistent.

The phenotype resembles that seen in AADC and BH₄ deficiencies (see earlier). Diagnosis requires molecular analysis of SLC18A2 (located on chromosome 10q25.3). Treatment with L -dopa/carbidopa caused exacerbation of symptoms, whereas pramipexole, a dopamine receptor agonist, resulted in a promising clinical response.

Histidine Decarboxylase Deficiency

Decarboxylation of histidine by histidine decarboxylase produces histamine, which functions as a neurotransmitter in the brain. Deficiency of this enzyme (expressed mainly in the posterior hypothalamus) results in deficiency of histamine in the CNS and in one family caused an autosomal dominant form of Tourette syndrome (see Chapter 103.13 ).

Hyperprolinemia

Intellectual disability and seizures are common findings in most patients with hyperprolinemia types I and II. Patients with type I hyperprolinemia typically show a benign clinical course but could have an increased risk of developing schizophrenia. The contribution of increased concentration of proline to the mechanisms of schizophrenia, however, remains unclear. The neurologic abnormalities observed in hyperprolinemia type II are mainly caused by development of vitamin B₆ dependency in this condition (see Chapter 103.9 ). Dietary intervention in hyperprolinemias type I and II is neither feasible nor recommended.

3-Phosphoglycerate Dehydrogenase Deficiency

See Chapter 103.8 .

Phosphoserine Aminotransferase Deficiency

See Chapter 103.8 .

Nonketotic Hyperglycinemia

See Chapter 103.7 .

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103.12

Urea Cycle and Hyperammonemia (Arginine, Citrulline, Ornithine)

Oleg A. Shchelochkov, Charles P. Venditti

Keywords

• urea cycle disorder
• hyperammonemia
• carbamoyl phosphate synthetase 1
• CPS1
• ornithine transcarbamylase
• OTC
• argininosuccinate synthetase 1
• classic citrullinemia
• ASS1
• citrin
• citrin deficiency
• SLC25A13
• neonatal intrahepatic cholestasis
• argininosuccinate lyase
• argininosuccinic aciduria
• ASL
• arginase 1
• hyperargininemia
• argininemia
• ARG1
• N -acetylglutamate
• NAG
• N -acetylglutamate synthetase
• NAGS
• sodium benzoate
• sodium phenylacetate
• sodium phenylbutyrate
• Ammonul
• ariginine
• citrulline
• orotate
• orotic acid
• argininosuccinate
• argininosuccinic acid
• acrodermatitis enteropathica
• valproic acid
• valproate
• transient hyperammonemia of the newborn
• THAN
• disorders of ornithine metabolism
• gyrate atrophy of the retina and choroid
• ornithine aminotransferase
• OAT
• hyperammonemia-hyperornithinemia-homocitrullinemia syndrome
• hyperammonemia-hyperornithinemia-homocitrullinuria syndrome
• HHH syndrome
• SLC25A15
• congenital glutamine deficiency
• glutamine synthetase
• GLUL

Catabolism of amino acids results in the production of free ammonia, which in high concentration is toxic to the CNS. Mammals detoxify ammonia to urea through a series of reactions known as the urea cycle (Fig. 103.12 ), which is composed of 5 enzymes: carbamoyl phosphate synthetase 1 (CPS1 ), ornithine transcarbamylase (OTC ), argininosuccinate synthetase (ASS ), argininosuccinate lyase (ASL ), and arginase 1. A 6th enzyme, N -acetylglutamate (NAG) synthetase (NAGS ), catalyzes synthesis of NAG, which is an obligatory activator (effector) of the CPS1 enzyme. Individual deficiencies of these enzymes have been observed and, with an overall estimated prevalence of 1 in 35,000 live births, they are the most common genetic causes of hyperammonemia in infants.

Fig. 103.12 Urea cycle: pathways for ammonia disposal and ornithine metabolism. Reactions occurring in the mitochondria are depicted in purple. Reactions shown with interrupted arrows are the alternate pathways for the disposal of ammonia. Enzymes: (1) Carbamyol phosphate synthetase type 1 (CPS1), (2) ornithine transcarbamylase (OTC), (3) argininosuccinate synthetase (ASS), (4) Argininosuccinate lyase (ASL), (5) arginase 1, (6) ornithine aminotransferase, (7) N -acetylglutamate (NAG) synthetase, (8) citrin, (9) ornithine transporter (ORNT1). HHH syndrome, Hyperammonemia-hyperornithinemia-homocitrullinemia.

Genetic Causes of Hyperammonemia

Hyperammonemia, sometimes severe, occurs in inborn errors of metabolism other than the urea cycle defects (Table 103.4 ; see also Table 102.5 ). The mechanisms of hyperammonemia in some of these conditions are diverse and include accumulation of toxic metabolites (e.g., organic acids), impaired transport of urea cycle intermediates (e.g., HHH syndrome), or depletion of urea cycle intermediates (e.g., lysinuric protein intolerance), leading to compromised function of the urea cycle.

Table 103.4

Inborn Errors of Metabolism Causing Hyperammonemia

• Deficiencies of the urea cycle enzymes
  • Carbamyl phosphate synthetase 1
  • Ornithine transcarbamylase
  • Argininosuccinate synthetase
  • Argininosuccinate lyase
  • Arginase 1
  • N -acetylglutamate synthetase
• Organic acidemias
  • Propionic acidemia
  • Methylmalonic acidemia
  • Isovaleric acidemia
  • β-Ketothiolase deficiency
  • Multiple carboxylase deficiencies
  • Medium-chain fatty acid acyl-CoA dehydrogenase deficiency
  • Glutaric acidemia type I
  • 3-Hydroxy-3-methylglutaric aciduria
• Lysinuric protein intolerance
• Hyperammonemia-hyperornithinemia-homocitrullinemia syndrome
• Transient hyperammonemia of the newborn
• Congenital hyperinsulinism with hyperammonemia

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 appears normal at birth but becomes symptomatic following the introduction of dietary protein. Refusal to eat, vomiting, tachypnea, and lethargy can quickly progress to a deep coma. Seizures are common. Physical examination may reveal hepatomegaly in addition to obtundation. Hyperammonemia can trigger increased intracranial pressure that may be manifested by a bulging fontanelle and dilated pupils.

In infants and older children , acute hyperammonemia is manifested by vomiting and neurologic abnormalities such as ataxia, confusion, agitation, irritability, combativeness, and psychosis. 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 caused by defects of the urea cycle enzymes. Blood urea nitrogen is usually low in these patients. Some patients may initially present with unexplained elevated serum alanine transaminase (ALT) and aspartate transaminase (AST) and even meet the criteria for acute liver failure. 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 may reveal microvesicular steatosis, mild cholestasis, and fibrosis of the liver. Thus, because of the nonspecific presentation or urea cycle disorders, it is imperative to measure plasma ammonia levels in any ill infant with severe sepsis, unexplained liver dysfunction, recurrent emesis, or progressive encephalopathy.

Diagnosis

The main criterion for diagnosis is hyperammonemia. Each clinical laboratory should establish its own normal values for blood ammonia. Normal newborn values are higher than those of the older child or adult. Levels as high as 100 µmol/L can occur in healthy term infants. An ill infant usually manifests a blood ammonia level >150 µmol/L. Fig. 103.13 illustrates an approach to the differential diagnosis of hyperammonemia in the newborn infant. Careful inspection of individual plasma amino acids usually reveals abnormalities that may help the diagnosis. In patients with deficiencies of CPS1, OTC, or NAGS, frequent findings include 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 helps differentiate this defect from CPS1 deficiency. Differentiation between the CPS1 deficiency and the NAGS deficiency may require an assay of the respective enzymes or molecular analysis of the relevant genes. Clinical improvement occurring after oral administration of carbamylglutamate, however, may suggest NAGS deficiency. Patients with a deficiency of ASS, ASL, or arginase 1 have marked increases in the plasma levels of citrulline, argininosuccinic acid, or arginine, respectively. The combination of hyperammonemia and marked hypercitrullinemia or argininosuccinic acidemia is virtually pathognomonic for these disorders. Children with urea cycle defects often self-select a low-protein, high-carbohydrate diet, especially those with late-onset disease or symptomatic females with partial OTC deficiency.

