Identification of two mutations in a compound heterozygous child with dihydrolipoamide dehydrogenase deficiency
Identification of two mutations in a compound heterozygous child with dihydrolipoamide dehydrogenase deficiencyYoung Soo Hong, Douglas S. Kerr1, William J. Craigen2, Jie Tan, Yanzhen Pan2, Marilyn Lusk1 and Mulchand S. Patel*
Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY 14214, USA, 1Departments of Biochemistry and Pediatrics and the Center for Inherited Disorders of Energy Metabolism, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA and 2Departments of Molecular and Human Genetics and Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA
Received June 28, 1996;Revised and Accepted September 23, 1996
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.
Eukaryotic dihydrolipoamide dehydrogenase (E3; dihydrolipoamide:NAD+ oxidoreductase, EC 1.8.1.4) is a flavoprotein component common to the three [alpha]-keto acid dehydrogenase multienzyme complexes, namely, pyruvate dehydrogenase complex (PDC), [alpha]-ketoglutarate dehydrogenase complex (KGDC), and branched-chain [alpha]-keto acid dehydrogenase complex (BCKDC) (for review see ref. 1 ). E3 is also a component (referred to as L protein) of the glycine cleavage system. It exists as a homodimer of 51 kDa subunits with four distinctive subdomain structures (FAD binding, NAD+ binding, central and interface domains) with non-covalently but tightly bound FAD in the holoenzyme (1 ). The catalytic mechanism of E3 involves two steps in which two electrons are transferred from the covalently linked dihydrolipoyl moiety of dihydrolipoamide acetyltransferase (E2) to E3, forming reduced E3 enzyme (E3H2), and subsequently transferred to NAD+ to form NADH and the oxidized form of the enzyme (2 ,3 ). The gene for human E3 has been localized to chromosome 7, within bands q31-q32, and the intron/exon organization of the gene has been characterized (4 ,5 ). The precursor form of E3 is translated in the cytosol, and contains a 35 amino acid leader sequence, which likely forms an amphiphilic [alpha]-helix that is cleaved upon transport of E3 into the mitochondrial matrix (6 ).
Since E3 is a shared component of the three [alpha]-keto acid dehydrogenase complexes, E3 deficiency results in a reduction in the activities of all three complexes. Clinically, E3 deficiency leads to lactic acidosis, increased concentrations of branched-chain amino acids in the plasma and increased urinary excretion of [alpha]-keto acids (7 ). E3 deficiency also causes neurological degeneration due to the sensitivity of the central nervous system to defects in oxidative metabolism. Previously, E3 deficiency has been documented in only seven patients (8 -14 ). Specific mutations have been identified in only a single patient (15 ). In this report, we describe two different mutations, one nonsense and one missense, causing E3 deficiency in a female patient and partial deficiency in her heterozygous parents.
The activities of PDC and E3 in the patient's blood lymphocytes were 26 and 2% of the mean of controls, respectively. PDC and E3 activities in cultured fibroblasts were 11 and 14% of controls, respectively. Activities of E1 and E2 in the patient's fibroblasts were normal (data not shown). PDC activity in blood lymphocytes from the father and mother was 54 and 64% of controls, while PDC activity in the parents' cultured fibroblasts was 55 and 52%, respectively (Table 1 ). E3 activity in blood lymphocytes as well as cultured fibroblasts from the father and mother ranged between 35 and 45% of controls (Table 1 ). KGDC activity in fibroblasts from the patient was 20% of controls; in fibroblasts from the father and mother, KGDC activity was within the normal range of controls (Table 1 ). This finding is interpreted as evidence that a partial reduction in E3 is not rate-limiting for KGDC activity in fibroblasts. The absence of hyperglycinemia in this patient and in previously reported cases of E3-deficiency (8 -14 ) remains unexplained. Possible explanations would include: (i) the residual activity may be adequate for glycine metabolism, and (ii) the presence of an immunologically distinct E3 for the glycine cleavage system could reflect a genetically distinct form of E3 which remains unidentified (16 ).
Western blot analysis revealed a reduced amount of E3 protein in fibroblasts from the patient and her father and a normal amount of E3 protein in the mother's fibroblasts (Fig. 1 A). Using the E1[alpha] bands as internal controls, the relative intensity ratios of E3 to E1[alpha] bands were measured by densitometry and compared with that of control fibroblasts. The ratios were 0.53 for the patient, 0.33 for the father and 0.89 for the mother. These results show a reduction in the amount of expressed E3 protein in fibroblasts from the patient and her father.
Northern blot analysis showed three E1[beta] mRNA bands (major, 1.6 and 1.3 kb; minor, 5.2 kb) and a doublet of E3 mRNA bands (2.2 and 2.4 kb). These results are in agreement with previously reported results obtained from cultured human fibroblasts (17 ,18 ). The relative intensity ratios of E3 (two bands) to E1[beta] (three bands) were measured by densitometry, and were compared with that of a control. The ratios were 1.02 for patient, 0.99 for the father and 1.04 for the mother, indicating that the patient's E3 deficiency was not caused by a lack of E3 mRNA (Fig. 1 B).
This infant had E3 deficiency due to compound heterozygosity of two mutations: a nonsense mutation in the leader sequence and a missense mutation in the carboxyl region of the protein. Two mutations were previously reported in another E3 deficient patient, both in the coding region; although compound heterozygosity was suggested, this was not firmly established because smaller E3 cDNA fragments were cloned and sequenced, and the parents' cells were not available for investigation (15 ).
