Glutaric acidemia type II (GA2), or multiple acyl CoA dehydrogenation deficiency, MIM 231680, is an inborn error characterized clinically by hypo- or nonketotic hypoglycemia and metabolic acidosis; pathologically by fatty degeneration of liver parenchymal cells, renal tubular epithelium, and myocardium; and biochemically by the accumulation of metabolites of compounds oxidized by enzymes that transfer electrons to ETF. In most cases the disorder is due to deficiency of either ETF or ETF-QO, but in some it may be due to an as yet undefined abnormality in flavin metabolism or transport. Complete deficiency of ETF-QO is often associated with congenital anomalies, the most frequent and characteristic being cysts and dysplasia of the kidneys. All forms of the disease are transmitted as autosomal recessive traits. Most patients with severe disease do not survive the first few weeks of life.
Patients with glutaric acidemia type II, which was first reported in 1976,52 fall into one of three groups, each consistent within a family. These have been designated (1) neonatal onset with congenital anomalies, (2) neonatal onset without anomalies, and (3) mild and/or later onset.53 The first two groups are sometimes said to have multiple acyl CoA dehydrogenation deficiency–severe (MADD:S) and the third to have multiple acyl CoA dehydrogenation deficiency–mild (MADD:M), or ethylmalonic-adipic aciduria.
Neonatal-onset patients with congenital anomalies are often premature and present during the first 24–48 hours of life with hypotonia, hepatomegaly, severe hypoglycemia and metabolic acidosis, and (often) an odor similar to that present in isovaleric acidemia (Chap. 93), i.e., the odor of sweaty feet. In some patients the kidneys are palpably enlarged, and there may be facial dysmorphism (high forehead and low-set ears, hypertelorism, hypoplastic midface, etc.), rocker-bottom feet, muscular defects of the anterior abdominal wall, and anomalies of the external genitalia, including hypospadias and chordee. Most such patients die within the first week of life.54– 58 In other patients, congenital anomalies are not noted on physical examination, and renal cysts are discovered only at autopsy.59– 61
Infants without congenital anomalies usually develop hypotonia, tachypnea and metabolic acidosis, hepatomegaly, hypoglycemia, and a “sweaty feet” odor within the first few days of life, many of them within the first 24 hours. The few patients with this form of the disease who have survived beyond the first week of life because of prompt diagnosis and treatment have died within a few months, usually with severe cardiomyopathy. A few other infants have been hypoglycemic in the newborn period and only later developed typical episodes of Reye syndrome-like illnesses; these patients have survived somewhat longer.52,54,62– 66
The course and age at presentation of later-onset glutaric acidemia type II is extremely variable. The first patient to be described with this form of the condition had intermittent episodes of vomiting, hypoglycemia, and acidosis beginning at 7 weeks of age,67,68 and another was totally symptom free during childhood, presenting in adult life with episodic vomiting, hypoglycemia, hepatomegaly, and proximal myopathy.69 Several other patients with episodic disease beginning during the first few years of life have been described,70– 72 as well as two others with progressive lipid storage myopathy and carnitine deficiency.51,73,74 A few patients have had a progressive extrapyramidal movement disorder much more typical of glutaryl CoA dehydrogenase deficiency.75
Routine laboratory evaluation shows severe metabolic acidosis, often with an anion gap, mild or moderate hyperammonemia (usually <300 μg/dl), and severe hypoglycemia without ketonuria or ketonemia. Serum transaminases may be elevated, and prothrombin and partial thromboplastin times may be prolonged. Lactic acid in serum is usually elevated. Chest x-ray may show cardiac enlargement, and echocardiography may show evidence of hypertrophic cardiomyopathy. MRI scans of two patients with macrocephaly during the first months of life showed the same symmetric hypoplasia of the temporal lobes seen in glutaryl CoA dehydrogenase deficiency.76,77 Abdominal ultrasound or CT scan may show renal cysts (Fig. 103-1).
