Differential phenotypic expression of a novel PDHA1 mutation in a female monozygotic twin pair.

Pyruvate dehydrogenase complex (PDC) deficiency caused by mutations in the X-linked PDHA1 gene has a broad clinical presentation, and the pattern of X-chromosome inactivation has been proposed as a major factor contributing to its variable expressivity in heterozygous females. Here, we report the first set of monozygotic twin females with PDC deficiency, caused by a novel, de novo heterozygous missense mutation in exon 11 of PDHA1 (NM_000284.3: c.1100A>T). Both twins presented in infancy with a similar clinical phenotype including developmental delay, episodes of hypotonia or encephalopathy, epilepsy, and slowly progressive motor impairment due to pyramidal, extrapyramidal, and cerebellar involvement. However, they exhibited clear differences in disease severity that correlated well with residual PDC activities (approximately 60% and 20% of mean control values, respectively) and levels of immunoreactive E1α subunit in cultured skin fibroblasts. To address whether the observed clinical and biochemical differences could be explained by the pattern of X-chromosome inactivation, we undertook an androgen receptor assay in peripheral blood. In the less severely affected twin, a significant bias in the relative activity of the two X chromosomes with a ratio of approximately 75:25 was detected, while the ratio was close to 50:50 in the other twin. Although it may be difficult to extrapolate these results to other tissues, our observation provides further support to the hypothesis that the pattern of X-chromosome inactivation may influence the phenotypic expression of the same mutation in heterozygous females and broadens the clinical and genetic spectrum of PDC deficiency.

Pyruvate dehydrogenase-E1α deficiency presenting as recurrent acute proximal muscle weakness of upper and lower extremities in an 8-year-old boy.

The mitochondrial pyruvate dehydrogenase enzyme complex (PDHC) plays an important role in aerobic energy metabolism and acid-base equilibrium. PDHC contains of 5 enzymes, 3 catalytic (E1, E2, E3) and 2 regulatory, as well as 3 cofactors and an additional protein (E3-binding protein) encoded by nuclear genes. The clinical presentation of PDHC deficiency ranges from fatal neonatal lactic acidosis to chronic neurologic dysfunction without lactic acidosis. Paroxysmal neurologic problems such as intermittent ataxia, episodic weakness, exercise-induced dystonia and recurrent demyelination may also be seen although they are rare. Here, we present an 8-year-old boy complaining of acute proximal muscle weakness of upper and lower extremities with normal mental status. He had a history of Guillain-Barré-like syndrome at the age of 2 years. Electrophysiologic studies showed sensorial polyneuropathy findings in the first attack and sensorimotor axonal polyneuropathy findings in the last attack. The genetic analysis revealed a previously reported hemizygote novel mutation of the PDHA1 gene (p.A353T/c.1057G > A), which encodes the E1α subunit of PDHC. Thiamine was ordered (15 mg/kg/day), dietary carbohydrates were restricted and clinical findings improved in a few weeks. This rare phenotype of PDHC deficiency is discussed.

Cardiac-Specific Deletion of the Pdha1 Gene Sensitizes Heart to Toxicological Actions of Ischemic Stress.

Pyruvate dehydrogenase (PDH) plays a key role in aerobic energy metabolism and occupies a central crossroad between glycolysis and the tricarboxylic acid cycle. We generated inducible cardiac-specific PDH E1α knockout (CreER(T2)-PDH(flox/flox)) mice that demonstrated a high mortality rate. It was hypothesized that PDH modulating cardiac glucose metabolism is crucial for heart functions under normal physiological and/or stress conditions. The myocardial infarction was conducted by a ligation of the left anterior descending coronary arteries. Cardiac PDH E1α deficiency caused large myocardial infarcts size and macrophage infiltration in the hearts (P < .01 vs wild-type [WT]). Wheat germ agglutinin and Masson trichrome staining revealed significantly increased hypertrophy and fibrosis in PDH E1α-deficient hearts (P < .05 vs WT). Measurements of heart substrate metabolism in an ex vivo working heart perfusion system demonstrated a significant impairment of glucose oxidation in PDH E1α-deficient hearts during ischemia/reperfusion (P < .05 vs WT). Dichloroacetate, a PDH activator, increased glucose oxidation in WT hearts during ischemia/reperfusion and reduced myocardial infarct size in WT, but not in PDH E1α-deficient hearts. Immunoblotting results demonstrated that cardiac PDH E1α deficiency leads to an impaired ischemic AMP-activated protein kinase activation through Sestrin2-liver kinase B1 interaction which is responsible for an increased susceptibility of PDH E1α-deficient heart to ischemic insults. Thus, cardiac PDH E1α deficiency impairs ischemic AMP-activated protein kinase signaling and sensitizes hearts to the toxicological actions of ischemic stress.