Fig. 103.13 Clinical approach to a newborn infant with symptomatic hyperammonemia. CPS1, Carbamoyl phosphate synthetase 1; HHH syndrome, hyperornithinemia-hyperammonemia-homocitrullinemia; NAGS, N -acetylglutamate synthetase; OTC, ornithine carbamoyltransferase.

Mass screening of newborn infants identifies patients with ASS, ASL, and arginase 1 deficiencies.

Treatment of Acute Hyperammonemia

Clinical outcome depends mainly on the severity and the duration of hyperammonemia. Serious neurologic sequelae are likely in newborns with severe elevations in blood ammonia (>300 µmol/L) for more than 12 hr. Thus, acute hyperammonemia should be treated promptly and vigorously. The goal of therapy is to lower the concentration of ammonia. This is accomplished by (1) removal of ammonia from the body in a form other than urea and (2) minimizing endogenous protein breakdown and favoring endogenous protein synthesis by providing adequate calories and essential amino acids (Table 103.5 ). Fluid, electrolytes, glucose (10–15%), and lipids (1-2 g/kg/24 hr) should be infused intravenously, together with minimal amounts of protein (0.25 g/kg/24 hr), preferably including essential amino acids. Oral feeding with a low-protein formula (0.5-1.0 g/kg/24 hr) through a nasogastric tube should be started as soon as sufficient improvement is seen.

Table 103.5

Treatment of Acute Hyperammonemia in an Infant

1. 1. Provide adequate calories, fluid, and electrolytes intravenously (10% glucose, NaCl* and intravenous lipids 1 g/kg/24 hr). Add minimal amounts of protein preferably as a mixture of essential amino acids (0.25 g/kg/24 hr) during the 1st 24 hr of therapy.
2. 2. Give priming doses of the following compounds:
   • (To be added to 20 mL/kg of 10% glucose and infused within 1-2 hr)
   • • Sodium benzoate 250 mg/kg ^†
   • • Sodium phenylacetate 250 mg/kg ^†
   • • Arginine hydrochloride 200-600 mg/kg as a 10% solution
3. 3. Continue infusion of sodium benzoate ^† (250-500 mg/kg/24 hr), sodium phenylacetate ^† (250-500 mg/kg/24 hr), and arginine (200-600 mg/kg/24 hr ^‡ ) following the above priming doses. These compounds should be added to the daily intravenous fluid.
4. 4. Initiate peritoneal dialysis or hemodialysis if above treatment fails to produce an appreciable decrease in plasma ammonia.

---

^* The concentration of sodium chloride should be calculated to be 0.45–0.9%, including the amount of the sodium in the drugs.

^† Sodium from these drugs should be included as part of the daily sodium requirement.

^‡ The higher dose is recommended in the treatment of patients with citrullinemia and argininosuccinic aciduria. Arginine is not recommended in patients with arginase deficiency and in those whose hyperammonemia is secondary to organic acidemia. Sodium benzoate and sodium phenylacetate should be used with caution in patients with organic acidemias.

Because the kidneys clear ammonia poorly, its removal from the body must be expedited by formation of compounds with a high renal clearance. An important advance in the treatment of hyperammonemia has been the introduction of acylation therapy by using an exogenous organic acid that is acylated endogenously with nonessential amino acids to form a nontoxic compound with high renal clearance. The main organic acids used for this purpose are sodium salts of benzoic acid and phenylacetic acid. Benzoate forms hippurate with endogenous glycine in the liver (see Fig. 103.12 ). 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. 103.12 ). Sodium phenylbutyrate, metabolized to phenylacetate, is the primary oral formulation. For intravenous (IV) use, a combined formulation of benzoate and phenylacetate (Ammonul) is commercially available.

Another valuable therapeutic adjunct is IV infusion of arginine , which is effective in all patients (except those with arginase deficiency). Arginine administration supplies the urea cycle with ornithine (see Fig. 103.12 ). In patients with citrullinemia, 1 mole of arginine reacts with 1 mole of ammonia (as carbamoyl phosphate) to form citrulline. In patients with argininosuccinic acidemia, 2 moles of ammonia (as carbamoyl phosphate and aspartate) react with arginine to form argininosuccinic acid. Citrulline and argininosuccinate are less toxic than ammonia and more readily excreted by the kidneys. In patients with CPS1 or OTC deficiencies arginine administration is indicated because this amino acid is not produced in sufficient amounts to enable endogenous protein synthesis. For enteral therapy, patients with OTC deficiency benefit from supplementation with citrulline (200 mg/kg/24 hr) because 1 mole of citrulline reacts with 1 mole of ammonia (through aspartic acid) to form arginine. Administration of arginine or citrulline is contraindicated in patients with arginase deficiency , a rare condition in which the usual presenting clinical picture is spastic diplegia rather than hyperammonemia. Arginine therapy is of no benefit if hyperammonemia is secondary to an organic acidemia. In a newborn infant with an initial episode of hyperammonemia, arginine should be used until the diagnosis is established (see Table 103.5 ).

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. Both benzoate and phenylacetate are usually supplied as concentrated solutions and should be properly diluted (1–2% solution) for IV use. The recommended therapeutic doses of both compounds deliver a substantial amount of sodium to the patient; this amount should be included in calculation of the daily sodiumn requirement. Benzoate and phenylacetate (or the combined formulation, Ammonul) should be used with caution in newborn infants with hyperbilirubinemia because they may displace bilirubin from albumin; however, there are no documented cases of kernicterus (see Chapter 123.4 ) reported in neonates with hyperammonemia who have received such therapies. In infants at risk, it is advisable to reduce bilirubin to a safe level while considering IV administration of benzoate or phenylacetate.

If the initial ammonia level is <500 µmol/L, and if the foregoing therapies fail within 4-6 hr to produce any appreciable change in the blood ammonia level, hemodialysis should be used. For patients presenting with an ammonia level >500 µmol/L, extracorporeal detoxification is the initial method of ammonia removal. 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 (see earlier). Hemodialysis dramatically lowers blood ammonia within a few hours, but if it is unavailable or technically unfeasible, peritoneal dialysis may be used as an alternative. When hyperammonemia is caused by an organic acidemia and hemodialysis is not available, peritoneal dialysis can be used to remove both the offending organic acid and ammonia.

Oral administration of neomycin limits growth of intestinal bacteria that can produce ammonia. However, this modality is of limited use in patients (e.g., affected neonates) in whom reduction of hyperammonemia is an urgent priority. Oral lactulose acidifies the intestinal lumen, thereby reducing the diffusion of ammonia across the intestinal epithelium. This agent is of limited applicability in newborns, who have a high risk of acidemia and dehydration.

There has been interest in the use of cooling as a therapeutic adjunct in newborn infants with metabolic encephalopathy such as that caused by hyperammonemia. Clinical studies are in progress to evaluate the efficacy of this approach. There may be considerable lag between the normalization of ammonia level and an improvement in the patient's neurologic status. Several days may be needed before the infant becomes fully alert.