Abdominal ultrasound showing renal cysts in a 2-day-old infant with glutaric acidemia type II due to ETF-QO deficiency. (Courtesy of Dr. C. Greenberg.)
Urine organic acid analysis often shows various combinations of short-chain volatile acids (e.g., isovaleric, isobutyric, 2-methylbutyric); glutaric, ethylmalonic, 3-hydroxyisovaleric, 2-hydroxyglutaric, 5-hydroxyhexanoic, adipic, suberic, sebacic, and dodecanedioic acids: and isovalerylglycine, isobutyrylglycine, and 2-methylbutyrylglycine (Fig. 103-2). 3-Hydroxybutyric and acetoacetic acids are either not seen or not prominent. Organic acids are also significantly elevated in serum and CSF. In some patients, especially those with episodic disease, urine organic acids are abnormal only during acute episodes.
Urine organic acids in an infant with glutaric acidemia type II. A = lactic, B = malonic (internal standard), C = ethylmalonic, D = glutaric, E = adipic, F = 2-hydroxyglutaric, G = suberic, H = sebacic, I = dodecanedioic. (From SI Goodman and SP Markey, Diagnosis of Organic Acidemias by Gas Chromatography-Mass Spectrometry, Alan R Liss Inc., New York, 1981. Used by permission.)
Generalized aminoacidemia and aminoaciduria, often with marked increases in proline and hydroxyproline, are common in neonatal-onset patients, and elevations of sarcosine in serum and urine are especially frequent in those with later onset.
Carnitine concentrations in serum may be normal or low, but acylcarnitine esters in urine may be significantly increased.78,79 Treatment of one patient with oral carnitine produced a large increase in several acylcarnitine esters in urine, including acetylcarnitine, isobutyrylcarnitine, isovalerylcarnitine, hexanoylcarnitine, butyrylcarnitine, and propionylcarnitine.51
All patients autopsied to date have shown severe microvesicular fatty changes in liver parenchymal cells, cells of the proximal renal tubule, and myocardium, but these changes are not diagnostic. Renal anomalies, when present, may be dramatic. The kidneys may be so large that they fill the abdomen, and cysts may occupy all of the cortex and medulla.57,80,81 Dysplastic changes are present in some cases, but not all (Fig. 103-3).
Kidney of a 19-week fetus with glutaric acidemia type II due to ETF-QO deficiency. Left: Not magnified. Right: Cysts derived from Bowman's capsule and renal tubules, with interstitial tissue resembling primitive mesenchyme. Both kidneys showed the same features. H & E; original magnification x50. (Courtesy of Dr Y. E. Hsia.)
Brain has been examined in several patients. In two cases there was focal dysplasia of the cerebral cortex with bilateral reduction in the number of gyri in the frontal, parietal, and temporal lobes, with numerous warty protrusions on the temporoparietal cortex, and microscopic evidence of abnormal neuronal migration.81 Abnormal neuronal migration was observed in three additional patients82,83 but not in a fourth,57 in whom the sole finding on routine examination was a reduced number of Purkinje cells in the cerebellar cortex. Electron microscopy in this patient showed moderately electron-dense, membrane-limited, cytoplasmic bodies, and it was suggested that these might be characteristic, if not pathognomonic. One patient with progressive neurologic disease, intermittent dystonic posturing, and ETF deficiency showed only neuronal loss and gliosis in the caudate nucleus and putamen,75 findings much more typical of glutaryl CoA dehydrogenase deficiency (Chap. 95).
Concentrations of glutaric acid are increased in several tissues, including liver and kidney, and detection of this compound in post-mortem tissue has established the diagnosis in several cases, one of them an infant with fatty changes in the liver that were prominent enough to suggest a diagnosis of Wolman's disease (Chap. 142).
