Part 3: Vitamins

VITAMIN B₁₂

Cobalamin (vitamin B₁₂) is a complex organometallic molecule that is synthesized by many bacteria and is obtained in the human diet from meat, fish, and dairy products. It is not present in plant foods, so strict vegetarians are at risk for dietary deficiency. Derivatives of cobalamin are required for the activity of 2 enzymes: methylcobalamin and 5′-deoxyadenosylcobalamin. Methylcobalamin is generated during the catalytic cycle of methionine synthase, a cytoplasmic enzyme that catalyzes methylation of homocysteine to methionine (see Chapter 133). 5′-Deoxyadenosylcobalamin (AdoCbl) is required for the mitochondrial enzyme methylmalonyl-coenzyme A (CoA) mutase to catalyze the conversion of methylmalonyl-CoA, formed during catabolism of branched-chain amino acids and odd-chain fatty acids, to succinyl-CoA (see Chapter 132). Therefore, inborn errors of cobalamin metabolism result in isolated methylmalonic aciduria, isolated homocystinuria, or combined methylmalonic aciduria and homocystinuria, depending on which step in cobalamin metabolism is affected. Without treatment, hyperhomocysteinemia is usually accompanied by hypomethioninemia. Significantly elevated homocysteine levels may be associated with an increased risk of thrombosis. Decreased methionine is associated with abnormalities of the white matter of the nervous system. Elevated levels of methylmalonic acid can lead to metabolic acidosis.

METABOLISM OF COBALAMIN

Cobalamin consists of a planar corrin ring with a central cobalt atom, a 5,6-dimethylbenzimidazole base, and an upper axial ligand attached to the cobalt atom that varies in different forms of cobalamin. Physiologically important cobalamins include hydroxycobalamin (OHCbl), methylcobalamin (MeCbl), and adenosylcobalamin (AdoCbl). The most common commercially available form of vitamin B₁₂ contains a cyano group in the upper axial position (CNCbl). The central cobalt can exist in the oxidized Co³⁺ state (cob[III]alamin), the Co²⁺ state (cob[II]alamin), or the fully reduced Co¹⁺ state (cob[I]alamin). Converting exogenous cobalamin, typically in the form of cob(III)alamin, to its biologically active coenzyme forms involves reducing the central cobalt to cob(I)alamin and then adding the appropriate upper axial ligand (Fig. 142-1).

Absorption of dietary cobalamin is a complex process that is dependent on several cobalamin-binding proteins. Cobalamin is released from food in the acidic environment of the stomach, where it becomes bound to transcobalamin I (TCN1) found in saliva and gastric juice. Transcobalamin I is broken down in the intestine by proteolytic enzymes, and cobalamin binds the gastric intrinsic factor (GIF). The GIF-cobalamin complex is taken up by enterocytes in the distal ileum in a process mediated by a receptor, cubam, composed of 2 proteins, cubilin (CUBN) and amnionless (AMN). After uptake by enterocytes, cobalamin is released into the circulation, where it binds the transport protein transcobalamin II (TCN2).

In blood, transcobalamin II–bound cobalamin is available for uptake by most cell types. Following endocytosis, mediated by the transcobalamin receptor (TCBLR encoded by CD320), the cobalamin–transcobalamin II complex dissociates in lysosomes, and free cobalamin is transferred to the cytoplasm with the participation of at least 2 proteins LMBRD1 (cblF) and ABCD4 (cblJ). Upon egress of the cobalamin from the lysosome, the vitamin begins the cytosolic pathway of metabolism. This process involves MMACHC (cblC), which removes axial cobalamin moieties, reduces Co³⁺ to Co²⁺, and facilitates the transfer of cobalamin to MMADHC (cblD). Subsequently, cobalamin becomes associated with methionine synthase in the cytoplasm, or it may be further transported to the mitochondria, where it is converted to 5’-deoxyadenosylcobalamin, the active cofactor for methylmalonyl-CoA mutase. The steps of cellular cobalamin metabolism common to synthesis of both coenzyme derivatives remain under study.

The catalytic cycle of methionine synthase involves transfer of a methyl group from 5-methyl-THF to an enzyme-bound cob(I)alamin molecule, forming methylcobalamin (MeCbl). The methyl group is then transferred from MeCbl to homocysteine, forming methionine and regenerating cob(I)alamin. Occasionally the cob(I)alamin is oxidized to cob(II)alamin; when this occurs, regeneration of MeCbl requires the activity of a second protein, methionine synthase reductase, which uses adenosylmethionine as its methyl group donor. Remethylation of homocysteine to form methionine thus requires the activity of both methionine synthase (cblG) and methionine synthase reductase (cblE).

The system by which cobalamin enters the mitochondria is unknown, as is the form of cobalamin that crosses the mitochondrial membrane. Mitochondrial reductases that can support reduction of cob(II)alamin to cob(I)alamin have been identified in vitro, but the enzyme that catalyzes this reaction in vivo is not yet known. Cob(I)alamin receives the adenosine base from ATP to form 5’-deoxyadenosylcobalamin (AdoCbl) in a reaction catalyzed by cob(I)alamin adenosyltransferase, encoded by the MMAB gene (cblB). Another protein, the product of the MMAA gene (cblA), appears to play a role in supporting the formation of holo-methylmalonyl-CoA mutase and in maintaining the enzyme-bound cobalamin in its active form. The presence of methylmalonyl-CoA mutase, cobalamin adenosyltransferase, and the MMAA protein, possibly in association with a cob(II)alamin reductase, is required for conversion of methylmalonyl-CoA to succinyl-CoA in vivo.

ISOLATED METHYLMALONIC ACIDURIA

METABOLIC DERANGEMENT

Isolated methylmalonic aciduria is seen in patients with methylmalonyl-CoA mutase deficiency (Online Mendelian Inheritance in Man [OMIM] no. 251000), cblA deficiency (OMIM no. 251100), cblB deficiency (OMIM no. 251110), and cblD deficiency variant 2 (OMIM no. 277410). Methylmalonyl-CoA mutase deficiency, also referred to as the mut disorder is caused by mutations in the MUT gene on chromosome 6p12.3. The mut disorder has been subdivided into 2 classes: (1) in mut⁰, there is no stimulation of mutase activity in cultured cells on incubation with hydroxycobalamin (OHCbl), and there is often no detectable mutase protein; (2) in mut^–, mutase activity is stimulated by addition of OHCbl. Deleterious mutations in the MMAA gene on chromosome 4q31.21 result in decreased synthesis of adenosylcobalamin (cblA deficiency). The cblB deficiency is caused by mutations in the MMAB gene on chromosome 12q24.11. Patients with mutations resulting in the loss of function of cblD required for the AdoCbl synthesis also experience isolated methylmalonic aciduria and are referred to as having cblD deficiency variant 2.

CLINICAL PRESENTATION

Patients with isolated methylmalonic aciduria are prone to episodes of life-threatening acidosis, often in response to infection or to increased protein intake. Initial presentation is most frequently during infancy, with vomiting, hypotonia, irritability, and lethargy, which can progress to coma and death if not treated. In the most severely affected individuals, there may be intractable acidosis during the neonatal period that leads to death, but in many patients, the disorder is characterized by recurrent acidotic crises characterized by high anion gap metabolic acidosis, ketoacidosis, lactic acidosis, and hyperammonemia. Anemia, neutropenia, thrombocytopenia, failure to thrive, optic nerve atrophy, acute and chronic kidney disease, pancreatitis, and basal ganglia injury occur variably, but are well-recognized complications of the disorder. Studies of a series of patients have shown that presentation is typically most severe in individuals with mut⁰. The clinical course of patients with cblB deficiency may resemble that of mut⁰ patients. cblA, cblD variant 2, mut^–, and rare OHCbl-responsive cblB patients have the least severe presentation and the best prognosis. Clinically unstable patients with severe methylmalonic acidemia may be candidates for an elective liver transplantation. Progression to end-stage renal failure will necessitate kidney or combined liver-kidney transplantation.

DIAGNOSTIC TESTS

Analysis of urinary organic acids from these patients reveals elevated levels of methylmalonic acid and propionyl-CoA derived metabolites such as methylcitric acid, 3-hydroxypropionic acid, and tiglylglycine. Amino acid analysis usually reveals elevated glycine in blood and urine, while the acylcarnitine carnitine profile shows elevated propionylcarnitine and methylmalonylcarnitine. Accumulation of CoA conjugates may result in secondary carnitine deficiency necessitating carnitine supplementation.

Elevation of methylmalonic acid levels may also be seen in individuals with methylmalonyl-CoA epimerase (racemase) deficiency and in patients with mutations affecting either subunit of succinyl-CoA ligase; however, in these orders, the levels are lower than in mut, cblA, and cblB patients. Mild elevations of methylmalonic acid need to be distinguished from dietary vitamin B₁₂ deficiency and other forms of abnormal cobalamin absorption in the gastrointestinal tract. Elevations of both malonic and methylmalonic acids on the urine organic acid assay can be seen in the combined malonic and methylmalonic aciduria (CMAMMA; OMIM no. 614265) due to mutations in ACSF3. Differentiation between the different forms of methylmalonic aciduria depends on molecular and biochemical methods. DNA-based molecular genetic testing has become the initial confirmatory step. If DNA analysis does not reveal the diagnosis, cellular biochemical testing using cultured fibroblasts becomes necessary.

GENETICS

All 4 disorders are inherited as autosomal recessive traits. Mutations in most families are private, but several mutations are relatively common within a specific ethnic group. Most pathogenic variants are missense or nonsense mutations, but copy number variants (deletions and insertions) have also been described. Genotype-phenotype correlations are difficult, but in general, patients with 2 nonsense, larger copy number variants and known severe missense mutations have a more severe course of the disease and should be considered for transplantation sooner. Patients with cblD deficiency variant 2 appear to have mutations in exon 3 and 4 of the MMADHC gene encoding a protein domain required for the AdoCbl synthesis.

