Identification of a novel mutation in patients with medium-chain acyl-CoA dehydrogenase deficiency

A novel mutation was identified in two unrelated patients with medium-chain acyl-CoA dehydrogenase deficiency. First, a 19-year-old Caucasian female presented with a devastating illness, resulting in sudden death in adulthood which is unusual. The second patient, now a 3.5-year-old male, presented at 17 months of age with a hypoglycemic seizure and dehydration. Sequence analysis revealed a novel mutation G617T in exon 8 resulting in an arginine to leucine substitution at codon 206 (R206L). Both patients were compound heterozygous for this G617T and the common mutation A985G.     

Clinical and molecular aspects of Japanese children with medium chain acyl-CoA dehydrogenase deficiency

We report the outcome of 16 Japanese patients with medium chain acyl-CoA dehydrogenase deficiency. Of them, 7 patients were diagnosed after metabolic crisis, while 9 were detected in the asymptomatic condition. Of the 7 symptomatic cases, 1 died suddenly, and 4 cases had delayed development. All 9 patients identified by neonatal or sibling screening remained healthy. Of 14 mutations identified, 10 were unique for Japanese, and 4 were previously reported in other nationalities. Presymptomatic detection including neonatal screening obviously improves quality of life of Japanese patients, probably regardless of the genotypes.     

Clinical and biochemical outcomes of patients with medium-chain acyl-CoA dehydrogenase deficiency

BackgroundMedium-Chain Acyl-CoA Dehydrogenase (MCAD) deficiency is a fatty acid oxidation disorder that can have variable clinical severity. There is still limited information on its clinical presentation and longitudinal history by genotype, and effectiveness of newborn screening (NBS).MethodsRetrospective data were collected from 90 patients (44 female, 46 male) to compare biochemical data with clinical outcomes. The frequency of adverse events (number of hypoglycemia-related ER visits and admissions) was assessed by genotype (homozygosity or not for the common pathogenic variant, p.Lys329Glu, in the ACADM gene), and method of diagnosis (NBS vs. clinical).ResultsMCAD deficiency in Utah was more frequent compared to the United States average (1: 9266 versus 1:17,759 newborns). With age, C8-carnitine did not change significantly whereas C2-carnitine decreased (p < .001), possibly reflecting reduced carnitine supplementation typically seen with age. Children with MCAD deficiency had normal growth. p.Lys329Glu homozygotes had higher NBS C8-carnitine (23.4 ± 19.6 vs. 6.6 ± 3.0 μmol/L) and lifetime plasma C8-carnitine levels (6.2 ± 5 vs. 3.6 ± 1.9 μmol/L) compared to patients with at least one other pathogenic variant (p < .001 for both) and higher transaminases compared to compound heterozygotes (ALT 41.9 ± 6.2 vs. 31.5 ± 3.7 U/L, AST 63.9 ± 5.8 vs. 45.7 ± 1.8 U/L, p < .05 for both). On average, p.Lys329Glu homozygotes had more hypoglycemic events than compound heterozygotes (1.44 versus 0.49 events/patient) as did patients diagnosed clinically compared to those diagnosed by NBS (2.15 versus 0.62 events/patient), though these differences were not statistically significant. Neonatal death was observed before results of newborn screening were available in one patient homozygous for the common p.Lys329Glu pathogenic variant, but severe neonatal complications (hypoglycemia, cardiac arrhythmia) were also seen in patients with other mutations. No irreversible complications were observed after diagnosis in any patient with MCAD deficiency.DiscussionHomozygosity for the common ACADM p.Lys329Glu pathogenic variant was associated with increased levels of C8-carnitine and transaminases. Newborn screening provides the opportunity to reduce morbidity and post-neonatal mortality in all patients with MCAD deficiency, regardless of genotype.     

Heterogeneity for mutations in medium chain acyl-CoA dehydrogenase deficiency in the UK population

MCAD is the commonest inherited disorder of fatty acid oxidation. We have sought for and studied 21 affected children from 18 families within the UK. In 14 families the children are homozygous for the G985 mutation. In three families the children are compound heterozygotes for G985 and thus carry another and unknown mutation. In one family the child does not carry the G985 mutation on either allele. The carrier incidence of the G985 mutation is 1 in 68, which suggests that the natural history of MCAD deficiency deserves further study.     