The inserted extra adenine in the last codon of the leader sequence (TAC -> TAAC) found in the patient and her father, results in the formation of a termination codon (TAA) at the end of the leader sequence, and a frameshift in the downstream reading frame. The predictable consequences of truncated translation are consistent with the immunoblot finding of reduction in total E3 protein, despite normal amounts of E3 mRNA (Fig. 1 B), confirming that the nonsense mutation was inherited from the father.
The missense mutation (AGA -> GGA) substituting glycine for the arginine at amino acid 460, was found in the patient and her mother's E3 cDNA. This single point mutation, which produces a significant change in both the charge and size of the amino acid residue, is associated with almost total loss of E3 activity in the patient and reduced E3 activity in the mother. The mechanism for the inactivation of E3 by this mutation in a critical region of the protein is most likely a change in the structure of the E3 dimer, since the active sites of E3 are composed of amino acid residues contributed by both subunits (20 ).
A detailed description of this case has been published separately (26 ). Briefly, this infant girl had a history of developmental delay and hypotonia, associated with metabolic acidosis. She was apparently normal at birth but had transient neonatal hypoglycemia and poor sucking. Plasma amino acid analysis initially showed increased leucine (444 [mu]M), isoleucine (134 [mu]M), valine (314 [mu]M) and alloisoleucine (46 [mu]M). Urine organic acid analysis showed mild to moderate increases of lactic, 2-hydroxybutyric, 3-hydroxybutyric, [alpha]-ketoglutaric, and 3-hydroxyisovaleric acids. Blood lactate was persistently increased (5.4-9.9 mM). She was treated initially with dietary restriction of branched-chain amino acids, which resulted in a lowering of her plasma branched-chain amino acids and disappearance of alloisoleucine. Subsequent treatment with dichloroacetate, thiamine, lipoic acid and carnitine was not effective, and she died at age 28 months. An autopsy was not performed.
Skin fibroblasts from the patient, her parents and controls were cultured and harvested as previously described (27 ). E3 activity was measured spectrophotometrically by reduction of NAD+ by reduced lipoamide (28 ). PDC and KGDC were assayed by decarboxylation of their respective [1-14C]keto acid substrates (28 ,29 ).
Immunoblotting was performed essentially as previously described (30 ), except the nitrocellulose membrane was blocked by incubation with 10% non-fat skim milk in Tris-buffered saline containing 2% Tween-20 (TTBS) for 1 h at room temperature after transfer. The membrane was then washed three times with TTBS and incubated for 1 h at room temperature with a mixture of rabbit antisera raised against bovine kidney pyruvate dehydrogenase (E1), bovine heart E2 and bovine heart E3. The membrane was washed again and incubated with horseradish peroxidase-conjugated anti-rabbit goat antibody, and the E1, E2 and E3 protein bands were detected by a chemiluminescence Western blotting system (DuPont). Antibodies were subsequently eluted from the membrane, and the immuno-hybridization assay was repeated using only the E3-specific antibody.
Total RNA was isolated from cultured fibroblasts from the patient, her parents, and a normal control using the `Perfect RNA' kit (5 Prime -> 3 Prime Inc.). RNA from each culture (20 [mu]g) was electrophoresed through formaldehyde containing agarose gel (31 ). RNA was transferred to Genescreen Nylon membrane (DuPont) according to the manufacturer's protocol. The membrane was baked at 80oC for 1 h. After pre- hybridization in Church buffer (32 ) for 1 h, the membrane was hybridized with 32P-labeled E1[beta]/E3 cDNA probe mixture overnight at 68oC (6 ,17 ). The membrane was then washed with 2* SSC/2% SDS twice for 30 min at 65oC, followed by 0.2* SSC/0.2% SDS twice for 30 min at 65oC and exposed to X-ray film overnight.
Total RNA from each cell line (1 [mu]g) was used for the synthesis of E3 cDNA using reverse transcription and the polymerase chain reaction (RT-PCR). In a 20 [mu]l reaction mixture, reverse transcription was carried out for 1 h at 43oC with 1 [mu]g of oligo d(T)16 primer, 0.5 nmol dNTP mixture, 20 mM dithiothreitol and 1 U SuperScript II reverse transcriptase (Gibco BRL), in the reaction buffer supplied by the manufacturer. Using the oligo dT primed first strand cDNAs as templates, E3 cDNAs for each specimen were specifically amplified by PCR using a pair of primers (sense, 5'-GCGCGCGGATCCGGAGGTGAAAGTATTGGCGG-3'; antisense, 5'-GCGCGCGGATCCTCAAAAGTTGATTGATTTGCC-3') that generate BamHI restriction sites at both ends of the full-length cDNA (33 ). After 35 cycles of PCR, the 1.5 kb fragment of E3 cDNA was isolated from a 1.2% agarose gel, and purified using the Wizard PCR preparation kit (Promega).
Purified E3-cDNAs and a pBluescript SK+ vector were digested with BamHI and ligated together before transforming into E.coli XL1-Blue competent cells (34 ). Plasmids containing the appropriate size DNA inserts were sequenced by Sequenase kit version 2.0 (US Biochemicals), using four pairs of overlapping primers (Table 2 ) and pBluescript primers for the entire E3 cDNA. To confirm the mutations that were identified, the RT-PCR and cloning of each subject's E3 cDNA was performed at least three times and subjected to repeated DNA sequencing to minimize any possible artifact caused by polymerase or sequenase errors.
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