TREATMENT

Patients with isolated methylmalonic aciduria require life-long monitoring and management. Episodes of acute metabolic decompensation are treated with intravenous fluids containing glucose, appropriate electrolytes, and if necessary, bicarbonate replacement. Dietary goals include a brief restriction of protein intake and supplementation of calories through carbohydrate and lipids. OHCbl responsiveness needs to be established in all patients. Acute mental status changes and new neurologic findings, even in the absence of acidosis, should be treated as metabolic emergencies. Chronic dietary management may require the judicious use of medical foods deficient in amino acids (ie, isoleucine, valine, threonine, and methionine), the precursors of methylmalonic acid. Due to poor palatability of protein-modified diet, frequent nausea, vomiting, and poor appetite, most patients need a gastrostomy tube to facilitate enteral feeding. Carnitine supplementation is used to prevent and treat secondary carnitine deficiency. Patients who experience frequent and severe decompensations may need to be evaluated for liver or combined liver-kidney transplantation. Liver and liver-kidney transplantation helps stabilize patients metabolically and reduce the number of hospitalizations secondary to acidotic decompensations and hyperammonemia. Even after liver and liver-kidney transplantation, patients need to continue a protein-restricted diet and should be monitored for secondary carnitine deficiency. Any changes in the neurologic status should prompt proactive management of metabolic status, as vision loss and brain injury can occur even after liver transplantation. The decision to undergo organ transplantation requires a consideration of many factors including the severity of the disorder, organ availability, rate of surgical complications, and the need for life-long immunosuppression.

ISOLATED HOMOCYSTINURIA

METABOLIC DERANGEMENT

Homocystinuria in the absence of methylmalonic aciduria is seen in patients with the cblE (OMIM no. 236270), cblG (OMIM no. 250940), and cblD variant 1 (OMIM no. 277410) deficiencies. In all 3 disorders, methionine synthase function is impaired; in the cblG deficiency, synthase-specific activity in cell extracts is decreased under all assay conditions, while in cblE deficiency, specific activity is decreased only in the presence of limiting concentrations of exogenous reducing agent. The cblG deficiency is caused by mutations affecting the MTR gene on chromosome 1q43, which encodes methionine synthase. The cblE deficiency is caused by mutations of the MTRR gene on chromosome 5p15.31, which encodes methionine synthase reductase. cblD variant 1 deficiency is caused by deleterious mutations in the gene MMADHC on chromosome 2q23.2 encoding a protein that participates in the intracellular conversion of cobalamin to adenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl).

CLINICAL PRESENTATION

Patients with these disorders manifest with megaloblastic anemia and neurologic problems, including developmental delay, cerebral atrophy, hypotonia, microcephaly, and seizures. Some patients may present with hemolytic-uremic syndrome, renal artery thrombosis, and pulmonary hypertension. In some patients, total plasma homocysteine can be as high as in patients with homocystinuria due to cystathionine β-synthase deficiency. Presentation is typically within the first 2 years of life, but it can also present in adulthood.

DIAGNOSTIC TESTS

Decreased methionine synthase activity results in increased total plasma homocysteine and in homocysteine in the urine, as well as reduced levels of methionine and elevated cystathionine. Serum cobalamin levels are normal. These disorders can be differentiated from homocystinuria due to cystathionine β-synthase deficiency, which presents with generally higher levels of homocysteine, elevated methionine, and decreased cystathionine levels (see Chapter 133). The diagnosis is confirmed either by DNA analysis of the implicated genes or by biochemical studies of patient’s fibroblasts. Affected fibroblasts are characterized by decreased methyl-THF incorporation with normal propionate incorporation and by decreased methylcobalamin synthesis with normal adenosylcobalamin synthesis. The disorders can be differentiated by the response of methionine synthase–specific activity to the titration of a reducing agent, but more typically, they are differentiated by complementation analysis.

GENETICS

All 3 disorders are inherited as autosomal recessive traits. Genotype-phenotype correlations are difficult to establish. Most described mutations are missense or nonsense. Patients with CblD variant 1 defect tend to have mutations in exons 6 and 8 of the MMADHC gene encoding a protein domain responsible for the MeCbl synthesis.

TREATMENT

Affected individuals can tolerate the recommended daily allowance of protein and need to avoid dietary methionine deficiency. For this reason, medical foods deficient in methionine are not recommended in this group of disorders. Patients respond to OHCbl injections. Supplementation with betaine (250 mg/kg/24 h) facilitates the conversion of homocysteine to methionine through an alternative pathway independent of methionine synthase, via hepatic betaine-homocysteine methyltransferase. To support remethylation reactions, patients with hyperhomocysteninemia are supplemented with folic or folinic acid. In patients with significant hyperhomocysteinemia, attention should be given to the management of risk factors predisposing to hypercoagulability.

COMBINED METHYLMALONIC ACIDURIA AND HOMOCYSTINURIA

METABOLIC DERANGEMENT

Five inborn errors of metabolism that affect early steps in cobalamin metabolism result in combined methylmalonic aciduria and homocystinuria. The cblC disorder (OMIM no. 277400) is caused by mutations in MMACHC on chromosome 1p34.1. The cblD disorder (OMIM no. 277410) is caused by mutations in MMADHC on chromosome 2q23.2. Products of these genes participate in the conversion of Co³⁺ to Co²⁺ and removal of the axial ligand. Although the original patients with the cblD disorder had combined methylmalonic acid and homocystinuria, additional patients with isolated homocystinuria (cblD variant 1) and isolated methylmalonic aciduria (cblD variant 2) have subsequently been recognized. The cblF (OMIM no. 277380) deficiency and cblJ (OMIM no. 614857) deficiency result in the inability to transfer endocytosed cobalamin from the lysosome to the cytoplasm. cblX deficiency (OMIM no. 309541) is a unique form of combined methylmalonic acidemia and homocystinuria. It is caused by mutations in the gene HCFC1 on chromosome X, which encodes a transcriptional coregulator involved in expression of MMACHC (cblC).

CLINICAL PRESENTATION

Most patients with combined methylmalonic aciduria and homocystinuria belong to the cblC class. Without treatment, individuals with this disorder typically present during the first year of life with megaloblastic anemia, failure to thrive, developmental delay, hypotonia, seizures, macrocephaly, and cerebral atrophy. Other patients present later in life or in adulthood with ataxia, dementia, or psychosis. Most patients with cblC deficiency develop a bull’s eye maculopathy and then optic nerve pallor. Retinal degeneration in cblC deficiency can be seen as early as 2 months of age and appears to be resistant to treatment with OHCbl. Noncompaction cardiomyopathy can be present in some patients, necessitating life-long surveillance of the cardiac function. Despite the presence of methylmalonic aciduria, metabolic decompensations are infrequent.

Presentation in classic cblD deficiency is similar to that of cblC. cblF and cblJ deficiencies are clinically variable but tend to show better response to treatment with parenteral hydroxocobalamin. Frequent findings have included feeding difficulties, failure to thrive, growth retardation, and persistent stomatitis. Due to the likely role of LMBRD1 (cblF) and ABCD4 (cblJ) in the intestinal uptake of dietary cobalamin, patients may present with low plasma cobalamin levels. The clinical picture of cblX disorder is dominated by developmental delays, intellectual disability, and intractable epilepsy.

DIAGNOSTIC TESTS

Patients with these disorders have the biochemical characteristics of both methylmalonic aciduria and homocystinuria, although methylmalonic acid levels are generally lower than in patients with isolated methylmalonic aciduria. Fibroblasts show decreased function of both methylmalonyl-CoA mutase and methionine synthase, and decreased synthesis of both adenosylcobalamin and methylcobalamin (Table 142-1). Fibroblasts from patients with the cblC or cblD disorder are unable to accumulate cobalamin in cells, reflecting the inability of cells to retain cobalamin that is not bound to one of the cobalamin-dependent enzymes. In cblF and cblJ disorder, fibroblasts accumulate large amounts of cobalamin, but most of this is unmetabolized cobalamin that is trapped within the lysosomes.

GENETICS

cblC, cblD, cblF, and cblJ deficiencies are inherited as autosomal recessive traits. Over 40 mutations in the MMACHC gene have been identified. The most common of these is a c.271dupA mutation that represents 40% of identified mutant alleles, primarily in patients of European or western Asian origin. It is associated with early-onset severe disease when homozygous. Several other mutations show evidence of clustering in specific ethnic groups, and genotype-phenotype correlations have been observed. A small number of mutations in the MMADHC gene have been identified in patients with the much rarer cblD disorder. The classical cblD disorder is associated with truncating mutations, while mutations at the C-terminal domain are asso­ciated with isolated homocystinuria (variant 1), and mutations in the N-terminal domain are associated with isolated methylmalonic aciduria (variant 2). Most known mutations in cblF are truncating, resulting in the loss of function of it respective protein. cblJ deficiency is very rare, and the full spectrum of mutations is under study. The HCFC1 gene implicated in the cblX disorder is located on the distal subband of chromosome Xq28 and is inherited as an X-linked trait. HCFC1 encodes a chromatin-associated protein playing a role in the transcriptional regulation of gene expression. Impaired function of HCFC1 results in dysregulated expression of MMACHC (cblC) and likely genes encoding components of the glycine cleavage complex, which may explain biochemical findings and intractable epilepsy observed in these patients.

TREATMENT

Patients with combined methylmalonic aciduria and homocystinuria are treated with large doses of cobalamin (up to 0.35 mg/kg/24 h, up to the maximum dose of 30 mg/24 h) administered subcutaneously or intramuscularly. Higher doses require concentrated forms of OHCbl to reduce the volume. Hydroxycobalamin is recommended over more widely available CNCbl; this may be true for other cobalamin disorders as well. Patients can tolerate the recommended daily allowance of protein. Medical foods deficient in methionine are not recommended in these patients. Carnitine supplementation is used to treat secondary carnitine deficiency. Supplementation with betaine, which supports conversion of homocysteine to methionine by the liver enzyme betaine-homocysteine methyltransferase, has proved effective in patients with homocystinuria. In patients with persistent hyperhomocysteinemia, attention should be given to the management of risk factors predisposing to hypercoagulability.