Long-Term Therapy

Once the infant is alert, therapy should be tailored to the underlying cause of the hyperammonemia. In general, all patients, regardless of the enzymatic defect, require protein restriction limited to age-adjusted recommended dietary allowance (RDA). In pediatric patients with defects in the urea cycle, chronic administration of sodium benzoate (250 mg/kg/24 hr), sodium phenylbutyrate (250-500 mg/kg/24 hr), and arginine (200-400 mg/kg/24 hr) or citrulline (in patients with OTC deficiency, 200-400 mg/kg/24 hr) is effective in maintaining blood ammonia levels within the normal range (shown doses are for patients who weigh <20 kg). Arginine and citrulline are contraindicated in patients with argininemia. Patients who have difficulty taking sodium phenylbutyrate can receive a trial of glycerol phenylbutyrate. This compound conceals the offensive odor of sodium phenylbutyrate and may help with patient adherence. Glycerol phenylbutyrate is not yet approved for use in children <2 months of age. Benzoate and phenylacetate may lower carnitine levels, but clinical signs of carnitine deficiency or benefit from carnitine supplementation have not yet been demonstrated. These compounds have been used during pregnancy without obvious teratogenic effect. However, experience is still limited, and appropriate caution should be exercised.

Growth parameters, especially head circumference, and nutritional indices (blood albumin, prealbumin, pH, electrolytes, amino acids, zinc, selenium) should be followed closely. Long-term care of these patients is best achieved by a team of experienced professionals (pediatrician, nutritionist, child neurologist, metabolic geneticist). Skin lesions resembling acrodermatitis enteropathica (see Chapter 691 ) have been noted in a few patients with different types of urea cycle defects, presumably from deficiency of essential amino acids, caused by overzealous dietary protein restriction. Catabolic states (infections, fasting) that may trigger hyperammonemia should be avoided. They must be treated vigorously if they occur. It is important that all children with urea cycle defects avoid valproic acid because this drug can elevate blood ammonia even in some healthy individuals. In patients with CPS1, OTC, or ASS deficiency, acute hyperammonemic attacks may be precipitated by valproate administration.

Carbamoyl Phosphate Synthetase 1 and N -Acetylglutamate Synthase Deficiencies

Deficiencies of these 2 enzymes produce similar clinical and biochemical manifestations (see Figs. 103.12 and 103.13 ). There is a wide variation in severity of symptoms and in the age at 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, coma). Increased intracranial pressure is frequent. Late forms (as late as the 4th decade of life) 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 common. Intermediate forms with intellectual disability and chronic subclinical hyperammonemia interspersed with bouts of acute hyperammonemia have also been observed.

Laboratory findings include hyperammonemia. The plasma amino acid analysis typically shows a marked increase of glutamine and alanine with relatively low levels of citrulline and arginine. These are nondiagnostic changes that occur in hyperammonemia of diverse cause. Urinary orotic acid is usually low or may be absent (see Fig. 103.13 ).

Treatment of acute hyperammonemic attacks and the long-term therapy of the condition are previously outlined (see Table 103.5 ). Patients with NAGS deficiency benefit from oral administration of carbamylglutamate. It is therefore important to differentiate between CPS1 and NAGS deficiencies by gene sequencing. Deficiency of NAGS is rare in North America.

CPS1 and NAGS deficiencies are inherited as an autosomal recessive trait; the CPS1 enzyme is normally present in liver and intestine. The gene (CPS1) is mapped to chromosome 2q34. The prevalence of the condition is approximately 1 : 1,300,000. The gene for NAG synthetase (NAGS) is located on chromosome 17q21.31. Neither of these conditions is identified by the mass screening of the newborn infants.

Ornithine Transcarbamylase Deficiency

In this X-linked disorder, the hemizygous males are more severely affected than heterozygous females (see Figs. 103.12 and 103.13 ). The heterozygous females may have a mild form of the disease, but the majority (approximately 75%) remain asymptomatic, although investigations indicate subtle neurologic defects even in women without a frank history of hyperammonemia. Ornithine transcarbamylase (OTC) deficiency is the most common form of all the urea cycle disorders, comprising approximately 40% of cases of urea cycle disorders.

Clinical manifestations in a male newborn are usually those of severe hyperammonemia (see earlier) occurring in the 1st few days of life. Mild forms , such as in some heterozygous females, characteristically have episodic manifestations, which may occur at any age (usually after infancy). Episodes of hyperammonemia, manifested by vomiting and neurologic abnormalities (e.g., ataxia, mental confusion, agitation, combativeness, frank psychosis), 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 one of these attacks. Cognitive development may proceed normally. Mild to moderate intellectual disability, 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. The blood level of urea is usually low. A marked increase in the urinary excretion of orotic acid differentiates this condition from CPS1 deficiency (see Fig. 103.13 ). Orotate may precipitate in urine as pink-colored gravel or stones. 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 (see Chapter 103.14 ) 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 is most conveniently confirmed by gene analysis. As many as 20% of affected patients demonstrate a normal sequence, perhaps because the pathogenic variant involves copy number variants and pathogenic variants involving introns or a promoter region. Copy number variants can be evaluated using a chromosomal microarray, and if positive, a contiguous gene deletion should be considered. If the molecular diagnostic approach is negative, a liver biopsy may be indicated. Prenatal diagnosis is feasible by analysis of DNA in amniocytes or chorionic villus samples. Increase in urinary excretion of orotidine after an allopurinol loading test can identify female carriers. Mild cerebral dysfunction may be present in asymptomatic 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 are previously outlined. For enteral use, citrulline is used in place of arginine in patients with OTC deficiency. Liver transplantation is a successful treatment for patients with severe OTC deficiency.

The gene for OTC has been mapped to the X chromosome (Xp21.1). Many disease-causing pathogenic variants (>300) have been identified. The prevalence of OTC deficiency is 1 : 56,000-1 : 77,000 live births. Genotype and the resulting degree of enzyme deficiency determine severity of the phenotype in most cases. Mothers of affected infants are expected to be carriers of the mutant gene unless a de novo pathogenic variant has occurred. A mother who gave birth to 2 affected male offspring was found to have a normal genotype, suggesting that gonadal mosaicism can be seen in some families. This condition is not identified by the mass screening of newborn infants.

Citrullinemia

Two clinically and genetically distinct forms of citrullinemia have been identified. The classic form (type I ) is caused by the deficiency of the ASS enzyme. Citrullinemia type II is caused by the deficiency of a mitochondrial transport protein named citrin . (See Figs. 103.12 and 103.13 .)

Citrullinemia Type I (Argininosuccinate Synthetase Deficiency, Classic Citrullinemia)

This condition is caused by the deficiency of ASS (see Fig. 103.12 ) and has variable clinical manifestations depending on the degree of the enzyme deficiency. Two major forms of the condition have been identified. The severe or neonatal form , which is most common, appears in the 1st few days of life with signs and symptoms of hyperammonemia (see earlier). In the subacute or mild form , clinical findings such as failure to thrive, frequent vomiting, developmental delay, and dry, brittle hair appear gradually after 1 yr of age. Acute hyperammonemia, triggered by an intercurrent catabolic state, may bring the diagnosis to light.

Laboratory findings are similar to those found in patients with OTC deficiency, except that the plasma citrulline concentration is greatly elevated (50-100 times normal) (see Fig. 103.13 ). Urinary excretion of orotic acid is moderately increased; crystalluria may also occur as a result of precipitation of orotates. The diagnosis is confirmed by DNA analysis or less frequently by assay of enzyme activity in cultured fibroblasts. Prenatal diagnosis is feasible with enzyme assay in cultured amniotic cells or by DNA analysis of cells obtained from chorionic villus biopsy.

Treatment of acute hyperammonemic attacks and long-term therapy are outlined earlier (see Table 103.5 ). Plasma concentration of citrulline remains elevated at all times and may increase further after administration of arginine. Patients can do well on a protein-restricted diet in conjunction with sodium benzoate, phenylbutyrate, and arginine therapy. Mild to moderate cognitive impairment is a common sequela, even in a well-treated patient.

Citrullinemia is inherited as an autosomal recessive trait. The gene (ASS1) is located on chromosome 9q34.11. The majority of patients are compound heterozygotes for 2 different alleles. The prevalence of the condition is 1 : 250,000 live births. The recent introduction of neonatal screening for urea cycle defects has shown that some affected patients are ostensibly asymptomatic even with ingestion of a regular diet. Long-term follow-up is needed to be certain that these individuals do not sustain neurologic sequelae.