Biochemical and Molecular Basis of Disease
In some patients glutaric acidemia type II is due to inherited deficiency of ETF-QO, and in others it is due to inherited deficiency of ETF. While all three clinical forms of the disease can be caused by a defect in either protein, patients with renal cystic dysplasia and other anomalies are much more likely to have ETF-QO deficiency. No single study has correlated the clinical phenotype with ETF and ETF-QO activity and mutations.
Deficiency of ETF-QO antigen was first demonstrated in membranes of liver mitochondria obtained from a female infant with polycystic kidneys and multiple congenital anomalies53 and then confirmed in fibroblasts from this child and several other infants with and without anomalies.46,84 Less complete deficiency of ETF-QO antigen and activity has now been demonstrated in patients with less severe and/or later-onset forms of the disease.46,51,85
cDNA encoding human ETF-QO has been isolated and expressed in S cerevisiae;47 the gene is on chromosome 4q32→ter and contains 13 exons.86 Two ETF-QO mutations, i.e., IVS12 +1g→t (c.1810 + 1g→t) and c.427-433del, have been described,50 and nine others are known to us. Of the mutations shown in Fig. 103-4, only c.427-433del, L334P, 1623delT, and P562L have been found in more than one patient. The genotypes of four patients with renal cystic dysplasia46,53,55 are c.427-433del/L334P, 36delA/36delA, c.427-433del/IVS12 + 1g→t, and L334P/L334P.
Schematic of the ETF-QO gene, showing nature and location of all mutations detected to date.84
IVS12 + 1g→t causes skipping of exon 12, which encodes the 5′-most cysteine residue (C561) of the iron sulfur cluster-binding domain at its 3′ end. When ETF-QO cDNA missing exon 12 is expressed in S. cerevisiae, the mutant protein can be reduced by ETF but can no longer reduce ubiquinone.50 It should be noted that P562 is one residue downstream of C561 and that proline in this position is conserved among 4Fe4S cluster-binding domains.47
Deficiency of ETF antigens was first noted in fibroblast lines from two affected infants without congenital anomalies84 and has now been confirmed by immunoblots or immunoprecipitation and pulse chase on several other patients;28,45,46,87,88 most of these did not have renal cystic dysplasia. Some cells are deficient in only one subunit, but in some lines there is rapid degradation of both, perhaps because the heterodimer is unstable. Pulse chase studies suggest that the primary defect in most of the latter lines, including one from an infant with renal cystic disease,87 is in the α-subunit. A primary defect of the β-subunit has been found only in two siblings.87,88 An almost total lack of ETF activity has been demonstrated in cells from patients with neonatal-onset disease, and partial deficiency has been noted in patients with late-onset disease.45,46
cDNAs encoding the α- and β-subunits of ETF have been isolated and expressed in E. coli. 18,24,25 The α- and β-ETF genes are on human chromosomes 15q23-25 and 19q13.3, respectively;89,90 gene structure has not been reported. α-ETF mutations delineated to date include 3-, 18-, and 66-bp deletions and V157G, G116R, and T266M.91,92 The last is common and in one study accounted for five mutant alleles in six patients.92 Only two β-ETF mutations, R164Q and an IVS12 + 1g→c mutation, have been described.93 The genotypes of five infants without renal cystic dysplasia46,64,70,87,92 are αT266M/G116R, αV157G/c.453-470del, αT266M/T266M, βR164Q/IVS12 + 1g→c, and αT266M/c.808-810del, respectively.
The αG116R and αT266M mutations have been expressed in E. coli. ETF containing the αG116R mutation is unstable and, even when forced to dimerize in the presence of the chaperonins GroEL and GroES, produces an inactive and abnormally folded protein.19 The hydroxyl group of αT266 is normally hydrogen bonded to the N(5) position of ETF FAD, and the backbone amide hydrogen is hydrogen bonded to C(4)O.26 ETF containing the αT266M mutation is stable in fibroblasts92 and E. coli,19 but the redox potentials of the oxidized/semiquinone and semiquinone/hydroquinone flavin couples is lowered to such a point that while the ETF can be reduced by MCAD, disproportionation of the semiquinone to ETF , the putative reductant of ETF-QO, is energetically unfavorable.19 The activity of purified αT266M ETF in the reaction with ETF-QO is about 8 percent of normal, which is in agreement with the 18 percent found in fibroblasts from a patient homozygous for this mutation.