INBORN ERRORS OF COBALAMIN UPTAKE AND TRANSPORT

Combined methylmalonic aciduria and homocystinuria is also seen in patients with defects affecting intestinal uptake of cobalamin, its transport in the serum, and cellular uptake. Decreased uptake occurs in individuals with intrinsic factor deficiency (OMIM no. 261000), caused by mutations in the GIF gene on chromosome 11q12.1, and in those with Imerslund-Gräsbeck syndrome (IGS; OMIM no. 261100). The latter disorder is the result of dysfunction of the vitamin B₁₂ receptor in the distal ileum, which can be caused by mutations at the CUBN gene on chromosome 10p13 or the AMN gene on chromosome 14q32, which encode cubilin and amnionless, the respective components of the cubam complex. Additional IGS families are not linked to either chromosome and represent further genetic heterogeneity in this disorder. Patients with these disorders usually present between 1 and 5 years of age with decreased serum cobalamin levels, megaloblastic anemia, and neurologic impairment. IGS patients often have proteinuria as well since cubilin and amnionless also mediate protein homeostasis in renal tubules. These disorders show an autosomal recessive inheritance. Clusters of IGS have been identified in Finland (CUBN mutations), Norway (AMN mutations), and the eastern Mediterranean (both genes). In the absence of readily available clinical tests to assess cobalamin uptake, specific diagnosis in these patients depends on molecular analysis of the GIF, CUBN, and AMN genes. Intrinsic factor deficiency and IGS require life-long treatment with parenteral hydroxocobalamin.

Patients with transcobalamin II deficiency (OMIM no. 275350) usually present in the first months of life with megaloblastic anemia, failure to thrive, weakness, and, frequently, immunologic dysfunction. Without treatment, neurologic impairment develops. Most cobalamin circulating in blood is bound to transcobalamin II. Cobalamin bound to transcobalamin II is not available for uptake by most types of cells; rather, transcobalamin II is the primary transporter of vitamin B₁₂ to peripheral tissues. Thus, total serum cobalamin levels may be normal, while the unsaturated vitamin B₁₂ binding capacity is reduced. The disorder is inherited as autosomal recessive trait. Mutations in the TCN2 gene on chromosome 22q12 have been identified in a small number of patients. Treatment involves maintaining very high serum levels of cobalamin by intramuscular route.

The uptake of cobalamin bound to transcobalamin II is mediated by the transcobalamin receptor (CD320, OMIM no. 606475). Known patients were identified on the newborn screen based on elevated propionylcarnitine (C3) in blood spots. Patients tend to have mildly elevated methylmalonic acidemia with normal total plasma homocysteine at birth and readily respond to hydroxocobalamin injections. Plasma vitamin B₁₂ levels can be elevated even without cobalamin supplementation. Studies of cellular vitamin B₁₂ metabolism using fibroblasts reveal normal propionate and methyltetrahydrofolate metabolism, normal conversion of cyanocobalamin to adenosylcobalamin and methylcobalamin, but reduced cobalamin binding and uptake. Methylmalonic acidemia appears to be transient; however, the long-term outcome and natural history of this condition are poorly understood.

FOLIC ACID

The folates are a class of molecules comprising folic acid and its derivatives. Biologically active folates consist of folic acid reduced to a tetrahydrofolate (THF) derivative through the attachment of 1-carbon groups to N⁵ and N¹⁰ of the pterin ring, and by addition of up to 6 additional glutamate residues by γ-peptide linkage. Folate is synthesized by many types of bacteria and is obtained in the diet from both plant and animal foods.

Within cells, folates accept 1-carbon units from various sources, primarily serine (via the reaction catalyzed by serine hydroxymethyltransferase) but also from glycine (the glycine cleavage system), formate (formyl-THF synthetase), and histidine (breakdown mediated by glutamate formiminotransferase and formiminotetrahydrofolate cyclodeaminase). Several reactions utilize 1-carbon units derived from folates (Fig. 142-2). Methyl-THF is required for methylation of homocysteine to form methionine, catalyzed by methionine synthase (described in the section on vitamin B₁₂; see also Chapter 133). Catalytic conversion of uridylate to thymidylate requires 5,10-methylene-THF for the activity of thymidylate synthase. Carbons 2 and 8 of the purine ring are added by the folate-dependent enzymes GAR transformylase and AICAR transformylase, both of which use 10-formyl-THF as a 1-carbon donor. Thus, synthesis of both purines and 1 of 2 pyrimidines required for DNA synthesis is dependent on folates.

Interconversion of 1-carbon substituted folates is an important aspect of cellular metabolism. 5,10-Methylene-THF can be reduced to 5-methyl-THF by the enzyme methylene-THF reductase (MTHFR). Alternatively, it can be oxidized to form 10-formyl-THF by sequential reactions catalyzed by methylene-THF dehydrogenase and methenyl-THF cyclohydrolase. The 2 activities are catalyzed by a single multifunctional protein called C1-THF synthase (OMIM no. 172460). The reaction catalyzed by MTHFR is irreversible under physiologic conditions. This means that when activity of methionine synthase, the only reaction that utilizes methyl-THF, is impaired (as in cobalamin deficiency or in the cblC, cblD, cblF, cblJ, cblE, and cblG disorders), cellular folate is trapped in its methylated form, resulting in deficiency of other folate coenzyme forms.

MTHFR DEFICIENCY

The most common inborn error of folate metabolism is MTHFR deficiency (OMIM no. 236250) due to mutations of the MTHFR gene on chromosome 1p36.22 (see Chapter 133). Decreased MTHFR activity results in deficiency of methyl-THF, the source of the methyl group used by methionine synthase. Thus, biochemically, MTHFR deficiency is characterized by hyperhomocysteinemia and homocystinuria with hypomethioninemia. Clinical presentation varies, but patients usually show symptoms during infancy or early childhood that include feeding difficulties, lethargy, hypotonia, developmental delay, and seizures. Cerebral atrophy and demyelination are often present. Patients with later onset may have intellectual disability, ataxia, or psychiatric problems. Unlike patients with inborn errors of cobalamin metabolism, in which methionine synthase activity is impaired, patients with MTHFR deficiency do not have megaloblastic anemia. Cultured fibroblasts have decreased incorporation of label from [¹⁴C]formate into cellular macromolecules, but incorporation of label from [¹⁴C]methyl-THF is normal. Diagnosis depends on molecular DNA analysis revealing deleterious mutations or enzyme assay of cell extracts demonstrating reduced MTHFR-specific activity.

MTHFR deficiency is inherited as an autosomal recessive trait. Almost all mutations have been restricted to 1 or 2 families—an exception is a c.1129C>T mutation that is present at a high frequency among the Old Order Amish.

Patients with MTHFR deficiency have been treated with a variety of agents, including methyl-THF and other folates, methionine, pyridoxine, cobalamin, carnitine, betaine, and riboflavin, individually and in various combinations. Of these, betaine appears to be the most effective. Treatment is most successful when the disease is diagnosed at an early stage, before irreversible neurologic damage occurs.

MTHFR deficiency needs to be differentiated from common polymorphism in the MTHFR gene Common polymorphisms of the MTHFR gene result in decreased enzyme activity less severe than that seen in patients with severe MTHFR deficiency. The most well studied of these is a c.665C>T (formerly c.677C>T) variant that results in “thermolability” of the enzyme. Homozygosity for the c.665C>T variant is associated with increased serum homocysteine levels in individuals with low folate intake, but the clinical significance of this variant is doubtful.

GLUTAMATE FORMIMINOTRANSFERASE DEFICIENCY

A small number of patients have been identified who have deficiency of the bifunctional enzyme that catalyzes the transfer of the formimino group from formiminoglutamate (FIGLU), generated during histidine catabolism, to THF to form 5-formimidoyl-THF, which is then converted to 10-formyl-THF. Patients with glutamate formiminotransferase deficiency (OMIM no. 229100) are characterized by elevated FIGLU levels either constitutively or in response to a histidine load. Because the affected enzyme is expressed only in liver and kidney, using enzyme assay to confirm the diagnosis is performed using molecular DNA analysis. Two classes of patients have been reported: one was characterized by intellectual disability, physical retardation, and cortical atrophy, while the second showed no intellectual disability but massive FIGLU excretion. Therefore, it has been suggested that the severe manifestations in the first group of patients were the result of ascertainment bias.

The gene encoding the bifunctional enzyme, FTCD, has been identified on chromosome 21q22.3, and causative mutations have been identified in several patients. It remains unclear whether there is any clinical phenotype beyond FIGLU excretion in this disorder. The disorder is inherited as autosomal recessive trait and is amenable to newborn screening.

HEREDITARY FOLATE MALABSORPTION

Mutations in a proton-coupled folate transporter result in reduced intestinal folate uptake (OMIM no. 229050), impaired transport across the blood-brain barrier, and ultimately intracellular folate deficiency. Therefore, presentation typically occurs in the first months of life with diarrhea, failure to thrive, developmental delay, megaloblastic anemia, and neurologic deterioration. Serum and cerebrospinal fluid folate levels are reduced. Patients with this autosomal recessive disorder have mutations in the SLC46A1 gene on chromosome 17q11.2. Optimal treatment involves maintaining adequate levels of folate in the central nervous system to avoid neurologic impairment; the appropriate level must be determined for each patient individually and may involve administration of very large doses of folate.

SUGGESTED READINGS

INTRODUCTION

Biotin (vitamin B₇ or vitamin H) is a water-soluble vitamin that functions as a carboxyl carrier in carboxylation, decarboxylation, and transcarboxylation reactions, and is attached to a lysine residue in a highly conserved domain common to all biotin-dependent carboxylases. The 5 biotin-dependent carboxylases in humans are mitochondrial propionyl-coenzyme A (CoA), β-methylcrotonyl-CoA, pyruvate carboxylases, and mitochondrial and cytosolic forms of acetyl-CoA carboxylase, with each carboxylase having specific roles in fatty acid, glucose, and amino acid metabolism. Biotin is covalently attached to carboxylases by the enzyme holocarboxylase synthetase (HLCS) encoded by the HLCS gene, while biotinidase, encoded by the BTD gene, provides free biotin following proteolysis of the carboxylase protein. Hence, biotinidase deficiency (Mendelian Inheritance in Man [MIM] no. 253260) reflects an inability to hydrolyze biocytin (biotinyllysine) or small biotin-containing peptide fragments from carboxylases, leading to a lack of free biotin, while holocarboxylase synthetase deficiency (MIM no. 253270) reflects a failure to incorporate biotin into the apoenzymes. The activity of HLCS involves a 2-step, adenosine triphosphate (ATP)-dependent reaction in which biotin is first activated to biotinyl-5’-adenosine monophosphate (AMP) and then transferred to the apocarboxylase substrate, with release of AMP. Failure to attach or recycle biotin leads to significantly reduced activity of these biotin-dependent carboxylases and results in multiple carboxylase deficiency. Humans cannot synthesize biotin, and they obtain it from exogenous sources via intestinal absorption. Absorption of biotin and transport of the vitamin into a variety of cell types occurs via a saturable, Na⁺-dependent, carrier-mediated mechanism that involves the human sodium-dependent multivitamin transporter, hSMVT, encoded by the SLC5A6 gene, which has been shown to also transport pantothenate and lipoate.