Citrin Deficiency (Citrullinemia Type II)

Citrin (aspartate-glutamate carrier protein) is a mitochondrial transporter encoded by a gene (SLC25A13) located on chromosome 7q21.3. One of this protein's functions is to transport aspartate from mitochondria into cytoplasm and replenish the cytosolic aspartate pool required for converting citrulline to argininosuccinic acid (see Fig. 103.12 ). If aspartate is unavailable to the cytoplasmic component of the urea cycle, urea will not be formed at a normal rate, and citrulline will accumulate. ASS activity is diminished in the liver of these patients, but no pathogenic variant in the ASS1 gene has been found. It is postulated that citrin deficiency interferes with translation of messenger RNA for ASS enzyme in the liver. The condition initially was reported in Japan, but non-Japanese patients have also been identified. Two clinical forms of citrin deficiency have been described.

Neonatal Intrahepatic Cholestasis (Citrullinemia Type II, Neonatal Form)

Clinical and laboratory manifestations, which usually start before 1 yr of age, include cholestatic jaundice with mild to moderate direct (conjugated) hyperbilirubinemia, marked hypoproteinemia, clotting dysfunction (increased prothrombin time and partial thromboplastin time), and increased serum γ-glutamyltransferase and alkaline phosphatase activities; liver transaminases are usually normal. Plasma concentrations of ammonia and citrulline are usually normal, but moderate elevations have been reported. There may be increases in plasma concentrations of methionine, tyrosine, alanine, and threonine. Elevated levels of serum galactose have been found, even though the enzymes of galactose metabolism are normal. The reason for hypergalactosemia is not known. Marked elevation in the serum level of α-fetoprotein is also present. These findings resemble those of tyrosinemia type I, but unlike the latter condition, urinary excretion of succinylacetone is not elevated (see Chapter 103.2 ). Liver biopsy shows fatty infiltration, cholestasis with dilated canaliculi, and a moderate degree of fibrosis. The condition is usually self-limiting, and the majority of infants recover spontaneously by 1 yr of age with supportive and symptomatic treatment. Hepatic failure requiring liver transplantation has occurred in a few cases. Although the condition is commonly seen in Japan, the diagnosis should be considered in any case of unexplained neonatal hepatitis with cholestasis. Data on the long-term prognosis and the natural history of the condition are limited; development into the adult form of the condition after several years of seemingly asymptomatic hiatus has been observed.

Citrullinemia Type II, Adult Form (Adult-Onset Citrullinemia; Citrullinemia Type II, Mild Form)

This form of citrullinemia type II starts acutely in a previously apparently normal individual and manifests with neuropsychiatric symptoms such as disorientation, delirium, delusion, aberrant behavior, tremors, and frank psychosis. Moderate degrees of hyperammonemia and hypercitrullinemia are present. The age at onset is usually between 20 and 40 yr (range: 11 to >100 yr). Patients who recover from the 1st episode may have recurrent attacks. Pancreatitis, hyperlipidemia, and hepatoma are major complications among the survivors. Medical treatment has been mostly ineffective for prevention of future attacks. Diet enriched for protein and lipids helps restore cytosolic aspartate pool and stimulate ureagenesis. Indeed, some have speculated that the administration of large amounts of glucose might even prove deleterious, because the citrin transporter is important to the glycolytic pathway. Although liver transplantation appears to be effective in preventing future episodes of hyperammonemia, enteral supplementation with arginine, pyruvate, and medium-chain triglycerides can be tried first to improve hyperammonemic episodes and growth.

Several disease-causing mutations of the gene have been identified in affected Japanese and non-Japanese families. Although the frequency of homozygosity is relatively high in Japan (1 : 20,000 people), the clinical condition has a frequency of only 1 : 100,000 to 1 : 230,000. This indicates that a substantial number of homozygous individuals remain asymptomatic.

Argininosuccinate Lyase Deficiency (Argininosuccinic Aciduria)

The severity of the clinical and biochemical manifestations varies considerably (see Figs. 103.12 and 103.13 ). In severe form of ASL deficiency, signs and symptoms of severe hyperammonemia (see earlier) develop in the 1st few days of life, and without treatment, mortality can be high. Clinical course of ASL deficiency in patients who survive the initial acute episode can be characterized by intellectual disability, failure to thrive, hypertension, gallstones, liver fibrosis, and hepatomegaly. A common finding in untreated patients is dry and brittle hair (trichorrhexis nodosa ). Acute attacks of severe hyperammonemia may occur during a catabolic state.

Laboratory findings include hyperammonemia, moderate elevations in liver enzymes, nonspecific increases in plasma levels of glutamine and alanine, a moderate increase in plasma levels of citrulline (less than in citrullinemia), and marked increase in the concentration of argininosuccinic acid in plasma, urine, and CSF. The CSF levels 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 pathogenic variants in the ASL 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 are outlined earlier in this chapter. Intellectual disability, persistent hepatomegaly with mild increases in liver enzymes, and bleeding tendencies as a result of abnormal clotting factors are common sequelae. This deficiency is inherited as an autosomal recessive trait with a prevalence of about 1 in 220,000 live births. The gene (ASL) is located on chromosome 7q11.21. Early detection is achieved through mass screening of newborn infants.

Arginase 1 Deficiency (Hyperargininemia)

This defect is inherited as an autosomal recessive trait (see Figs. 103.12 and 103.13 ). There are 2 genetically distinct arginases in humans. One is cytosolic (ARG1) and is expressed in the liver and erythrocytes, and the other (ARG2) is found in renal and brain mitochondria. The gene for ARG1, the enzyme that is deficient in patients with arginase 1 deficiency, is mapped to chromosome 6q23.2. The role of the mitochondrial enzyme is not well understood; its activity increases in patients with argininemia but has no protective effect.

Clinical manifestations of this rare distal urea cycle disorder are somewhat different from those of other urea cycle enzyme defects, although acute neonatal form with intractable seizures, cerebral edema, and death has also been reported. The onset arginase 1 deficiency often is insidious; the infant can remain asymptomatic in the 1st few mo or yr of life. A progressive spastic diplegia with scissoring of the lower extremities, choreoathetotic movements, loss of developmental milestones, and failure to thrive in a previously normal infant may suggest a degenerative disease of the CNS. Some children were treated for years as cases of cerebral palsy before their arginase 1 deficiency was confirmed. Intellectual disability is progressive; seizures are common, but episodes of severe hyperammonemia are not as frequent as in the more proximal urea cycle defects. Hepatomegaly may be present.

Laboratory findings include marked elevations of arginine in plasma and CSF (see Fig. 103.13 ). Urinary orotic acid can be increased. Determination of amino acids in plasma is a critical step in the diagnosis of argininemia. The guanidino compounds (α-keto-guanidinovaleric acid and α-keto-argininic acid) are markedly increased in urine. The diagnosis is confirmed by assaying arginase activity in erythrocytes or by the identification of the mutant gene.

Treatment consists of a low-protein diet providing the RDA. The composition of the diet and the daily intake of protein should be monitored by frequent plasma amino acid determinations. Sodium benzoate or sodium phenylbutyrate are also effective in controlling hyperammonemia and lowering plasma arginine levels. Intellectual disability is a common sequela of the condition. One patient developed type 1 diabetes by 9 yr of age while his argininemia was under good control. Liver transplantation has produced promising results, but experience with long-term outcome is limited. Early detection is feasible through mass screening of newborn infants.

Transient Hyperammonemia of the Newborn

The blood concentration of ammonia in full-term infants may be as high as 100 µmol/L, or 2-3 times greater than that of the older child or adult. Blood levels approach the adult normal values after a few weeks of life (see Fig. 103.13 ).