Fibroblasts from some patients with clinical and biochemical features of GA2 have normal ETF and ETF:QO activities,94 and since severe riboflavin deficiency can produce a quite similar organic aciduria in rats,95 the disease in such patients might be due to a defect in FAD biosynthesis or transport. There are, however, no instances in which such defects have been conclusively demonstrated. The central role of flavoproteins in cytosolic, microsomal, mitochondrial, and peroxisomal function suggests that early or complete blocks in FAD biosynthesis would be lethal, but the effects of a less severe block in FAD biosynthesis, or those of an as yet undescribed transport system that might move FAD from the cytoplasm into mitochondria, are more difficult to predict. A defect in cytoplasmic FAD biosynthesis could lead to FAD deficiency in both peroxisomes and mitochondria and might explain why certain patients with glutaric acidemia type II resemble those with Zellweger syndrome,94 in which peroxisomal assembly is defective (Chap. 129). Defects of this type should affect only mitochondrial (and peroxisomal) processes that are flavoprotein dependent and should especially affect proteins that have low affinity for FAD or, like ETF and glutaryl CoA dehydrogenase, are unstable in its absence.
Deficiency of ETF or ETF-QO in severely affected patients is virtually complete, resulting in functional deficiency of several enzymes and accumulation of their substrates and metabolites. Some metabolites, like glutaric acid, derive from simple hydrolysis of the accumulated coenzyme A ester, but most have a more complex origin that involves carboxylation, ω- or (ω-1)-oxidation in microsomes, β-oxidation in peroxisomes, or glycine conjugation, either alone or in combination. For example, microsomal ω-oxidation of accumulated long-chain acyl CoAs probably creates long-chain dicarboxylic esters, with subsequent β-oxidation in peroxisomes generating the C12 (dodecanedioic), C10 (sebacic), C8 (suberic), and C6 (adipic) acids.
The origin of 2-hydroxyglutaric acid, which is now confirmed to be the D-isomer,63,96 is not clear but suggests that there is yet another flavin dehydrogenase that acts on 2- or 4-D-hydroxyglutaryl CoA and transfers electrons to the respiratory chain via ETF and ETF-QO. A primary defect of this dehydrogenase might cause isolated D-2-hydroxyglutaric acidemia.97– 99
Sarcosine is frequently found in serum and urine of less severely affected patients but not in patients with acute neonatal onset, perhaps for the following reason. Sarcosine is synthesized by dimethylglycine dehydrogenase (DMGDH) and metabolized by sarcosine dehydrogenase (SDH). Both enzymes transfer flavin-bound electrons to ETF, and sarcosine biosynthesis might be blocked in complete ETF (or ETF-QO) deficiency. With less severe deficiency of ETF or ETF-QO, sarcosine would accumulate if its rate of oxidation were slower than its rate of biosynthesis. The apparent Km values of DMGDH and SDH for their substrates are 0.5 and 1.0 mM, respectively,100 which would favor sarcosine accumulation if the enzyme turnover numbers were similar.
Limited availability of acetyl CoA from β-oxidation of fatty acids, with decreased synthesis of N-acetylglutamate (Chap. 85) and reduced allosteric activation of pyruvate carboxylase (Chap. 100), may be important in causing hyperammonemia and hypoglycemia. Decreased generation of NADH could also limit gluconeogenesis by decreasing glyceraldehyde phosphate dehydrogenase activity. Hyperammonemia may contribute to the encephalopathy that occurs during acute episodes, but high circulating levels of toxic short-chain fatty acids and reduced levels of ketone bodies may also play a role. The heart's inability to oxidize long-chain fatty acids and the decreased availability of ketone bodies deprive it of preferred energy sources, perhaps causing cardiomyopathy.