More recently, HLCS has also been shown to have an additional role as a nuclear protein that may catalyze the binding of biotin to distinct lysine residues in chromatin proteins such as histones, although the specific mechanisms at play remain under debate. HLCS-dependent epigenetic marks are reported to be abundant in genomic regions that are subject to transcriptional repression. HLCS has been proposed to be a member of a multi-protein gene repression complex that directs its localization within chromatin. HLCS has also been linked to the regulation of histone deacetylases in a biotin-independent manner and to the regulation of heat shock proteins and inflammatory cytokines. Hence, HLCS appears to be a multifaceted protein involved in intermediary metabolism, as a transcriptional repressor, and in gene regulation of biotin-dependent and independent pathways.

BIOTINIDASE AND HOLOCARBOXYLASE SYNTHETASE DEFICIENCY

Deficiencies of biotinidase and HLCS are autosomal recessive disorders with considerable clinical variability. Newborn screening for biotinidase deficiency has been carried out since the 1980s using direct enzyme testing. HLCS deficiency is screened for by tandem mass spectroscopy using elevated propionylcarnitine (C3) and hydroxypentanoylcarnitine (C5-OH) on dried blood spots. The urine organic acid profile may demonstrate elevated lactic, 3-OH isovaleric, 3-OH propionic, 3-methylcrotonyl, methylcitric, and tiglylglycine consistent with loss of function of the above carboxylases.

Prior to the advent of universal newborn screening in the United States, the age of onset of symptoms was used in the initial diagnostic evaluation to differentiate between HLCS deficiency and biotinidase deficiency, with biotinidase generally presenting after 3 months. However, milder forms of HLCS deficiency are now recognized, and this distinction is not so clear. The typical triad of multiple carboxylase deficiency is of alopecia, dermatitis, and encephalopathy. Clasically HLCS deficiency presents in the neonatal period with emesis, hypotonia, lethargy, seizures, metabolic ketolactic acidosis, hyperammonemia, developmental delay, skin rash, and alopecia. Left untreated, infants will progress to profound metabolic acidosis, cerebral edema, coma, and death. However, milder cases have been observed, with ketolactic acidemia associated with infections. Untreated, biotinidase deficiency usually presents later in infancy with periorificial dermatitis resembling acrodermatitis enteropathica, patchy alopecia, and neurologic abnormalities such as ataxia, neurosensory defects, developmental delay, and epilepsy. Cerebral calcifications may be seen. In both conditions, the rash may be complicated by superinfection with monilia.

DIAGNOSIS

Two forms of biotinidase deficiency are recognized: profound biotinidase deficiency with enzymatic activity below 10% of control values, and partial biotinidase deficiency with activity between 10% and 30% of control values. Treatment is universally accepted for profound deficiency; however, the need to treat partial deficiencies with biotin remains unsettled, with only anecdotal reports of a rash, hypotonia, and hair loss in untreated patients. The potential benefits of avoiding any ectodermal complications and preventing potential seizures can be discussed with parents; however, partial biotinidase patients generally have no biochemical abnormalities in blood and urine and have a benign natural course. The carrier frequency in European populations for a partial loss of function missense variant p.D444H is about 4%, and partial deficiency cases typically have this variant in conjunction with a severe allele. Biochemical testing in symptomatic patients with profound biotinidase or HLCS deficiency by urinary organic acid analysis shows increased 3-methylcrotonylglycine and 3-hydroxyisovaleric acid, often with methylcitric, 3-hydroxypropionic, and lactic acids. Acylcarnitine analysis by tandem mass spectroscopy shows propionyl- and 3-hydroxyisovalerylcarnitine, along with lactic acidemia. Holocarboxylase synthetase deficiency can be demonstrated in fibroblasts or leukocytes by measuring individual carboxylase activities. Biotinidase activity is typically assayed in serum, although the enzyme is labile, and this may lead to falsely reduced activity. DNA testing can confirm the diagnosis.

TREATMENT

The mainstay of treatment for both biotinidase and HLCS deficiency is biotin supplementation in an effort to alleviate the symptoms of the enzyme deficiency or prevent symptoms from developing in asymptomatic individuals. The former responds promptly to 5 to 10 mg of biotin, whereas much higher doses may be required for the latter disorder. Effectiveness can be monitored by acylcarnitine profiling, determining lactate concentrations, and analysis of urinary organic acids.

BIOTIN-RESPONSIVE BASAL GANGLIA DISEASE

Biotin-responsive basal ganglia disease (BBGD), also known as biotin-thiamine–responsive basal ganglia disease (BTBGD), is an autosomal recessive disorder that presents in early childhood with confusion, dysarthria, external ophthalmoplegia, and dysphagia, often in conjunction with intercurrent febrile illness and, if untreated, progresses to dystonia, quadriplegia, and eventual death. Cerebral magnetic resonance imaging shows bilateral central necrosis of the caudate head with variable involvement of the putamen. In the initial reports, although symptoms disappeared within a few days of biotin administration (5–10 mg/kg/d) and reappeared within 1 month if biotin was discontinued, it has subsequently been reported that one-third of patients who were initially responsive to biotin alone subsequently relapsed, but when thiamine was added, further improvement was observed, with no further episodes of decompensation. A recent comparison of thiamine-only (40 mg/kg/d) versus thiamine plus biotin treatment observed minimal difference in outcome. The basis for biotin responsiveness is currently unknown. To date, almost 90 patients have been described, many from consanguineous Middle Eastern families. The defect is in a thiamine transporter (hTHTR2) on the cell surface encoded by the SLC19A3 gene that has no demonstrated involvement with biotin transport. It has been speculated that the higher level of biotin after administration may increase the activity of biotin-dependent enzymes, resulting in an overall improvement of energy production, which is impaired in thiamine deficiency.

Finally, a single child has been described with biallelic variants in the human sodium-dependent multivitamin transporter, hSMVT, encoded by the SLC5A6 gene. The child exhibited failure to thrive, microcephaly and brain anomalies, spasticity, developmental delay, variable immunodeficiency, osteoporosis, and pathologic bone fractures.

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VITAMIN B₁ (THIAMINE)

INTRODUCTION

Thiamine has long been recognized as an essential nutrient. Its minimal essential requirement is about 0.5 mg/1000 kcal, which is usually obtained through a normal, well-balanced diet (recommended daily thiamine intake ranges from 0.2 mg in neonates to 1–1.2 mg in adults). However, requirements are variable and increase in parallel with carbohydrate intake and during pregnancy, lactation, and hypermetabolic states. Thiamine is physiologically active in its phosphorylated form, thiamine pyrophosphate (TPP), which is a coenzyme for pyruvate dehydrogenase (PDH), α-ketoglutarate dehydrogenase, and branched-chain α-ketoacid dehydrogenase (BCKDH) in the mitochondrion; 2-hydroxyacyl-CoA lyase in the peroxisome; and transketolase (pentose phosphate pathway) in the cytosol. Being placed at these highly regulated enzymatic steps, thiamine plays a crucial role in carbohydrate metabolism and in the metabolic switch from the fed to the fasting state.

Acute acquired thiamine deficiency states (as occurs with total parenteral nutrition without thiamine supplementation or beriberi) are life-threatening emergencies and present with cardiac failure, Gayet-Wernicke encephalopathy, and/or lactic acidosis. Metabolic markers include elevated serum lactate and pyruvate with normal lactate-to-pyruvate ratio, slight elevations of branched-chain amino acids in plasma, the presence of α-keto acids (ketoglutarate, pyruvate, branched-chain keto acids) in urine with a positive dinitrophenylhydrazine (DNPH) reaction, and low transketolase activity in red blood cells. However, these markers are rarely available in an emergency, and the diagnosis relies on the primary care or emergency physician recognizing the disorder and administering the lifesaving therapeutic test of thiamine 5 mg/kg/d. There is no significant risk of adverse effects from supplementation.

METABOLISM

Through the diet, thiamine is consumed as phosphate derivatives and converted into free thiamine by phosphatases present in the lumen of the small intestine. The epithelia of the small intestine contain two thiamine transporters, THTR1 and THTR2, which are localized to the basal and apical membranes of the polarized cell, respectively (these transporters are variably expressed in other tissues). Within the cell, free thiamine is converted into TPP in the cytosol by thiamine pyrophosphokinase (TPK). A mitochondrial TPP carrier (TPC) imports TPP from the cytosol into the mitochondrion, allowing TPP to associate with mitochondrial dehydrogenases.

GENETICS

Thiamine-dependent inborn errors of metabolism are rare. They can be caused by: (1) mutations in subunits of PDH or BCKDH that directly or indirectly interfere with the binding of TPP and thus activation of the apoenzyme, (2) a defect of conversion of thiamine into TPP, or (3) defects of specific cellular or mitochondrial transporters of thiamine and TPP, respectively. THTR1 is encoded by SLC19A2 (chromosome 1q24.2; Mendelian Inheritance in Man [MIM] no. 603941). THTR2 is encoded by SLC19A3 (chromosome 2q36.3; MIM no. 606152). TPK is encoded by TPK1 (chromosome 7q35; MIM no. 606370). TPC is encoded by SLC25A19 (chromosome 17q25.1; MIM no. 606152). Pathogenic variants in each of these genes cause autosomal recessive syndromes with predominant neurologic signs and symptoms (deficiency of THTR1 also causes thiamine-responsive megaloblastic anemia) as summarized in Table 144-1.