Severe transient hyperammonemia is observed in some newborn infants. The majority of affected infants are premature and have 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 µmol/L) with moderate increases in plasma levels of glutamine and alanine. Plasma concentrations of urea cycle intermediate amino acids are usually normal except for citrulline, which may be moderately elevated. The cause of the disorder is unknown. Urea cycle enzyme activities are normal. Treatment of hyperammonemia should be initiated promptly and continued vigorously. Recovery without sequelae is common, and hyperammonemia does not recur even with a normal protein diet.

Disorders of Ornithine Metabolism

Ornithine, a key intermediate of the urea cycle, is not incorporated into natural proteins. Rather, it is generated in the cytosol from arginine and must be transported into mitochondria, where it becomes a substrate for the reaction catalyzed by OTC that forms citrulline. Excess ornithine is catabolized by 2 enzymes, ornithine aminotransferase, which is a mitochondrial enzyme converting ornithine to a proline precursor, and ornithine decarboxylase, which resides in the cytosol and converts ornithine to putrescine (see Fig. 103.12 ). Two genetic disorders feature hyperornithinemia : gyrate atrophy of the retina and hyperammonemia-hyperornithinemia-homocitrullinemia syndrome.

Gyrate Atrophy of the Retina and Choroid

This rare, autosomal recessive disorder is caused by deficiency of ornithine aminotransferase (see Fig. 103.12 ). Approximately 30% of the reported cases are from Finland. Clinical manifestations may include hyperammonemia in the 1st mo of life in some patients. Findings that define the phenotype of ornithine aminotransferase deficiency include night blindness, myopia, loss of peripheral vision, and posterior subcapsular cataracts. These eye changes start between 5 and 10 yr of age and progress to complete blindness by the 4th decade of life. Atrophic lesions in the retina resemble cerebral gyri. These patients usually have normal intelligence. Besides the characteristic 10-20–fold increase in plasma levels of ornithine (400-1,400 µmol/L), plasma levels of glutamate, glutamine, lysine, creatine, and creatinine can be moderately decreased. Some patients respond partially to high doses of pyridoxine. An arginine-restricted diet in conjunction with supplemental lysine, proline, and creatine has been successful in reducing plasma ornithine concentration and has produced some clinical improvement. The gene for ornithine aminotransferase (OAT) is mapped to chromosome 10q26.13. Many (at least 60) pathogenic variants have been identified in different families.

Hyperammonemia-Hyperornithinemia-Homocitrullinemia Syndrome

In this rare autosomal recessive 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 a depletion of this amino acid in mitochondria. The former causes hyperornithinemia, and the latter results in disruption of the urea cycle and hyperammonemia (see Fig. 103.12 ). Homocitrulline is presumably formed from the reaction of mitochondrial carbamoyl phosphate with lysine, which can become a substrate for the OTC reaction when ornithine is deficient. 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 (see Fig. 103.13 ). Acute episodes of hyperammonemia should be treated promptly (see earlier). Restriction of protein intake improves hyperammonemia. Oral supplementation with arginine (or citrulline) has produced clinical improvement in some patients. The gene for this disorder (SLC25A15) is located on chromosome 13q14.11.

Congenital Glutamine Deficiency

Glutamine is synthesized endogenously from glutamate and ammonia by a ubiquitous enzyme, glutamine synthetase (see Fig. 103.11 ). Glutamine is known to be involved in several important functions, including detoxification of ammonia. Deficiency of this enzyme, resulting in glutamine deficiency, has been reported in 3 infants from 3 unrelated families. All affected infants manifested multiorgan involvement, including significant brain malformations (abnormal gyrations, hypomyelination), facial abnormalities (broad nasal root, low-set ears), hypotonia and seizures at birth. Two of the patients died from multiorgan failure (respiratory and heart failure) in the neonatal period. One child was alive at 3 yr of age with severe developmental delay. Glutamine was absent in plasma, urine, and CSF, but plasma levels of glutamic acid were normal. Genetic defects of this enzyme underline the critical role of glutamine in embryogenesis, especially for normal brain development. The condition is inherited as an autosomal recessive trait; the gene for glutamine synthetase (GLUL) is mapped to chromosome 1q25.3.

Bibliography

Adam S, Almeida MF, Assoun M, et al. Dietary management of urea cycle disorders: European practice. Mol Genet Metab . 2013;110:439–445.

Bergmann KR, McCabe J, Smith RT, et al. Late-onset ornithine transcarbamylase deficiency: treatment and outcome of hyperammonemic crisis. Pediatrics . 2014;133:e1072–e1076.

Camacho J, Rioseco-Camacho N. Hyperornithinemia-hyperammonemia-homocitrullinuria syndrome. Pagon RA, Adam MP, Ardinger HH, et al. GeneReviews [Internet] . University of Washington: Seattle; 2012 http://www.ncbi.nlm.nih.gov/books/NBK97260/ [1993–2014].

Cederbaum S, LeMons C, Lee B. New developments and future directions for urea cycle disorders. Mol Genet Metab . 2010;100(Suppl):1–106.

Diaz GA, Krivitzky LS, Mokhtarani M, et al. Ammonia control and neurocognitive outcome among urea cycle disorder patients treated with glycerol phenylbutyrate. Hepatology . 2013;57:2171.

Enns GM, Berry SA, Berry GT, et al. Survival after treatment with phenylacetate and benzoate for urea-cycle disorders. N Engl J Med . 2007;356:2282–2292.

Ficicioglu C, Mandell R, Shih VE. Argininosuccinate lyase deficiency: long-term outcome of 13 patients detected by newborn screening. Mol Genet Metab . 2009;98:273–277.

Gallagher RC, Lam C, Wong D, et al. Significant hepatic involvement in patients with ornithine transcarbamylase deficiency. J Pediatr . 2014;164(4):720–725.

Gardeitichik T, Humphrey M, Nation J, et al. Early clinical manifestations and eating patterns in patients with urea cycle disorders. J Pediatr . 2012;161:328–332.

Gropman AL, Seltzer RR, Yudkoff M, et al. 1H MRS allows brain phenotype differentiation in sisters with late onset ornithine transcarbamylase deficiency (OTCD) and discordant clinical presentations. Mol Genet Metab . 2008;94:52–56.

Häberle J, Boddaert N, Burlina A, et al. Suggested guidelines for the diagnosis and management of urea cycle disorders. Orphanet J Rare Dis . 2012;7:32.

Häberle J, Görg B, Toutain A, et al. Inborn error of amino acid synthesis; human glutamine synthetase deficiency. J Inherit Metab Dis . 2006;29:352–358.

Häberle J, Shahbeck N, Ibrahim K, et al. Natural course of glutamine synthetase deficiency in a 3 year old patient. Mol Genet Metab . 2011;103:89–91.

Häberle J, Shahbeck N, Ibrahim K, et al. Glutamine supplementation in a child with inherited GS deficiency improves the clinical status and partially corrects the peripheral and central amino acid imbalance. Orphanet J Rare Dis . 2012;7:48.

Kim SZ, Song WJ, Nyhan WL, et al. Long-term follow-up of four patients affected by HHH syndrome. Clin Chim Acta . 2012;413:1151–1155.

Kobayashi K, Saheki T, Song YZ. Citrin deficiency. Pagon RA, Adam MP, Ardinger HH, et al. GeneReviews [Internet] . University of Washington: Seattle; 2005 [updated 2012] http://www.ncbi.nlm.nih.gov/books/NBK1181/ [1993–2014].

Lichter-Konecki U, Caldovic L, Morizono H, et al. Ornithine transcarbamylase deficiency. Pagon RA, Adam MP, Ardinger HH, et al. GeneReviews [Internet] . University of Washington: Seattle; 2013 http://www.ncbi.nlm.nih.gov/books/NBK154378/ [1993–2014].