Pathologic characterization of short-chain acyl-CoA dehydrogenase deficiency in BALB/cByJ mice

BALB/cByJ mice have a deficiency of short-chain acyl-CoA dehydrogenase (SCAD) and are a useful model for studying the inborn errors of fatty acid metabolism which affect humans. Patients with some of these disorders present with hypoglycemia, hyperamonemia, and microvesicular fatty change of hepatocytes. In the present study we examined pathogen-free, SCAD deficient BALB/cByJ mice and control BALB/cBy mice for biochemical and tissue changes following fasting or salicylate challenge. We observed mitochondrial swelling and microvesicular fatty changes in hepatocytes in mutant mice, especially severe following a fast. However, fasting did not alter their blood ammonia and there was no apparent clinical disease. Similarly, salicylates did not produce disease in the BALB/cByJ mice. We did detect in mice an alternative pathway for salicylate metabolism, by-passing glycine conjugation which is the principal metabolic pathway in humans. © 1993 Wiley-Liss, Inc.     

Gestational, pathologic and biochemical differences between very long-chain acyl-CoA dehydrogenase deficiency and long-chain acyl-CoA dehydrogenase deficiency in the mouse

Although many patients have been found to have very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency, none have been documented with long-chain acyl-CoA dehydrogenase (LCAD) deficiency. In order to understand the metabolic pathogenesis of long-chain fatty acid oxidation disorders, we generated mice with VLCAD deficiency (VLCAD(-/-)) and compared their pathologic and biochemical phenotypes of mice with LCAD deficiency (LCAD(-/-)) and wild-type mice. VLCAD(-/-) mice had milder fatty change in liver and heart. Dehydrogenation of various acyl-CoA substrates by liver, heart and skeletal muscle mitochondria differed among the three genotypes. The results for liver were most informative as VLCAD(-/-) mice had a reduction in activity toward palmitoyl-CoA and oleoyl-CoA (58 and 64% of wild-type, respectively), whereas LCAD(-/-) mice showed a more profoundly reduced activity toward these substrates (35 and 32% of wild-type, respectively), with a significant reduction of activity toward the branched chain substrate 2,6-dimethylheptanoyl-CoA. C(16) and C(18) acylcarnitines were elevated in bile, blood and serum of fasted VLCAD(-/-) mice, whereas abnormally elevated C(12) and C(14) acylcarnitines were prominent in LCAD(-/-) mice. Progeny with the combined LCAD(+/+)//VLCAD(+/-) genotype were over-represented in offspring from sires and dams heterozygous for both LCAD and VLCAD mutations. In contrast, no live mice with a compound LCAD(-/-)//VLCAD(-/-) genotype were detected.     

Proteomic investigation of cultivated fibroblasts from patients with mitochondrial short-chain acyl-CoA dehydrogenase deficiency

Short-chain acyl-CoA dehydrogenase (SCAD) deficiency is a rare inherited autosomal recessive disorder with not yet well established mechanisms of disease. In the present study, the mitochondrial proteome of five symptomatic patients homozygous for missense variations in the SCAD gene ACADS was investigated in an extensive large-scale proteomic study to map protein perturbations linked to the disease.Fibroblast cultures of patient cells homozygous for either c.319C>T/p.Arg107Cys (n = 2) or c.1138C>T/p.Arg380Trp (n = 3) in ACADS, and healthy controls (normal human dermal fibroblasts), were studied. The mitochondrial proteome derived from these cultures was analyzed by label free proteomics using high mass accuracy nanoliquid chromatography tandem mass spectrometry (nanoLC–MS/MS).More than 300 mitochondrial proteins were identified and quantified. Thirteen proteins had significant alteration in protein levels in patients carrying variation c.319C>T in ACADS compared to controls and they belonged to various pathways, such as the antioxidant system and amino acid metabolism. Twenty-two proteins were found significantly altered in patients carrying variation c.1138C>T which included proteins associated with fatty acid β-oxidation, amino acid metabolism and protein quality control system. Three proteins were found significantly regulated in both patient groups: adenylate kinase 4 (AK4), nucleoside diphosphate kinase A (NME1) and aldehyde dehydrogenase family 4 member A1 (ALDH4A1). Proteins AK4 and NME1 deserve further investigation because of their involvement in energy reprogramming, cell survival and proliferation with relevance for SCAD deficiency and related metabolic disorders.     