VITAMIN B₆ (PYRIDOXINE)

INTRODUCTION

Vitamin B₆ is present in the human body as 6 vitamers that all share a 2-methyl-3-hydroxypyridine structure but differ in the nature of their C4 and C5 functional groups. The C4 carbon bears a CH₂OH group in pyridoxine, a CHO group in pyridoxal, and a CH₂NH₂ group in pyridoxamine. All 3 of these C4 variants can exist with the C5 substituent esterified to phosphate: the pyridoxal 5′-phosphate (PLP) is a highly reactive chemical. The requirement for B₆ is primarily related to the fact that PLP is the cofactor for over 160 enzyme-catalyzed reactions that occur in humans. PLP is required for the metabolism of many amino acids and neurotransmitters including decarboxylation of aromatic amino acid, glutamate, and histidine; transamination of branched-chain amino acid, tyrosine, and γ-aminobutyric acid (GABA); the glycine cleavage system; threonine and serine dehydratase; cystathionine β-synthase; and aminolevulinate synthase. Consequently, there are many biochemical markers for B₆ deficiency or dependency states.

METABOLISM

The phosphorylated B₆ vitamers in the diet are thought to be hydrolyzed by intestinal phosphatases before absorption. The absorbed vitamers are rapidly cleared by uptake into the liver, where they are phosphorylated by pyridoxal kinase. Pyridoxine phosphate and pyridoxamine phosphate are then converted to PLP by pyridox(am)-5′-phosphate oxidase (PNPO). PLP reenters the circulation bound to the lysine-190 residue of albumin. Delivery of PLP to the tissues requires hydrolysis of circulating PLP to pyridoxal by the ectoenzyme tissue nonspecific alkaline phosphatase. Only pyridoxal is able to cross the blood-brain barrier and enter other tissues, but intracellularly, pyridoxal needs to be rephosphorylated by pyridoxal kinase to regenerate active cofactor PLP.

GENETICS

There are several mechanisms that can lead to an increased requirement for pyridoxine or PLP: (1) inborn errors affecting the pathways of B₆ vitamer metabolism (PNPO and alkaline phosphatase defects); (2) inborn errors that lead to accumulation of small molecules that react with PLP and inactivate it (hyperprolinemia type II and pyridoxine-responsive epilepsy); (3) inborn errors affecting specific PLP-dependent enzymes (X-linked sideroblastic anemia, classical homocystinuria, and gyrate atrophy of the choroid); (4) celiac disease, which is thought to lead to malabsorption of B₆ vitamers, or renal dialysis, which leads to increased losses of B₆ vitamers from the circulation; and (5) drugs that affect the metabolism of B₆ vitamers (enzyme-inducing anticonvulsants, methyl xanthines) or that react with PLP. Thus isoniazid inhibits pyridoxine phosphokinase and isoniazid metabolites bind to and inactivate B₆ vitamers; therefore, isoniazid can induce a pyridoxine-dependent peripheral neuropathy in patients who are slow isoniazid inactivators or who have renal insufficiency. Pyridoxine-dependent inborn errors are summarized in Table 144-1. Pyridoxine-dependent epilepsy is an autosomal recessive disorder caused by biallelic pathogenic variants in ALDH7A1 (chromosome 5q23.2; MIM no. 107323). ALDH7A1 encodes α-amino-adipic acid dehydrogenase, an enzyme of lysine and pipecolic acid metabolism. A deficiency of this enzyme results in the accumulation of δ-1-piperideine-6-carboxylic acid (P6C), which reacts with PLP and inactivates it. Treatment involves provision of large amounts of pyridoxine (10–30 mg/kg/d), along with a lysine-restricted diet supplemented with arginine (300–400 mg/kg/d) to compete with lysine uptake at the blood-brain barrier. PLP-responsive epilepsy is an autosomal recessive disorder caused by pyridoxal 5′-phosphate oxidase (PNPO) deficiency due to biallelic pathogenic variants in PNPO (chromosome 17q21.32; MIM no. 610090). Historically, it was thought that treatment of this disorder requires provision of PLP; however, it is clear that a subset of patients respond to pharmacologic doses of pyridoxine. Another disorder associated with a functional reduction in PLP is a defect in the enzyme Δ¹-pyrroline 5-carboxylate dehydrogenase (P5CDh), which normally converts Δ¹-pyrroline 5-carboxylate (P5C) into glutamate as part of a proline degradation pathway; hence, this disorder is known as hyperprolinemia type II, with high concentrations of both proline and P5C. This is a complex neurometabolic disorder caused by biallelic mutations in the gene ALDH4A1 (chromosome 1p36, MIM no. 606811), but it has been shown that, much like the condensation of P6C with PLP, P5C can similarly react with PLP and create a PLP deficiency state. Finally, a more recently described form of pyridoxine-dependent epilepsy is an autosomal recessive disorder caused by biallelic pathogenic variants in PROSC (chromosome 8p11.23, MIM no. 604436). The role of PROSC in pyridoxine metabolism is not yet fully understood, but it is thought to be involved in intracellular homeostatic regulation of PLP levels as it binds PLP. Disorders reviewed in more detail in other chapters that may respond to pyridoxine are listed in Table 144-1.

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INTRODUCTION

Mitochondrial fatty acid oxidation provides the main source of energy for heart and skeletal muscle and, through generation of acetyl-coenzyme A (CoA), for tricarboxylic acid (TCA) cycle function and ketone body production; it also provides energy for other tissues when the supply of glucose is limited. Long-chain fatty acids entering the cell are esterified with carnitine before being transported across the mitochondrial membrane through a series of steps known as the carnitine cycle. The free CoA esters then undergo β-oxidation in the mitochondrial matrix (Fig. 145-1). Disorders that interfere with any of these steps limit energy production in heart and skeletal muscle at rest and reduce the ability of other tissues, including the brain, to tolerate a low-glucose milieu during times of increased energy demand.

Disorders of long-chain fatty acid oxidation (FAO) include defects in the cellular carnitine transporter, carnitine palmitoyltransferase (CPT) I and II, carnitine-acylcarnitine translocase (CACT), very-long-chain acyl-CoA dehydrogenase (VLCAD), mitochondrial trifunctional protein (TFP), and isolated long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD). They can present at any age, largely dependent on the residual activity of the defective enzyme. Complete deficiencies typically manifest in infancy or early childhood; about 25% in the first week of life. Neonates may present with cardiac arrhythmias or sudden death, and occasionally with facial dysmorphism and malformations, including renal cystic dysplasia. Infants and young children may show involvement of liver, heart, or skeletal muscle, with life-threatening fasting- or stress-related hypoketotic hypoglycemia or Reye-like syndrome. Conduction abnormalities, arrhythmias, or dilated or hypertrophic cardiomyopathy and muscle weakness or fasting- and/or exercise-induced rhabdomyolysis are common findings. Onset in adolescence and early adulthood is usually with episodes of recurrent rhabdomyolysis without hypoglycemia. Cardiomyopathy may be present. Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency historically presented with hypoketotic hypoglycemia with or without hyperammonemia or sudden death with fasting or illness. Most patients with recurrent Reye syndrome ultimately proved to have MCAD deficiency. Once the disease is identified in a patient (possibly by newborn screening), the outlook is excellent as long as care is taken during times of illness. Short-chain acyl-CoA dehydrogenase (SCAD) deficiency has been reported in a wide variety of clinical settings but is now generally recognized as a benign biochemical phenotype rather than a disease. Short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD/ACADSB) deficiency is anomalous in this group of disorders in that it typically presents as an insulin hypersensitivity syndrome with hypoglycemia.

The diagnosis of an FAO disorder is straightforward when a patient is clinically symptomatic, with characteristic metabolites easily identifiable in blood and urine by tandem mass spectrometry (MS-MS) and urine organic acid analysis by gas chromatography–mass spectrometry (GC-MS). However, in many cases, these metabolites may disappear when a patient is well. Probably the most important single diagnostic test is analysis of the acylcarnitine ester profile in serum or plasma by MS-MS, which identifies characteristic esters in most disorders, even when patients are symptom-free. Confirmation of the diagnosis is most often accomplished through gene sequencing. Other tests that may be useful include analysis of urine organic acids, free and total carnitine in serum and urine, and enzyme assays or acylcarnitine profiling in leukocytes or fibroblasts. Newborn screening by MS-MS is an effective tool for early detection of FAO disorders in asymptomatic infants and is now routinely performed in the United States and many other countries. Combined, approximately 2 to 3 per 10,000 newborns in the United States have an FAO disorder.

Treatment of acute symptoms including encephalopathy associated with hypoketotic hypoglycemia is with 10% glucose (usually with electrolytes) given intravenously to deliver at least 8 mg/kg/min. Intravenous L-carnitine may be helpful if low and is lifesaving in carnitine transporter deficiency. Long-term therapy involves replenishing carnitine stores with oral L-carnitine and preventing hypoglycemia. This may be accomplished by providing a snack before bedtime, but continuous intragastric feeding may be required. Supplementation with medium-chain triglycerides in long-chain FAO disorders provides a fat source that patients can metabolize. Except for MCAD deficiency and carnitine transporter defect, the long-term prognosis for most of these conditions remains guarded even when identified by newborn screening, with sudden death often occurring due to a conduction defect, arrhythmias, and cardiomyopathy.

CARNITINE UPTAKE DEFECT (PRIMARY CARNITINE DEFICIENCY)

This disorder is caused by a defect in the sodium-dependent high-affinity carnitine transporter (OCTN2) in the cellular plasma membrane, which ultimately limits β-oxidation by reducing entry of long-chain acyl-CoA esters into mitochondria (see Fig. 145-1). The resultant absorptive defect of carnitine in the kidney and intestine leads to very low levels of free carnitine in serum, and the disorder responds dramatically to L-carnitine. It is inherited as an autosomal recessive trait.

CLINICAL FEATURES

The condition may present in early infancy or in late childhood, usually with dilated cardiomyopathy or recurrent episodes of encephalopathy and hypoketotic hypoglycemia. Skeletal muscle involvement may be apparent as hypotonia or proximal limb weakness. Conduction defects and arrhythmias are rare. Sudden death is observed, with autopsy showing fat in the heart, liver, renal tubules, and skeletal muscle. Adult presentations with cardiomyopathy and arrhythmias have been reported, as has identification of asymptomatic affected females identified through a carnitine deficiency in a newborn.