While loss of carnitine esters of organic acids in the urine might cause depletion of carnitine stores and hypocarnitinemia, its role in pathogenesis is not clear. In severely affected infants, for example, complete deficiency of ETF or ETF-QO would preclude mitochondrial β oxidation of fatty acids, even in the presence of normal carnitine stores. Carnitine deficiency is more likely to be of significance in patients with milder disease. In these patients, who presumably have marginally adequate rates of β oxidation, carnitine depletion could further impair β oxidation by impairing uptake of long-chain fatty acids into skeletal muscle mitochondria, leading to fat accumulation and myopathy.
The cause of abnormal fetal development in some patients with ETF or ETF-QO deficiency is not known, but the presence of congenital anomalies suggests a specific toxic effect of one of the accumulated metabolites or the need for this part of the electron transport chain in certain developmental processes. The similarity of the renal lesions to those seen in riboflavin deficiency, Zellweger syndrome, and neonatal carnitine palmitoyltransferase II deficiency has been noted82,101 but remains unexplained.
Many of the first glutaric acidemia type II patients described were males, prompting speculation that the disorder was inherited as an X-linked trait,60 but additional pedigree data and gene localization studies make it clear that all forms of the disease are transmitted as autosomal recessive traits. Deficiency of ETF-QO in patient fibroblasts, with enzyme activity values in parents intermediate between patient and control values,46,84 establishes inheritance of ETF-QO deficiency as an autosomal recessive trait, and the gene for ETF-QO is on chromosome 4q32→ter.86
The occurrence of defects in α-ETF biosynthesis in males and females, even in the same family, suggests that α-subunit deficiency is also inherited as an autosomal recessive trait, in accord with data indicating that the gene for the β-subunit is on human chromosome 15q23-25.89 The two siblings with primary defects in the β-ETF subunit are male, but the gene encoding the β-subunit is on chromosome 19q13.3,90 establishing inheritance as an autosomal trait.
The disorder is not screened for in the newborn period, and there are no accurate figures on its incidence. Many reports of the condition have appeared since its first description in 1976,52 and it is probably one of the more common inborn errors. It is certainly not rare.
The presence of the characteristic organic acid pattern in urine from a newborn with nonketotic hypoglycemia and metabolic acidosis, with or without congenital anomalies, establishes the diagnosis as glutaric acidemia type II. A very similar organic aciduria occurs in Jamaican vomiting sickness,102 which occurs after ingestion of unripe ackees—and thus only in areas of the world in which the fruit is eaten—and (probably) also in severe riboflavin deficiency.95
Diagnosis in late-onset cases may be considerably more difficult, because metabolic acidosis, the usual indication for examining urine organic acids, may not be present. Further, the organic aciduria in such patients is considerably less pronounced and often intermittent, being present only during acute episodes. The finding of 2-hydroxyglutaric aciduria in such patients is a useful diagnostic point, serving to distinguish the condition from glutaric acidemia (glutaryl CoA dehydrogenase deficiency), in which 3-hydroxyglutaric acid is excreted. Some patients with mild glutaric acidemia type II bear a strong phenotypic resemblance to children with von Gierke's disease (Chap. 71), and liver biopsy will be avoided only if the absence of ketonemia and ketonuria is recognized.