Martinelli D, Diodato D, Ponzi E, et al. The hyperprnithinemia-hyperammonemia-homocitrullinuria syndrome. Orph J Rare Dis . 2015;10:29.

The Medical Letter. Glycerol phenylbutyrate (Ravicti) for urea cycle disorders. Med Lett . 2014;56:77–78.

Mew NA, Krivitzky L, McCarter R, et al. Clinical outcomes of neonatal onset proximal versus distal urea cycle disorders do not differ. J Pediatr . 2013;162:324–329.

Nagamani SC, Lee B, Erez A. Optimizing therapy for argininosuccinic aciduria. Mol Genet Metab . 2012;107:10–14.

Smith W, Diaz GA, Lichter-Konecki U, et al. Ammonia control in children ages 2 months through 5 years with urea cycle disorders comparison of sodium phenylbutyrate and glycerol phenylbutyrate. J Pediatr . 2013;162:1228–1234.

Summar ML, Koelker S, Freedenberg D, et al. The incidence of urea cycle disorders. Mol Genet Metab . 2013;110:179–180.

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Van Karnebeek C, Häberle J. Carbonic anhydrase VA deficiency. Adam MP, Ardinger HH, Pagon RA, et al. GeneReviews [Internet] . University of Washington: Seattle; 2015 https://www.ncbi.nlm.nih.gov/books/NBK284774 [1993–2017].

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103.13

Histidine

Oleg A. Shchelochkov, Charles P. Venditti

Keywords

• histidine
• L -histidine decarboxylase
• urocanic acid

Histidine is degraded through the urocanic acid pathway to glutamic acid. Several genetic biochemical aberrations involving the degradative pathway of histidine have been reported, but the clinical significance of elevated histidine levels has not been established.

Decarboxylation of histidine by histidine decarboxylase produces histamine. Deficiency of this enzyme has been implicated in the familial form of Tourette syndrome (see Chapter 103.11 ).

Bibliography

Castellan Baldan L, Williams KA, et al. Histidine decarboxylase deficiency causes Tourette syndrome: parallel findings in humans and mice. Neuron . 2014;81:77–90.

Ercan-Sencicek AG, Stillman AA, Ghosh AK, et al. L-histidine decarboxylase and Tourette's syndrome. N Engl J Med . 2010;362:1901–1908.

103.14

Lysine

Oleg A. Shchelochkov, Charles P. Venditti

Keywords

• lysine
• saccharopine
• hyperlysinemia-saccharopinuria
• antiquitin
• ALDH7A1
• pyridoxine-dependent epilepsy
• α-aminoadipic semialdehyde dehydrogenase deficiency
• pyridoxal 5′-phosphate
• pyridoxine 5′-phosphate oxidase deficiency
• PNPO deficiency
• PNPO
• piperideine-6-carboxylic acid
• P6C
• hypophosphatasia
• glutaric acid
• glutarylcarnitine
• 3-OH-glutaric acid
• glutaric aciduria type 1
• glutaryl-CoA dehydrogenase deficiency
• GCDH
• lysinuric protein intolerance
• SLC7A7

Lysine is catabolized through 2 pathways. In the 1st pathway, lysine is condensed with α-ketoglutaric acid to form saccharopine. Saccharopine is then catabolized to α-aminoadipic semialdehyde and glutamic acid. These 1st 2 steps are catalyzed by α-aminoadipic semialdehyde synthase, which has 2 activities: lysine-ketoglutarate reductase and saccharopine dehydrogenase (Fig. 103.14 ). In the 2nd pathway, lysine is first transaminated and then condensed to its cyclic forms, pipecolic acid and piperideine-6-carboxylic acid (P6C ). P6C and its linear form, α-aminoadipic semialdehyde, are oxidized to α-aminoadipic acid by the enzyme antiquitin . This is the major pathway for D -lysine in the body and for the L -lysine in the brain.

Fig. 103.14 Pathways in the metabolism of lysine. Enzymes: (1) Lysine ketoglutarate reductase, (2) saccharopine dehydrogenase, (3) α-aminoadipic semialdehyde/piperidine-6-carboxylic acid (P6C) dehydrogenase (antiquitin), (4) α-aminoadipic acid transferase, (5) α-ketoadipic acid dehydrogenase, (6) glutaryl-CoA-dehydrogenase. NE, Nonenzymatic; PDE, pyridoxine-dependent epilepsy.

Hyperlysinemia-saccharopinuria and α-aminoadipic-α-ketoadipic acidemia are biochemical conditions caused by inborn errors of lysine degradation. Individuals with these conditions are usually asymptomatic.

Pyridoxine (Vitamin B₆ )–Dependent Epilepsy

Pyridoxal 5′-phosphate (P5P ), the active form of pyridoxine, is the cofactor for many enzymes including those involved in the metabolism of neurotransmitters. Intracellular P5P deficiency in the brain may result in a seizure disorder that is refractory to common anticonvulsant agents but is responsive to high doses of pyridoxine. These pyridoxine-responsive phenotypes are seen in the following genetic metabolic conditions:

Antiquitin (α-Aminoadipic Semialdehyde Dehydrogenase) Deficiency

This is the most common cause of pyridoxine-dependent epilepsy. Deficiency of antiquitin results in accumulation of P6C in brain tissue (see Fig. 103.14 ); P6C reacts with P5P and renders it inactive. Large doses of pyridoxine are therefore needed to overcome this inactivation. The condition is inherited as an autosomal recessive trait; the gene for antiquitin (ALDH7A1) is on chromosome 5q31.

Pyridox(am)ine 5′-Phosphate Oxidase (PNPO) Deficiency

PNPO deficiency clinically overlaps with antiquitin deficiency. PNPO-deficient patients often present with neonatal-onset seizures, developmental delays, spastic tetraplegia, and nonspecific findings on brain imaging (delayed myelination, cerebral atrophy, and abnormal signals in basal ganglia). Developmental regression, optic disc pallor, and retinopathy have been reported infrequently. Plasma and CSF amino acid analysis may reveal elevated glycine, prompting evaluation for nonketotic hyperglycinemia (see Chapter 103.7 ) and lead to a delay in initiating treatment with P5P. CSF neurotransmitter assay revealed inconsistent changes in the levels of 3-O -methyldopa, homovanillic acid, and 5-hydroxyindoleacetic acid. Normal CSF level of P5P was reported in one patient, suggesting that a therapeutic trial with P5P and molecular analysis may be a prudent strategy in some patients irrespective of the CSF studies. The lowest effective dose of P5P should be used to avoid toxicity. The disorder is caused by autosomal recessive pathogenic variants in PNPO .

Sulfite Oxidase Deficiency and Molybdenum Cofactor Deficiency

In this rare condition (see Chapter 103.4 ), accumulation of sulfites causes inhibition of enzymatic activity of antiquitin and accumulation of P6C, which in turn causes inactivation of P5P and vitamin B₆ dependency.

Hyperprolinemia Type II

In this condition, accumulation of Δ1-pyrroline-5-carboxylate (P5C) in brain tissue causes inactivation of P5P, leading to pyridoxine dependency (see Chapter 103.9 and Fig. 103.9 ).

Hypophosphatasia

Pyridoxal-5′-phosphate is the main circulating form of pyridoxine. Alkaline phosphatase (ALP) is required for dephosphorylation of P5P to generate free pyridoxine, which is the only form of vitamin B₆ that can cross the blood-brain barrier and enter the brain cells. Pyridoxine is rephosphorylated intracellularly to form P5P. In the infantile form of hypophosphatasia, P5P cannot be dephosphorylated to free pyridoxine because of marked deficiency of tissue-nonspecific ALP. This results in deficiency of pyridoxine in the brain and pyridoxine-dependent epilepsy (see Chapters 611 and 724 ).