DIAGNOSIS

The diagnosis is based on finding extremely low levels of carnitine in serum and tissues; serum carnitine may be 1 μmol/L or undetectable (normal 30–70 μmol/L). Organic acid and acylcarnitine analysis are usually normal. The gene encoding the carnitine transporter is localized to chromosome 5 (5q31), and many disease-causing mutations have been described. Diagnosis is most easily confirmed through gene sequencing. Carnitine transport may also be demonstrated to be deficient in fibroblasts, and fetal diagnosis is possible based on the same assay on amniocytes.

TREATMENT

The response to L-carnitine supplementation is dramatic and lifesaving; 100 to 300 mg/kg per day can be given intravenously in emergency situations and then administered orally chronically.

DEFECTS OF THE CARNITINE CYCLE

Short- and medium-chain fatty acids can enter mitochondria directly, but fatty acids longer than C₁₂ must be esterified with coenzyme A and transported across the mitochondrial membrane by CPT I, CPT II, and carnitine-acylcarnitine translocase (see Fig. 145-1). CPT I in the mitochondrial outer membrane first transfers the acyl moieties from CoA to carnitine, the translocase moves the carnitine esters across the inner membrane, and CPT II on the matrix side of the inner membrane transfers the carnitine esters to CoA and releases the CoA esters, which enter the β-oxidation spiral. Defects of CPT I, CPT II, and translocase are inherited as autosomal recessive traits.

CLINICAL FEATURES

CPT I deficiency usually presents in infancy with episodes of fasting-induced hypoketotic hypoglycemia. There are 2 distinct phenotypes of CPT II deficiency, dictated by residual enzyme activity. The more common muscular form presents with exercise-induced muscle pain and rhabdomyolysis in adolescence or early adult life. More rarely, a severe hepatocardiomuscular presentation occurs in infants and children. Those who present as neonates die in 1 to 2 weeks with hepatomegaly, cardiomyopathy, and encephalopathy, and they often have renal cystic dysplasia. Those presenting later usually exhibit hypoketotic hypoglycemia, cardiomyopathy, and muscle disease and are at risk for sudden death due to conduction defects or arrhythmias.

CACT deficiency usually presents as a life-threatening disease in the newborn period, with hypoglycemia, hyperammonemia, conduction defects, arrhythmias, and evidence of skeletal muscle involvement, with high creatine phosphokinase (CPK) values. The ultimate prognosis of this condition is good if the child survives the neonatal period, although cardiomyopathy remains a chronic problem in some patients.

DIAGNOSIS

The serum acylcarnitine profile is usually normal in CPT I deficiency, although the concentrations of all species may be relatively low, and free carnitine may be high. C₁₆ esters are usually elevated in CPT II and CACT deficiency. There are no changes in amino acids (eg, high or low citrulline) to indicate a urea cycle defect as the cause of the hyperammonemia in translocase deficiency, and urine organic acids are normal or show only mild dicarboxylic aciduria. Free carnitine concentration in serum is 2 to 3 times normal in CPT I deficiency and is typically very low in CPT II and translocase deficiency.

All 3 enzymes can be assayed in fibroblasts and leukocytes, and while prenatal diagnosis by enzyme assay on amniocytes should be possible in all of these disorders, it has been accomplished only in CPT II and translocase deficiency. In some instances, a prenatal diagnosis of CPT II can also be made by ultrasonography that shows enlarged echogenic kidneys. Gene sequencing is now the easiest way to confirm a diagnosis. Disease-causing mutations have been identified in all the genes. All 3 diseases can be identified through newborn screening with acylcarnitine profiling, but milder, late-onset forms are usually missed.

TREATMENT

Acute episodes of hypoketotic hypoglycemia should be treated with intravenous glucose-containing fluids, and treatment of hyperammonemia may require dialysis. Preventing fasting may be sufficient in CPT I deficiency, but continuous nighttime intragastic feeding may be necessary in CPT II and translocase deficiency. Carnitine should be given when the serum carnitine level is low to avoid secondary carnitine deficiency.

DEFECTS OF β-OXIDATION

Once in the mitochondrial matrix, acyl-CoA esters enter the β-oxidation spiral, where a series of 4 reactions remove the 2-carbon compound acetyl-CoA. Flavin adenine dinucleotide (FAD)-containing acyl-CoA dehydrogenases oxidize the acyl-CoA to 2,3-enoyl-CoA, which then becomes hydrated to a 3-hydroxyacyl-CoA by hydratases. Oxidation to a 3-ketoacyl-CoA by nicotinamide adenine dinucleotide (NAD)-requiring hydroxyacyl-CoA dehydrogenases and removal of acetyl-CoA by 3-ketothiolases follow, and the acyl-CoA, now 2 carbons shorter, reenters the spiral.

All these reactions are catalyzed by enzymes with distinct (but often overlapping) chain-length specificities. For example, different FAD-containing acyl-CoA dehydrogenases act on very-long-chain (C_12–24), long-chain (C_6–20), medium-chain (C_4–14), and short-chain (C_4–6) acyl-CoAs. Similar specificities exist for the hydratases, hydroxyacyl-CoA dehydrogenases, and thiolases.

Inherited disorders in most of these enzymes have been described. As a rule, defects in long-chain–specific enzymes block β-oxidation more completely and cause more severe clinical disease. Most of these conditions are rare, but VLCAD deficiency, MCAD deficiency, and LCHAD deficiency are relatively common and therefore merit further discussion. All 3 are inherited as autosomal recessive traits. Short-chain acyl-CoA dehydrogenase deficiency is frequently suspected on neonatal screening on the basis of ethylmalonic and butyryl carnitine accumulation, but the relevance is questionable, as many individuals remain asymptomatic without treatment.

VERY-LONG-CHAIN ACYL-CoA DEHYDROGENASE (VLCAD) DEFICIENCY

CLINICAL FEATURES

VLCAD deficiency can present in the newborn period with arrhythmias and sudden death, or with hepatic, cardiac, or muscle presentations later in infancy or childhood. The hepatic presentation is characterized by fasting-induced hypoketotic hypoglycemia, encephalopathy, and mild hepatomegaly, often with mild acidosis, hyperammonemia, and elevated liver transaminases. Some patients present with arrhythmias or dilated or hypertrophic cardiomyopathy in infancy or childhood, and a few patients present with exercise-induced muscle pain, rhabdomyolysis, elevated CPK levels, and myoglobinuria. Cardiomyopathy may present (or recur) at any age. Adolescent- or adult-onset episodes of recurrent rhabdomyolysis have been described. Patients are at greatest risk of hypoglycemia in the first few years of life, with a transition to predominantly muscular symptoms thereafter.

DIAGNOSIS

Analysis of serum acylcarnitines by MS-MS usually reveals elevations of saturated and unsaturated C_14–18 esters, even between episodes. Urine organic acid analysis during acute illness often shows increased C₆ (adipic), C₈ (suberic), and C₁₀ (sebacic) dicarboxylic acids. However, because very similar changes can be seen in resolving ketosis and after ingesting medium-chain triglycerides, this will not raise suspicion of disease unless C₁₂ and C₁₄ dicarboxylic acids are also present. Serum concentration of free carnitine is usually low. Newborn screening is possible by MS-MS and routine in the United States and many other countries. The incidence of VLCAD deficiency is approximately 1:40,000 live births.

VLCAD deficiency and accumulation of characteristic acylcarnitines can be demonstrated in fibroblasts or leukocytes, and enzyme assay in cultured amniocytes can be used for prenatal diagnosis. The gene encoding the enzyme has been cloned and localized to chromosome 17p13. Many disease-causing mutations have been described. Confirmation of the diagnosis is now most commonly obtained by DNA sequencing.

TREATMENT

Treatment of acute episodes includes institution of a high glucose intravenous (IV) infusion rate (8–10 mg/kg/min) and addressing any underlying intercurrent illnesses. Chronic management involves avoidance of fasting and supplementation with medium-chain triglycerides as a souce of fatty acid derived energy. Carnitine supplementation may be helpful if blood free carnitine level is low. Continuous intragastric feeding may be necessary. Fasting in the first year of life should be limited to 4 hours up to age 6 months, 6 hours up to age 12 months, and 8 hours thereafter.

MEDIUM-CHAIN ACYL-CoA DEHYDROGENASE (MCAD) DEFICIENCY

CLINICAL FEATURES

MCAD deficiency is the most common FAO defect, with a frequency of 1:10,000 to 1:20,000 in individuals of western European ancestry. It is much less common in Asian and African populations. When identified through newborn screening, MCAD deficiency is an easily treated disorder that nonetheless can still be life threatening. In countries in which MCAD deficiency is not screened for at birth, symptoms usually develop between 18 and 36 months of life with episodes of fasting-induced vomiting, lethargy progressing to coma and seizures, hypoketotic hypoglycemia, and hepatomegaly, often with mild hyperammonemia. The liver may appear fatty by imaging. Misdiagnosis as Reye syndrome or, because the initial episode is fatal in about 25% of patients, sudden infant death syndrome is common. Levels of uric acid, liver transaminases, and CPK are often elevated during the acute episode, and liver biopsy shows microvesicular steatosis. Autopsy shows fatty infiltration of the liver, renal tubules, and heart and skeletal muscle. Enzyme-deficient individuals may not develop symptoms.

DIAGNOSIS

Analysis of serum acylcarnitines by MS-MS shows elevations of C₈, C_8:1, C₁₀, and C_10:1 esters, even between episodes. Urine organic acids during acute illness often show increased C₆ (adipic), C₈ (suberic), and C₁₀ (sebacic) dicarboxylic acids, together with hexanoylglycine, suberylglycine, and phenylpropionylglycine. The dicarboxylic aciduria also occurs during resolving physiologic ketosis and after ingestion of medium-chain triglycerides. It will not be perceived as abnormal unless the glycine esters, or unsaturated or longer-chain dicarboxylic acids, are also present. Free carnitine level in serum is usually low.

The enzyme defect can be demonstrated in several tissues, including fibroblasts and leukocytes, but molecular diagnosis is often more readily available. The gene encoding MCAD has been localized to chromosome 1p31. Many disease-causing variants have been identified, but the K304E mutation, which changes the lysine residue at position 304 of the mature enzyme to glutamic acid, accounts for 90% of mutant alleles found in Caucasian populations. Ninety percent of affected Caucasians have at least 1 copy of this mutation (81% are homozygous and 18% are compound heterozygous), and analysis for this mutation will confirm the diagnosis in many patients. MCAD deficiency can be identified through newborn screening with MS-MS.