Whole fibroblast metabolism of a variety of radiolabeled compounds may be examined in an attempt to solidify the diagnosis, and this can be done by measuring either oxidation of various 14C-compounds, e.g., [1-14C]palmitate, [1-14C]octanoate, [1-14C]butyrate, [l,5-14C]glutarate, 2-14C]lysine, [2-14C]leucine to 14CO2 or incorporation of 14C from appropriate labeled precursors into lipid or protein. Another published method assays oxidation of [l,5-14C]glutaryl CoA to 14CO2 in the absence of artificial electron acceptor, when oxidation is totally dependent on the presence of endogenous ETF and ETF-QO.103,104 However, since there is more than one cause of glutaric acidemia type II, the specific diagnosis can be established only by demonstrating deficiency of ETF or ETF-QO in fibroblasts or other appropriate tissues, e.g., liver, or by showing that cells from a particular patient do not complement those of patients with a known biochemical defect.
In some cases the specific defect can be demonstrated by showing deficient ETF or ETF-QO antigen in appropriate tissues, either by Western blot analysis46 or by radiolabeling of cells followed by immunoprecipitation.28 In other cases, usually those with mild disease or late onset, direct assay of ETF or ETF-QO activity is necessary.
Glutaric acidemia type II fibroblasts also show a defect in the ability to release tritium from [9,10(n)-3H]-palmitic acid into cell water, and mutant fibroblasts may be assigned to complementation groups based on their ability to correct the defect in another cell line after polyethylene glycol-induced cell fusion.105 Such procedures can quickly and accurately identify the enzyme defect in a cell line without growing the large number of cells needed to assay ETF or ETF-QO. An added advantage is that if there are forms of glutaric acidemia type II due to mutations of proteins other than ETF and ETF-QO, cells with such defects can be identified as such without identifying the defective protein.
Prenatal diagnosis of glutaric acidemia type II has been established on several occasions by demonstrating increased glutaric acid in amniotic fluid, acylcarnitine esters in maternal urine, and/or impaired substrate oxidation by whole cultured amniocytes.64,65,106– 109 Confirmation of predictions that the fetus would be affected has been obtained by showing deficient substrate oxidation in whole fetal fibroblasts, the presence of large cystic kidneys in ETF-QO-deficient fetuses (Fig. 103-1), or, in other instances, by showing that fetuses that had been allowed to proceed to term indeed had the disease.
Analysis of organic acids in amniotic fluid and substrate oxidation in amniocytes might not clearly indicate fetal status when the proband had mild disease or slight or intermittent organic aciduria. Further, methods of measuring ETF or ETF-QO antigen or activity require amounts of tissue that are not easily obtained by amniocentesis or CVS. It is thus possible that in utero diagnosis of patients with mild and variant forms of this condition will increasingly rely on mutation identification in the probands.
Most patients who present within a few days of birth, even those without multiple anomalies, die within the first few months of life. Diets low in fat and protein, with supplementation of carnitine and riboflavin, have been tried without success. Intravenous administration of methylene blue (2 mg/kg per dose) has been tried in one instance, with apparent clinical improvement and almost total clearing of organic aciduria, but the child died soon after the therapeutic trial was terminated.108 Such treatment assumes that the artificial electron acceptor will enter mitochondria, remove flavin-bound electrons from acyl CoA dehydrogenases, and lessen substrate accumulation behind the metabolic block.
Treatment with riboflavin, carnitine, and diets low in protein and fat have been somewhat more successful when applied to patients with milder or later-onset disease. Treatment with oral riboflavin (100–300 mg/day) has been particularly effective in a few patients, including a woman who developed organic aciduria and sarcosinuria only during pregnancy and intercurrent infection and who without treatment had had six consecutive offspring die by 3 months of age.71–73,110 With riboflavin treatment during pregnancy, she delivered two normal offspring.110 The primary defects in these patients have not been identified. Patients with defects in flavin metabolism or transport should be excellent candidates for such treatment, but there may also be patients whose defects in ETF and ETF-QO, both of which contain ionically bound FAD, might respond to higher intramitochondrial FAD concentrations. This might overcome a defect in coenzyme binding, or the FAD might stabilize the mutant electron transferase just enough to increase its activity above a required threshold.