The main clinical manifestation of pyridoxine-dependent epilepsy caused by antiquitin deficiency is generalized seizures, which usually occur in the first days of life and are unresponsive to conventional anticonvulsant therapies. Some mothers of affected fetuses report abnormal intrauterine fluttering movements. The seizures are usually tonic-clonic in nature but can be almost any type. Other manifestations such as dystonia, respiratory distress, and abdominal distention with vomiting, hepatomegaly, hypoglycemia and hypothermia may be present. Learning problems and speech delay are common sequelae. Late-onset forms of the condition (as late as 5 years of age) have been reported. Consequently, a trial with vitamin B₆ is recommended in any infant with intractable convulsions (see Chapters 611.04 and 611.06 ).

Laboratory findings show increased concentrations of α-aminoadipic semialdehyde and pipecolic acid in the CSF, plasma, and urine. EEG abnormalities may normalize after treatment. Neuroimaging may be normal but cerebellar and cerebral atrophy, periventricular hyperintensity, intracerebral hemorrhage, and hydrocephalus have been reported.

Treatment with vitamin B₆ (50-100 mg/day) usually results in a dramatic improvement of both seizures and the EEG abnormalities. High doses of pyridoxine can result in peripheral neuropathy, and doses >500 mg/day should be avoided. The pyridoxine dependency and thus the therapy are lifelong. The therapeutic benefit of a lysine-restricted diet is being evaluated.

Glutaric Aciduria Type 1 (Glutaryl-CoA Dehydrogenase Deficiency)

Glutaric acid is an intermediate in the degradation of lysine (see Fig. 103.14 ), hydroxylysine, and tryptophan. Glutaric aciduria type 1 , a disorder caused by a deficiency of glutaryl-CoA dehydrogenase, should be differentiated from glutaric aciduria type 2 , a distinct clinical and biochemical disorder caused by defects in the mitochondrial electron transport chain (see Chapter 104.1 ).

Clinical Manifestations

Macrocephaly is a common but nonspecific finding in patients with glutaric aciduria type 1. It develops in the 1st yr of life but can also be present at birth and precede the onset of neurologic manifestations. Some affected infants may also show subtle neurologic symptoms, such as delayed onset of motor milestones, irritability, and feeding problems, during this seemingly asymptomatic period. The onset of the condition is usually heralded by acute encephalopathic findings , such as loss of normal developmental milestones (head control, rolling over, or sitting), seizures, generalized rigidity, opisthotonos, choreoathetosis, and dystonia caused by acute striatal injury. These symptoms may occur suddenly in an apparently normal infant after a minor infection. Brain imaging reveals increased extraaxial (particularly frontal) fluid with stretched bridging veins, striatal lesions, dilated lateral ventricles, cortical atrophy (mainly in frontotemporal region), and fibrosis. Recovery from the 1st attack usually occurs slowly, but some residual neurologic abnormalities may persist, especially dystonia and choreoathetosis. Without treatment, additional acute attacks resembling the first can occur during subsequent episodes of intercurrent infections or catabolic states. In some patients these signs and symptoms may develop gradually in the 1st few yr of life. Hypotonia and choreoathetosis may gradually progress into rigidity and dystonia (insidious form ). Acute episodes of metabolic decompensation with vomiting, ketosis, seizures, and coma also occur in this form after infection or other catabolic states. Without treatment, death may occur in the 1st decade of life during one of these episodes. Affected infants are prone to development of subdural hematoma and retinal hemorrhage following minor falls and head traumas. This can be misdiagnosed as child abuse. The intellectual abilities usually remain relatively normal in most patients.

Laboratory Findings

During acute episodes, mild to moderate metabolic acidosis and ketosis may occur. Hypoglycemia, hyperammonemia, and elevations of serum transaminases are seen in some patients. High concentrations of glutaric acid are usually found in urine, blood, and CSF. 3-Hydroxyglutaric acid may also be present in the body fluids. Acylcarnitine profile shows elevated glutarylcarnitine (C5-DC) in blood and urine. Plasma concentrations of amino acids are usually within normal limits. Laboratory findings may be unremarkable between attacks. Glutaric aciduria type 1 can be identified on the newborn screen by measuring glutarylcarnitine levels in blood spots. The sensitivity of this screening method depends on the cutoff value used by a newborn screen program, and some patients can be missed. For example, it can happen in a subset of patients with glutaric aciduria type 1 who may present with normal plasma and urinary levels of glutaric acid and variably elevated plasma glutarylcarnitine. This type of glutaric aciduria type 1 referred to as a “low-excretor” phenotype carries the same risk of developing brain injury as in a “high-excretor” phenotype. In some low-excreting patients, glutaric acid is elevated only in CSF. Urinary glutarylcarnitine appears to be a more sensitive screening method to identify affected low-excreting patients. In any child with progressive dystonia and dyskinesia, activity of the enzyme glutaryl-CoA dehydrogenase and molecular analysis of GCDH should be performed.

Treatment

Patients require lysine- and tryptophan-restricted diet while meeting physiologic requirements for protein, micronutrients, and vitamins. Increased dietary arginine may decrease cellular uptake of lysine and decrease the endogenous formation of glutaryl-CoA. Patients should be routinely evaluated for lysine and tryptophan deficiency by monitoring plasma amino acids and growth. L -Carnitine supplementation (50-100 mg/kg/24 hr orally) is recommended in all cases. Emergency treatment during acute illness, including temporary cessation of protein intake for 24 hr, replacement of lost calories using carbohydrates or lipids, IV L -carnitine, IV dextrose, prompt treatment of infection, and control of fever, is critical to decreasing the risk of striatal injury. All patients should be provided with an emergency letter describing the underlying diagnosis, recommended evaluation, and treatment. Early diagnosis through newborn screening with prevention and aggressive treatment of intercurrent catabolic states (infections) can help minimize striatal injury and ensure a more favorable prognosis. Patients with movement disorder and spasticity may require treatment with baclofen, diazepam, trihexyphenidyl, and injectable botulinum toxin A.

Glutaric aciduria type 1 is inherited as an autosomal recessive trait. The prevalence is estimated at 1 : 100,000 live births worldwide. The condition is more prevalent in some ethnic populations (Canadian Oji-Cree Indians, Irish Travelers, black South Africans, Swedes, and the Old Order Amish population in the United States). The gene for glutaryl-CoA dehydrogenase (GCDH) is located on chromosome 19p13.2. Molecular analysis of GCDH can aid in identifying patients with a low-excretor phenotype associated with specific pathogenic variants (e.g., p.M405V, p.V400M, p.R227P). High prevalence of known pathogenic variants in specific ethnic populations can enable a cost-effective molecular evaluation and counseling.

Prenatal diagnosis can be accomplished by demonstrating increased concentrations of glutaric acid in amniotic fluid, by assay of the enzyme activity in amniocytes or chorionic villus samples, or by identification of the known pathogenic variants in GCDH .

Lysinuric Protein Intolerance (Familial Protein Intolerance)

This rare autosomal recessive disorder is caused by a defect in the transport of the cationic amino acids lysine, ornithine, and arginine in both intestine and kidneys. Deficiency of the transporter protein (Y+L amino acid transporter 1) in this condition causes multisystem manifestations, which start initially with gastrointestinal (GI) symptoms. 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.

Refusal to feed, nausea, aversion to protein, vomiting, and mild diarrhea, which may result in failure to thrive, wasting, and hypotonia, may be seen shortly after birth. Breastfed infants usually remain asymptomatic until soon after weaning, possibly because of the low-protein content of breast milk. Episodes of hyperammonemia may occur after ingestion of a high-protein meal. 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. Neurocognitive status is usually normal, but moderate intellectual disability has been observed in some patients.

Progressive interstitial pneumonitis with bouts of acute exacerbation often occurs in these patients. This usually progresses to severe alveolar proteinosis. Clinical manifestations include progressive exertional dyspnea, fatigue, cough, diminished breath sound, and inspiratory rales; cyanosis may develop in older patients. 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.