TREATMENT

Acute episodes should be treated with IV glucose at an infusion rate of 8 to 10 mg/kg/min. Long-term treatment consists of avoiding fasting, usually by providing carbohydrate snacks at bedtime. Oral carnitine (50–100 mg/kg/d) is used by some, but not all, clinicians. Developmental delay, behavioral problems, and other chronic central nervous system problems may occur if there are episodes of hypoglycemia, but if such an injury does not occur, prognosis is quite good. As a rule, fasting should be avoided, especially in younger children.

LONG-CHAIN 3-HYDROXYACYL-CoA DEHYDROGENASE (LCHAD) DEFICIENCY

NAD-dependent 3-hydroxyacyl-CoA dehydrogenases catalyze oxidation of 3-hydroxyacyl-CoA esters to their 3-keto analogues. LCHAD, the long-chain-specific enzyme, acts on acyl groups longer than C₈ and exists in mitochondria as part of a protein complex with 3 activities. This mitochondrial trifunctional protein (MTP) is an α₄β₄ octamer, with the α subunit harboring LCHAD and long-chain enoyl-CoA hydratase activities, and the β subunit bearing long-chain β-ketothiolase activity. LCHAD deficiency can exist alone or with deficiency of the other 2 enzymes. Isolated LCHAD deficiency occurs in 1:40,000 to 1:80,000 newborns.

CLINICAL FEATURES

Patients with isolated LCHAD deficiency often present in infancy with fasting-induced hypoketotic hypoglycemia; however, patients may also present with neonatal cardiomyopathy or, later in life, with exercise-induced rhabdomyolysis. A clinical history often reveals a pregnancy complicated by acute fatty liver or HELLP (hemolysis, elevated liver enzymes, and low platelets). Unlike most other fatty acid oxidation disorders, severe and progressive cholestatic liver disease is common, and many patients develop retinopathy with hypopigmentation or focal pigmentary aggregations. Many patients die in early childhood. The presentation of individuals with MTP deficiency is similar to that of isolated LCHAD deficiency, but it is usually earlier and more severe.

DIAGNOSIS

Acylcarnitine analysis by MS-MS is usually diagnostic and shows elevated saturated and unsaturated C₁₆ and C₁₈ hydroxyacylcarnitines. Organic acid analysis often shows elevated C_6–14 3-hydroxydicarboxylic acids, but the same abnormalities can be seen in patients with respiratory chain defects and glycogenoses and are not specific. The enzyme defect can be demonstrated in fibroblasts and leukocytes and, for prenatal diagnosis, in amniocytes. The genes encoding the α and β subunits are localized to chromosome 2p23.3. The E510Q mutation, which changes the glutamic acid residue at position 510 to glutamine, accounts for nearly 90% of mutant alleles in Europeans. LCHAD and MTP deficiency can be identified through newborn screening with MS-MS.

TREATMENT

Therapy is much the same as that for VLCAD, CPT, and CACT deficiencies. As in VLCAD deficiency, it may be necessary to completely eliminate fasting by continuous intragastric feeding, and medium-chain triglycerides may be used to provide calories. Carnitine may be useful to prevent depletion of stores. Oral supplements of docosahexaenoic acid, a polyunsaturated C₂₀ acid, appear useful in slowing the progression of retinopathy.

GLUTARIC ACIDEMIA TYPE II

Electrons from the acyl-CoA dehydrogenases involved in fatty acid and amino acid oxidation are transferred from their FAD coenzymes into the respiratory chain via redox centers in electron transfer flavoprotein (ETF) and ETF:ubiquinone oxidoreductase (ETF:QO). Recessively inherited defects in ETF and ETF:QO cause glutaric acidemia type II (GA2), also known as multiple acyl-CoA dehydrogenase deficiency (MADD). Several additional gene defects presenting with a biochemical pattern of GA2 have recently been described, including defects of cellular riboflavin transport and FAD synthesis. Historically, prior to the molecular characterization of the disorders, they were clinically described as Brown-Vialetto-Van Laere syndrome and Fazio Londe syndrome—progressive neuromuscular disorders with variable features of myopathy, hearing loss, optic atrophy, and ataxia. Treatment with high-dose riboflavin can be lifesaving.

CLINICAL FEATURES

GA2 may present in the neonatal period with severe hypoglycemia, metabolic acidosis, hyperammonemia, and the odor of sweaty feet typical of isovaleric acidemia, often with cardiomyopathy, facial dysmorphism, and severe renal cystic dysplasia. Most such patients die within the first days or weeks of life, often of conduction defects or arrhythmias. Fatty infiltration of the liver, renal tubules, and heart and skeletal muscle are consistent autopsy findings. Milder disease, sometimes called ethylmalonic adipic aciduria, may present with episodic vomiting, hypoketotic hypoglycemia, and hepatomegaly in childhood, or simply as hypoglycemia in adult life. Leukodystrophy and cardiomyopathy may also be present, along with an elevated CPK.

DIAGNOSIS

Acylcarnitine analysis by MS-MS shows glutarylcarnitine, isovalerylcarnitine, and straight-chain esters of chain lengths C₄, C₈, C₁₀, C_10:1, and C₁₂. Urine organic acids show increased ethylmalonic, glutaric, 2-hydroxyglutaric, and 3-hydroxyisovaleric acids, together with C₆, C₈, and C₁₀ dicarboxylic acids and isovalerylglycine. The serum carnitine concentration is usually low. Elevated serum sarcosine on amino acid analysis is common in patients with mild disease.

Assays using fibroblasts show that some GA2 patients are deficient in ETF and, more commonly, that others are deficient in ETF:QO. Disease-causing mutations in the 3 genes encoding ETF:QO and the α and β subunits of ETF have been identified in many patients. Additional molecular defects in the cellular riboflavin transporter and FAD synthesis have recently been described; thus, gene sequencing of all of these genes is typically the easiest way to make a diagnosis when a metabolite pattern is suggestive of GA2. GA2 is identifiable through newborn screening by MS-MS.

TREATMENT

Patients with complete defects die during the first weeks of life, usually of conduction defects or arrhythmias, but those with incomplete defects can survive well into adult life. As in other fatty acid oxidation disorders, treatment relies on the avoidance of fasting, sometimes with continuous intragastric feeding, and carnitine to replenish lost stores. High-dose riboflavin supplementation can benefit a subset of patients, and the use of D/L-3-hydroxybutyrate has been shown to be effective in reversing the leukodystrophy in a few patients not responsive to riboflavin.

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INTRODUCTION

Hepatic biosynthesis of ketone bodies involves the condensation of acetyl-coenzyme A (CoA) and acetoacetyl-CoA to hydroxymethylglutaryl-CoA (HMG-CoA) by HMG-CoA synthase, followed by hydrolysis of HMG-CoA to acetyl-CoA and acetoacetic acid (AcAc) by HMG-CoA lyase as the final step in leucine degradation. AcAc is reduced to 3-hydroxybutyric acid (3HB), and extrahepatic tissues use the 2 ketone bodies as energy sources during fasting (ketolysis). Recessively inherited defects of HMG-CoA synthase (HMGCS2) and HMG-CoA lyase (HMGCL) cause hypoketotic hypoglycemia during fasting, and defects of ketolysis cause persistent or episodic ketoacidosis.

HYDROXYMETHYLGLUTARYL-CoA SYNTHASE DEFICIENCY

Deficiency of HMG-CoA synthase (MIM no. 605911) causes hypoketotic hypoglycemia, typically during intercurrent illnesses, along with encephalopathy and hepatomegaly. Serum free fatty acids are very elevated, urine and serum ketones are inappropriately low or absent, transaminases are elevated, urine organic acid analysis shows severe dicarboxylic aciduria with unsaturated and 3-hydroxy derivatives, and serum acylcarnitines are normal. When the deficiency is suspected, mutation analysis can confirm the diagnosis. Treatment involves avoiding fasting and administering glucose during episodes of hypoglycemia, along with bedtime carbohydrates such as uncooked cornstarch.

HYDROXYMETHYLGLUTARYL-CoA LYASE DEFICIENCY

Deficiency of 3-hydroxy-3-methylglutaryl-CoA lyase (MIM no. 246450) can present in the newborn period with severe hypoketotic hypoglycemia, elevated transaminases, metabolic acidosis, and hyperammonemia, or later with episodes of hypoglycemia, hepatomegaly, and encephalopathy following intercurrent infection. The latter form of the disease is often mistaken for Reye syndrome. If not promptly treated, cerebral atrophy, neurologic effects, and developmental disabilities may follow. Adolescent-and adult-onset patients have been described and may also have a leukoencephalopathy. It is a common organic aciduria in the Saudi population but is otherwise rare.

DIAGNOSIS

Urine organic acid analysis shows increased 3-hydroxy-3-methylglutaric, 3-methylglutaconic, and 3-methylglutaric acids, and tandem mass spectrometry (MS/MS) shows increased 3-hydroxy-3-methylglutarylcarnitine. The disorder can also be identified by MS/MS on newborn screening blood spots with elevations typically reported as “C5-hydroxyacylcarntine,” a designation shared by several other isobaric metabolites. Due to the possibility of a catastrophic presentation, rapid additional diagnostic testing by urine organic acids is necessary. Liver function tests may be abnormal at times of acute illness. The enzyme defect is apparent in many tissues, including fibroblasts and peripheral leukocytes, but is usually not necessary to measure due to the availability of DNA testing. Fetal disease can be diagnosed by enzyme assay on cultured amniocytes or chorionic villus samples, by demonstrating the characteristic organic acids in amniotic fluid, or by DNA analysis if a familial mutation is known.

TREATMENT

Acute management requires intravenous fluids, electrolytes, and glucose. Long-term management is directed at avoiding fasting and the resulting hypoglycemia, and bedtime carbohydrates may be warranted. The possible life-threatening consequences of fasting make it imperative that parents bring the child to the hospital as soon as possible when oral intake is compromised.