Expression and characterization of mutations in human very long-chain acyl-CoA dehydrogenase using a prokaryotic system

Very long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the first enzymatic step in the mitochondrial beta-oxidation of fatty acids 14-20 carbons in length. More than 100 cases of VLCAD deficiency have been reported with the disease varying from a severe, often fatal neonatal form to a mild adult-onset form. VLCAD is distinguished from matrix-soluble acyl-CoA dehydrogenases by its unique C-terminal domain, homodimeric structure, and localization to the inner mitochondrial membrane. We have for the first time expressed and purified VLCAD using a bacterial system. Recombinant VLCAD had similar biochemical properties to those reported for native VLCAD and the bacterial system was used to study six previously described disease-causing missense mutations including the two most common mild mutations (T220M, V243A), a mutation leading to the severe disease phenotype (R429W), and three mutations in the C-terminal domain (A450P, L462P, and R573W). Of particular interest was the finding that the A450P and L462P bacterial extracts had normal or increased amounts of VLCAD antigen and activity. In the pure form L462P had roughly 30% of wild-type activity while A450P was normal. Using computer modeling both mutations were mapped to a predicted charged surface of VLCAD that we postulate interacts with the mitochondrial membrane. In a membrane pull down assay both mutants showed greatly reduced mitochondrial membrane association, suggesting a mechanism for the disease in these patients. In summary, the bacterial expression system developed here will significantly advance our understanding of both the clinical aspects of VLCAD deficiency and the basic biochemistry of the enzyme.     

Identification of two mutations in a compound heterozygous child with dihydrolipoamide dehydrogenase deficiency

An infant girl with elevated blood lactate, pyruvate, and plasma branched-chain amino acids was diagnosed with dihydrolipoamide dehydrogenase (E3; dihydrolipoamide: NAD+ oxidoreductase, EC 1.8.1.4) deficiency. Activities of the pyruvate dehydrogenase complex and E3 from patient were 26 and 2% of controls in blood lymphocytes, and 11 and 14% in cultured skin fibroblasts, respectively. Western blot analysis demonstrated that the amount of E3 protein in fibroblasts from the patient and her father was about half of controls, while Northern blot analysis showed normal amounts of E3 RNA. DNA sequencing of cloned full-length E3 cDNAs from the patient revealed two mutations in separate alleles. One is a single base insertion of an extra adenine in the last codon of the leader peptide sequence (TAC-->TAAC) leading to a nonsense mutation which results in the premature termination of the precursor E3 polypeptide (Y35X). The other is a missense mutation due to substitution of guanine for adenine, causing an Arg-->Gly substitution at amino acid 460 of the mature protein (R460G) which triggers the loss of E3 activity probably by structural change in the E3 dimer. DNA sequencing of E3 cDNAs from the parents demonstrated that the nonsense mutation was inherited from the father and the missense mutation was inherited from the mother.      

Identification of the human mitochondrial FAD transporter and its potential role in multiple acyl-CoA dehydrogenase deficiency

Multiple acyl-CoA dehydrogenase deficiency (MADD) or glutaric aciduria type II (GAII) is most often caused by mutations in the genes encoding the alpha- or beta-subunit of electron transfer flavoprotein (ETF) or electron transfer flavoprotein dehydrogenase (ETF-DH). Since not all patients have mutations in these genes, other as yet unidentified genes are predicted to be involved as well. Because all affected mitochondrial flavoproteins in MADD have FAD as a prosthetic group, the underlying defect in these patients may be due to a thus far undisclosed disturbance in the metabolism of FAD. Since a proper mitochondrial flavin balance is maintained by a mitochondrial FAD transporter, a defect of this transporter could also cause an MADD-like phenotype. In yeast, FAD is transported across the mitochondrial inner membrane by the FLX1 protein. An FLX1-mutated Saccharomyces cerevisiae strain exhibits a decreased activity of several mitochondrial flavoproteins. In the present study, we report the identification of the human mitochondrial FAD transporter. Based on sequence similarity to FLX1, we identified two human candidate genes (MFT and N111), which were cloned and characterized by functional expression in an FLX1-mutated yeast strain. Of the two candidate genes, only the previously described mitochondrial folate transporter (MFT) was able to functionally complement the FLX1 mutant. Candidates for mutations in the MFT gene are patients with a clinical suspicion of MADD but without any mutation in the alpha- or beta-subunit of ETF or ETF-DH.     