Renal involvement is manifested initially by proteinuria, hematuria, and elevation of serum creatinine, which may progress to end-stage renal failure. Renal tubular involvement with laboratory findings of renal Fanconi syndrome may also be present. Renal biopsy reveals pathologic findings consistent with glomerulonephritis and tubulointerstitial nephritis. Hematologic findings of anemia, leukopenia, thrombocytopenia, and elevated ferritin may also be present. A condition resembling hemophagocytic lymphohistiocytosis/macrophage activation syndrome has also been reported. Immunologic abnormalities (impaired lymphocyte function, abnormalities in immune globulins, hypocomplementemia) and acute pancreatitis are frequent features of lysinuric protein intolerance.

Laboratory findings may reveal hyperammonemia and an elevated concentration of urinary orotic acid, which develop after high-protein feeding. Plasma concentrations of lysine, arginine, and ornithine are usually mildly decreased, but urinary levels of these amino acids, especially lysine, are greatly increased. The pathogenesis of hyperammonemia is likely related to the depletion of urea cycle intermediates caused by poor absorption and the increased renal loss of ornithine and arginine. Plasma concentrations of alanine, glutamine, serine, glycine, and proline are usually increased. Anemia, increased serum levels of ferritin, lactate dehydrogenase (LDH), thyroxine-binding globulin, hypercholesterolemia, and hypertriglyceridemia are common findings. This condition should be differentiated from hyperammonemia caused by urea cycle defects (see Chapter 103.12 ), especially in heterozygous females with OTC deficiency, in whom increased urinary excretion of lysine, ornithine, and arginine is not seen.

Treatment with a low-protein diet providing the RDA of protein and supplemented with oral citrulline (50-100 mg/kg/day) can produce biochemical and clinical improvements. Episodes of hyperammonemia should be treated promptly (see Chapter 103.12 ). Supplementation with lysine (10-30 mg/kg/day) given in small and frequent doses helps improve plasma levels. The dose of lysine should be titrated down if patients develop abdominal pain and diarrhea. Treatment with high doses of prednisone has been effective in the management of acute pulmonary complications in some patients. Bronchopulmonary lavage is the treatment of choice for patients with alveolar proteinosis. The condition is more prevalent in Finland and Japan, where the prevalence is 1 : 60,000 and 1 : 57,000 live births, respectively.

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

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103.15

N -Acetylaspartic Acid (Canavan Disease)

Reuben K. Matalon, Joseph M. Trapasso

Keywords

• Canavan disease
• atypical Canavan disease
• N -acetylaspartic acid
• NAA
• ASPA
• aspartoacylase
• recombinant adeno-associated viruses
• rAAVs
• blood-brain barrier
• leukodystrophy
• progressive macrocephaly

N -Acetylaspartic acid (NAA ), a derivative of aspartic acid, is synthesized in the brain and is found in a high concentration similar to glutamic acid. Studies suggest that NAA has multiple functions, such as serving as an acetate reservoir for myelin synthesis and being an organic osmolyte that helps regulate cerebral osmolality. However, the complete function of NAA is not yet fully understood. Aspartoacylase cleaves the N -acetyl group from NAA. Deficiency of aspartoacylase leads to Canavan disease , a severe leukodystrophy characterized by excessive excretion of NAA and spongy degeneration of the white matter of the brain. Canavan disease is an autosomal recessive disorder and is more prevalent in individuals of Ashkenazi Jewish descent than in other ethnic groups. The defective gene for Canavan disease (ASPA) is located on chromosome 17, and genetic testing can be offered for patients, family members, and at-risk populations.

Etiology and Pathology

The deficiency of the enzyme aspartoacylase leads to NAA accumulation in the brain, especially in white matter, and massive urinary excretion of this compound. Excessive amounts of NAA are also present in the blood and CSF. Brain biopsies of patients with Canavan disease show spongy degeneration of the myelin fibers, astrocytic swelling, and elongated mitochondria. There is striking vacuolization and astrocytic swelling in white matter. Electron microscopy reveals distorted mitochondria. As the disease progresses, the ventricles enlarge because of cerebral atrophy.

Clinical Manifestations

The severity of Canavan disease covers a wide spectrum. Infants usually appear normal at birth and may not manifest symptoms of the disease until 3-6 mo of age, when they develop progressive macrocephaly , severe hypotonia, persistent head lag, and delayed milestones. As the disease progresses, there is spasticity, joint stiffness, and contractures. Optic atrophy and seizures develop. Feeding difficulties, poor weight gain, and gastroesophageal reflux may occur in the 1st yr of life; swallowing deteriorates, and nasogastric feeding or permanent gastrostomy may be required. In the past, most patients died in the 1st decade of life, but with the advances in medical technology and improved supportive care, now they often survive to the 2nd or 3rd decade.

Atypical Canavan Disease

Juvenile or mild Canavan disease is less common than infantile Canavan disease and is most prevalent in non-Ashkenazi Jews. Affected patients with juvenile Canavan disease usually present with mild speech and motor delay and may have retinitis pigmentosa . The other typical features of Canavan disease are usually not present. These children have moderately increased urinary excretion of NAA, which suggests Canavan disease. Brain MRI demonstrates increased signal intensity in the basal ganglia rather than global white matter disease, sometimes leading to confusion with mitochondrial disease.

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. 103.15 ). Repeated evaluations may be required. MRS performed at the time of MRI can be done to show the high peak of NAA, suggesting Canavan disease. The diagnosis can also be established by finding elevated amounts of NAA in the urine or blood. NAA 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 NAA can also be detected in plasma, CSF, and brain tissue. Aspartoacylase in fibroblasts is often used to confirm the diagnosis but is not necessary. The activity of aspartoacylase in the fibroblasts of obligate carriers is half or less the activity found in normal individuals. Genotyping of patients with Canavan disease should always be done and will show mutations of ASPA . The differential diagnosis of Canavan disease should include Alexander disease , which is another leukodystrophy associated with macrocephaly. Alexander disease is caused by a defect in the synthesis of glial fibrillary acidic protein, and the diagnosis can be ruled out by molecular diagnosis on blood lymphocytes.

Fig. 103.15 Axial T-weighted MRI of a 2 yr old patient with Canavan disease. Extensive thickening of the white matter is seen.

There are 2 predominant pathogenic variants leading to Canavan disease in the Ashkenazi Jewish population. The first 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 2nd common pathogenic variant is a change from tyrosine to a nonsense mutation, leading to a stop in the coding sequence (Y231X). This accounts for 13% of mutant alleles. In the non-Jewish population, more diverse pathogenic variants have been observed, and the 2 variants common in Jewish people are rare. A different mutation (A305E), the substitution of alanine for glutamic acid, accounts for 40% of 62 mutant alleles in non-Jewish patients. More than 50 pathogenic variants are 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 NAA level in the amniotic fluid. In Ashkenazi Jewish patients, the carrier frequency can be as high as 1 : 40, which is close to that of Tay-Sachs disease. Carrier screening for Canavan disease is available for Jewish individuals. Genotype phenotype correlation and aspartoacylase expression show that expression studies may aid in understanding the disease.

Patients with juvenile or mild forms of Canavan disease have been compound heterozygotes with a mild pathogenic variant on one allele and a severe variant on the other allele. Mild variants include p.Tyr288Cys and p.Arg71His.

Treatment and Prevention

No specific treatment is currently available. Recent studies of gene therapy using recombinant adeno-associated viruses (rAAVs ) have shown some positive results in rescuing knockout mice but have yet to be tested in humans. Feeding problems and seizures should be treated on an individual basis. Genetic counseling, carrier testing, and prenatal diagnosis are the only methods of prevention. Gene therapy attempts in children with Canavan disease have shown lack of long-term adverse events, some decrease in the brain elevation of N -acetylaspartic acid, improved seizure frequency, and stabilization of overall clinical status.

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