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INTRODUCTION

Ketolysis involves esterification of acetylacetate (AcAc) to AcAc-coenzyme A (CoA) by succinyl-CoA:3-oxoacid transferase (SCOT, OXCT1 gene) and hydrolysis of AcAcCoA by mitochondrial acetoacetyl-CoA thiolase (T2, ACAT1 gene) to form acetyl-CoA. Inherited disorders of ketolysis involve these 2 enzymes and cause persistent or episodic ketoacidosis. If SCOT is entirely lacking, ketolysis is completely blocked, but if functional T2 is completely absent, some ketolysis is still possible, likely due to the presence of another mitochondrial enzyme, medium chain 3-ketoacyl-CoA thiolase, that has some activity for hydrolyzing AcAcCoA. This latter enzyme may explain in part why permanent ketosis is often observed in SCOT deficiency but not in T2 deficiency. Ketone body production is regulated by the hormones glucagon and catecholamines, which induce free fatty acid (FFA) mobilization from adipose tissue, fatty acid oxidation, and ketogenesis in the liver, while insulin suppresses these steps. Ketogenic stresses including fasting, prolonged exertion, febrile illnesses, and vomiting and diarrhea, leading to both FFA oxidation and ketone body synthesis. The enzymes of ketogenesis (see Chapter 146); hydroxymethylglutaryl-CoA synthase and lyase, generate AcAc, which is then reduced by mitochondrial D-β-hydroxybutyrate (D-βOHB)-dehydrogenase (BDH1) in the liver to D-βOHB. Ketone bodies then circulate to peripheral tissues, where BDH1 regenerates AcAc, allowing SCOT and T2 to generate acetyl-CoA in order to maintain cellular energy production via the tricarboxylic acid (TCA) cycle and spare glucose utilization.

SUCCINYL-CoA TRANSFERASE DEFICIENCY

SCOT deficiency (Online Mendelian Inheritance in Man [MIM] no. 245050) is characterized by persistent or exaggerated episodic ketoacidosis, often beginning in infancy, with increased levels of ketone bodies in the blood even in the fed state. A diagnosis can be established by enzyme assay in fibroblasts or by mutation analysis, and prenatal diagnosis can be accomplished in the same manner.

BETA-KETOTHIOLASE DEFICIENCY

Mitochondrial 3-ketothiolase releases acetyl-CoA from AcAcCoA and from 2-methylacetoacetyl-CoA, an intermediate in isoleucine oxidation (see Chapter 132). Mitochondrial 3-ketothiolase deficiency (MIM no. 203750) can present in infancy with hypoglycemia, elevated transaminases, hyperuricemia, metabolic acidosis, and severe ketosis, or later with fasting- or protein-induced episodes of vomiting, hepatomegaly, ketoacidosis, and encephalopathy.

DIAGNOSIS

In SCOT deficiency, levels of ketones in the blood may be persistently elevated or show episodic increases during illness. Urine organic acid analysis shows increased 3-hydroxybutyrate and acetoacetate levels. In beta-ketothiolase deficiency, urine organic acid analysis shows increased 2-methyl-3-hydroxybutyric acid, 2-methylacetoacetic acid, and tiglylglycine levels, but they may be obscured during acute illnesses by 3-hydroxybutyrate (3HB) and AcAc and may be detectable only between episodes or after an oral load of isoleucine. Glycine levels are often elevated in blood and urine. In blood acylcarnitine analysis, C5:1 acylcarnitine (tiglylcarnitine) and C5-OH acylcarnitine (2-methyl-3-hydroxybutyrylcarnitine) may be elevated, but this is not a consistent finding, and the disorder is not readily detectable by newborn screening. While usually not necessary to establish a diagnosis, the enzyme defects can be demonstrated in fibroblasts and leukocytes, and in amniocytes for prenatal diagnosis. Disease-causing mutations can be identified by clinically available DNA sequencing.

TREATMENT

Treatment of acute episodes in SCOT deficiency is supportive, providing IV fluids and glucose for hydration and calories until normal caloric intake can be re-established. Chronic bicarbonate administration may be necessary if chronic ketosis is present. Anecdotally, long-term outcomes appear good, but formal longitudinal studies have not been performed. Beta-ketothiolase deficiency should be treated with intravenous glucose and sodium bicarbonate. A low-protein/isoleucine-restricted diet, coupled with avoidance of fasting, decreases the frequency and severity of acute episodes and permits normal growth and development if irreversible neurologic damage has not already occurred.

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INTRODUCTION

Leukotrienes (LTs) are inflammatory lipid mediators synthesized from arachidonic acid (C20:4), a 20-carbon polyunsaturated fatty acid that is present in membrane phospholipids. LTs consist of several biochemically distinct forms (LTA₄, LTB₄, LTC₄, LTD₄, LTE₄) that act through interactions with specific cellular receptors. LTC₄, LTD₄, and LTE₄ are distinct from the other LT species because they are formed by the incorporation of cysteine into their structures and are referred to as cysteinyl-LTs. These lipid mediators are biologically active in allergic and immune reactions, bronchoconstriction, neutrophil and macrophage chemotaxis, and T-cell differentiation. Emerging data also implicate their involvement in neurologic processes.

PATHOGENESIS AND EPIDEMIOLOGY

Several rare disorders of LT metabolism have been identified. They can be classified into those that either block LT synthesis or prevent its degradation (Fig. 148-1). All are rare and have an unknown incidence.

Disorders of Leukotriene Synthesis

The biosynthesis of LT begins with 5-lipooxygenase acting on arachidonic acid to produce the short-lived LTA₄, which is the metabolic precursor for the other LTs. LTA₄ is enzymatically converted to LTB₄, a potent proinflammatory mediator, and is also acted on by LTC₄ synthase, which uses glutathione as a co-substrate, to generate LTC₄ and the subsequent series of cysteinyl-LTs (LTD₄ and LTE₄). Deficient LTC₄ synthase activity has been described in 2 children who had severely reduced levels of LTC₄, LTD₄, and LTE₄. Since glutathione is a co-substrate for this enzyme, patients with genetic defects in the production of glutathione (eg, glutathione synthetase deficiency) also exhibit reductions of all cysteinyl-LT species. LTC₄ is converted to LTD₄ by γ-glutamyl transpeptidase, and patients with deficiency of this enzyme accumulate LTC₄ and have profound reductions in LTD₄ and LTE₄. The subsequent metabolic step uses dipeptidase to convert LTD₄ to LTE₄. One 15-year-old patient with suspected dipeptidase deficiency accumulated LTD₄ and had undetectable LTE₄.

Disorders of Leukotriene Degradation

The degradation of LTB₄ (and LTE₄) proceeds by ω-oxidation, which adds a hydroxyl group to the C20-carbon of LTB₄. This reaction is catalyzed by the P450 enzyme CYP4F3A. 20-Hydroxy-LTB₄ is subsequently oxidized via an aldehyde intermediate to 20-carboxy-LTB₄ (see Fig. 148-1). Patients with Sjögren-Larsson syndrome have deficient fatty aldehyde dehydrogenase and cannot carry out this alcohol-acid conversion and thus accumulate LTB₄ and 20-hydroxy-LTB₄. In neutrophils, CYP4F3A can also catalyze this alcohol-acid conversion, but its overall contribution to LT degradation is comparatively minor in other tissues. Subsequent catalytic steps in 20-carboxy-LTB₄ require peroxisomal β-oxidation to shorten the carbon chain. Consequently, patients with peroxisome biogenesis disorders (Zellweger spectrum disorders) and isolated defects in peroxisomal β-oxidation exhibit increased LTB₄ (and LTE₄) metabolites (see Fig. 148-1).

How these biochemical abnormalities contribute to the pathogenesis of the LT diseases is not known. Since all of the disorders with impaired synthesis of the cysteinyl-LTs have significant neurologic symptoms, it is likely that these LTs are critical for normal brain function. Moreover, the disorders of LT degradation (Sjögren-Larsson syndrome and peroxisomal disorders) have additional biochemical abnormalities and exhibit multiorgan involvement, which complicates identifying the specific contributions of LTs to the total phenotype. Symptoms of inflammation, asthma, and immune dysfunction are not prominent among the LT disorders. Curiously, animal studies indicate that LTB₄ can induce pruritus when administered subcutaneously.

CLINICAL MANIFESTATIONS

Clinical features of the LT disorders vary widely, but most patients have serious neurologic symptoms. Hypotonia, psychomotor retardation, microcephaly, and failure to thrive are reported in LTC₄ synthase deficiency. Patients with γ-glutamyl transpeptidase deficiency have exhibited variable intellectual disability, seizures, behavior problems, poor coordination, and/or dysmorphisms. The patient with suspected dipeptidase deficiency had intellectual disability, mild motor deficits, and hearing impairment. Diseases of glutathione production are associated with hemolytic anemia, metabolic acidosis, intellectual disability, and progressive neurologic symptoms. Sjögren-Larsson syndrome has a characteristic triad of pruritic ichthyosis, intellectual disability, and spasticity. Peroxisome biosynthesis disorders and isolated peroxisome β-oxidation defects share features of hypotonia, psychomotor retardation, seizures, deafness, and visual impairment.

DIAGNOSIS

LT disorders are undoubtedly underrecognized. In light of very limited knowledge of their clinical phenotype, the suspicion of a cysteinyl-LT disorder should arise after eliminating more common neurologic diseases. Unfortunately, diagnostic testing is problematic. Although screening urine, cerebrospinal fluid, or blood for LT metabolites is feasible in a research setting, the analytical method is demanding, and a diagnostic test is not yet universally available for the practicing physician. Consequently, genetic testing is probably the most convenient, but still unproven, method for diagnosis, since disease-causing mutations in most of the primary disorders of LT biosynthesis are still unknown. In contrast, disorders of LT degradation (Sjögren-Larsson syndrome, peroxisome defects) are readily diagnosed using conventional biochemical and molecular tests.

TREATMENT

Effective therapy for the LT biosynthetic disorders has not been developed. Zileuton, which inhibits 5-lipooxygenase and lowers LTB₄ levels, was anecdotally reported to improve the pruritus in several patients with Sjögren-Larsson syndrome, but no convincing benefit was seen in a recent double-blind clinical trial. No reports have been published on the use of cysteinyl-LT receptor antagonists (eg, montelukast) in the LT synthetic disorders.

PREVENTION

As suspected genetic diseases, prevention of LT disorders can theoretically be achieved by prenatal diagnosis.

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