Novel mutations in five Japanese patients with 3-methylcrotonyl-CoA carboxylase deficiency

Isolated 3-methylcrotonyl-CoA carboxylase (MCC) deficiency appears to be the most frequent organic aciduria detected in tandem mass spectrometry (MS/MS) screening programs in the United States, Australia, and Europe. A pilot study of newborn screening using MS/MS has recently been commenced in Japan. Our group detected two asymptomatic MCC deficiency patients by the pilot screening and collected data on another three MCC deficiency patients to study the molecular bases of the MCC deficiency in Japan. Molecular analyses revealed novel mutations in one of the causative genes, MCCA or MCCB, in all five of the patients: nonsense and frameshift mutations in MCCA (c.1750C > T/c.901_902delAA) in patient 1, nonsense and frameshift mutations in MCCB (c.1054_1055delGG/c.592C > T) in patient 2, frameshift and missense mutations in MCCB (c.1625_1626insGG/c.653_654CA > TT) in patient 3, a homozygous missense mutation in MCCA (c.1380T > G/ 1380T > G) in patient 4, and compound heterozygous missense mutations in MCCB (c.569A > G/ c.838G > T) in patient 5. No obvious clinical symptoms were observed in patients 1, 2, and 3. Patient 4 had severe neurological impairment and patient 5 developed Reye-like syndrome. The increasing use of MS/MS newborn screening in Japan will further clarify the clinical and genetic heterogeneity among patients with MCC deficiency in the Japanese population.     

A novel molecular aspect of Japanese patients with medium-chain acyl-CoA dehydrogenase deficiency (MCADD): c.449-452delCTGA is a common mutation in Japanese patients with MCADD

We studied 11 Japanese patients with medium-chain acyl-CoA dehydrogenase deficiency (MCADD) and found a common mutation, c.449-452delCTGA, which accounted for 45% of the mutations. Seven of 10 independent patients carried at least one copy of this mutation. Phenotypes of homozygous patients with the c.449-452delCTGA mutation varied from asymptomatic to life-threatening metabolic decompensation in Japanese patients with MCADD, similar to the phenotypic variations in Caucasians. This study suggests the genotypic difference between those of Caucasians and Japanese regarding MCADD.     

Molecular characterization of galactokinase deficiency in Japanese patients

Galactokinase (GALK) deficiency is an autosomal recessive disorder, which causes cataract formation in children not maintained on a lactose-free diet. We characterized the human GALK gene by screening a Japanese genomic DNA phage library, and found that several nucleotides in the 5′-untranslated region and introns 1, 2, and 5 in our GALK genomic analysis differed from published data. A 20-bp tandem repeat was found in three places in intron 5, which were considered insertion sequences. We identified five novel mutations in seven unrelated Japanese patients with GALK deficiency. There were three missense mutations and two deletions. All three missense mutations (R256W, T344M, and G349S) occurred at CpG dinucleotides, and the T344M and G349S mutations occurred in the conserved region. The three missense mutations led to a drastic reduction in GALK activity when individual mutant cDNAs were expressed in a mammalian cell system. These findings indicated that these missense mutations caused GALK deficiency. The two deletions, of 410delG and 509–510delGT, occurred at the nucleotide repeats GGGGGG and GTGTGT, respectively, and resulted in in-frame nonsense codons at amino acids 163 and 201. These mutations arose by slipped strand mispairing. All five mutations occurred at hot spots in the CpG dinucleotide for missense mutations and in short direct repeats for deletions. These five mutations in Japanese have not yet been identified in Caucasians. We speculate that the origin of GALK mutations in Japanese is different from that in Caucasians. Received: May 19, 1999 / Accepted: August 21, 1999     

Morbidity and mortality among exclusively breastfed neonates with medium-chain acyl-CoA dehydrogenase deficiency

PurposeDespite greatly improved morbidity and mortality among infants with medium-chain acyl-CoA dehydrogenase deficiency (MCAD) since the implementation of universal newborn screening (NBS), a population of neonates still becomes ill before their positive screen results are available. Exclusive breastfeeding is a proposed risk factor in this group. Since initial studies of MCAD NBS, breastfeeding rates have increased substantially. In this study, we quantify the current risk of early decompensation in neonates with MCAD and identify factors associated with poor outcomes.MethodsWe completed a retrospective analysis of neonates with MCAD referred to our center between 2010 and 2015.ResultsOf 46 infants with MCAD, 11 (23.9%) were symptomatic before the return of the NBS results. Four died or had cardiac arrest; the remaining seven had lethargy and hypoglycemia. All symptomatic patients were exclusively breastfed; only 40.6% of asymptomatic patients were exclusively breastfed. Breastfeeding rates increased from 45.5% in 2010–2011 to 64.7% in 2012–2013 and 87.5% in 2014–2015. Over these same periods, rates of early decompensation increased from 9.09% to 23.5% and 75%, respectively.ConclusionsExclusively breastfed neonates with MCAD are at risk for early metabolic decompensation. As breastfeeding rates increase, close management of feeding difficulties is essential for all neonates awaiting NBS results.Genet Med 18 12, 1315–1319.     

Identification and characterization of three novel mutations in the CASQ1 gene in four patients with tubular aggregate myopathy

Here, we report the identification of three novel missense mutations in the calsequestrin‐1 (CASQ1) gene in four patients with tubular aggregate myopathy. These CASQ1 mutations affect conserved amino acids in position 44 (p.(Asp44Asn)), 103 (p.(Gly103Asp)), and 385 (p.(Ile385Thr)). Functional studies, based on turbidity and dynamic light scattering measurements at increasing Ca²⁺ concentrations, showed a reduced Ca²⁺‐dependent aggregation for the CASQ1 protein containing p.Asp44Asn and p.Gly103Asp mutations and a slight increase in Ca²⁺‐dependent aggregation for the p.Ile385Thr. Accordingly, limited trypsin proteolysis assay showed that p.Asp44Asn and p.Gly103Asp were more susceptible to trypsin cleavage in the presence of Ca²⁺ in comparison with WT and p.Ile385Thr. Analysis of single muscle fibers of a patient carrying the p.Gly103Asp mutation showed a significant reduction in response to caffeine stimulation, compared with normal control fibers. Expression of CASQ1 mutations in eukaryotic cells revealed a reduced ability of all these CASQ1 mutants to store Ca²⁺ and a reduced inhibitory effect of p.Ile385Thr and p.Asp44Asn on store operated Ca²⁺ entry. These results widen the spectrum of skeletal muscle diseases associated with CASQ1 and indicate that these mutations affect properties critical for correct Ca²⁺ handling in skeletal muscle fibers.     

Biochemical characterization of two GALK1 mutations in patients with galactokinase deficiency

Galactokinase (GALK1) deficiency is an autosomal recessive disorder, which causes cataract formation in children not maintained on a lactose-free diet. Galactokinase deficiency results from mutation in the GALK1 gene mapped on 17q24. Since GK1 cDNA was cloned about 20 mutations (prevalently deletions and missense) have been reported to date. Most of these reported mutations are confined to single families, and only one of them, P28T, has been referred as the founder Romani mutation. In this paper we report two novel missense mutations in GALK1 gene, identified in two unrelated patients with galactokinase deficiency. One mutation, g.575G>A, substitutes a valine for a methionine at amino acid 32 (p.V32M), while the other mutation, g.2839G>A, results in the arginine to glutamine substitution p.R239Q (GenBank sequence L76927). Biochemical studies demonstrate that these mutations led to a drastic modification in GALK activity when individual mutant cDNAs were expressed in an E. coli system. These findings indicate the pathogeneticity of these mutations causing GALK deficiency.     

Identification of novel mutations in Spanish patients with muscle carnitine palmitoyltransferase II deficiency

Carnitine palmitoyltransferase II (CPT II) deficiency is the most common recessively inherited disorder of lipid metabolism affecting skeletal muscle and the most frequent cause of hereditary myoglobinuria. We studied 5 Spanish patients with CPT II deficiency from four unrelated families. Four patients had the typical clinical phenotype of muscle CPT II deficiency with recurrent episodes of myoglobinuria, triggered by prolonged exercise, fasting, or fever, and marked elevation of creatine kinase values during metabolic crisis. One patient had exercise-related myalgia, cramps and moderate elevation of serum CK values, but had never had myoglobinuria. Molecular analysis showed that three patients were heterozygous for the S113L mutation and one patient heterozygous for the P50H substitution. To identify the mutations in the other alleles of our patients we amplified and sequenced genomic DNA fragments encompassing the entire coding region and intron/exon boundaries of the CPT2 gene. We found the recently reported 178 insT/del 25 bp in one patient. Three novel mutations were identified: a Y120C substitution that leads to a nonconservative amino acid replacement; a 36-38 insGC mutation that results in premature termination of the translation; and an I502T substitution that affects a conserved amino acid residue in the CPT II protein. Our data confirm the molecular heterogeneity of patients with CPT II deficiency, and suggest that the ethnic origin has to be taken into account before performing mutation analysis in these patients.