A new approach to generate a safe double-attenuated Plasmodium liver stage vaccine.

Recently it has been shown in rodent malaria models that immunisation with genetically attenuated Plasmodium parasites can confer sterile protection against challenge with virulent parasites. For the mass production of live attenuated Plasmodium parasites for vaccination, safety is a prerequisite. Knockout of a single gene is not sufficient for such a strategy since the parasite can likely compensate for such a genetic modification and a single surviving parasite is sufficient to kill an immunised individual. Parasites must therefore be at least double-attenuated when generating a safe vaccine strain. Genetic double-attenuation can be achieved by knocking out two essential genes or by combining a single gene knockout with the expression of a protein toxic for the parasite. We generated a double-attenuated Plasmodium berghei strain that is deficient in fatty acid synthesis by the knockout of the pdh-e1α gene, introducing a second attenuation by the liver stage-specific expression of the pore-forming bacterial toxin perfringolysin O. With this double genetically attenuated parasite strain, a superior attenuation was indeed achieved compared with single-attenuated strains that were either deficient in pyruvate dehydrogenase (PDH)-E1 or expressed perfringolysin O. In vivo, both single-attenuated strains resulted in breakthrough infections even if low to moderate doses of sporozoites (2,000-5,000) were administered. In contrast, the double genetically attenuated parasite strain, given at moderate doses of 5,000 sporozoites, did not result in blood stage infection and even when administered at 5- to 20-fold higher doses, only single and delayed breakthrough infections were observed. Prime booster immunisation with the double genetically attenuated parasite strain completely protected a susceptible mouse strain from malaria and even a single immunisation conferred protection in some cases and lead to a markedly delayed onset of blood stage infection in others. Importantly, premature rupture of the parasitophorous vacuole membrane by liver stage-specific perfringolysin O expression did not induce host cell death and soluble parasite proteins, which are released into the host cell cytoplasm, have the potential to be processed and presented via MHC class I molecules. This, in turn, might support immunological responses against Plasmodium-infected hepatocytes.

A case of pyruvate dehydrogenase E1α subunit deficiency with antenatal brain dysgenesis demonstrated by prenatal sonography and magnetic resonance imaging.

Prenatal depiction of brain dysgenesis in patients with pyruvate dehydrogenase complex (PDHc) deficiencies has been infrequently reported. As PDHc plays a critical role in the brain that obtains all of the energy from the aerobic oxidation of glucose, its deficiency is a severe inborn disorder of metabolism, which predominantly affects the nervous system. This report describes a case of PDHc deficiency with antenatal brain dysgenesis depicted in detail by fetal ultrasound and magnetic resonance imaging. This is the first case report clearly demonstrating the developing mechanism and time course of antenatal brain lesions in a patient with PDHc deficiency.

MRI features of 4 female patients with pyruvate dehydrogenase E1 alpha deficiency.

Pyruvate dehydrogenase complex is a key intramitochondrial multienzyme complex required for the conversion of pyruvate to acetyl-CoA. Most patients with pyruvate dehydrogenase deficiency have a defect in the E1 alpha subunit, associated with mutations in the PDHA1 gene. In this report, we submit detailed magnetic resonance images in 4 affected female patients with PDHA1 mutations who had with severe cortical atrophy, dilated ventricles, and an incomplete corpus callosum. In one of these patients, the magnetic resonance imaging pattern prompted molecular diagnostic testing when enzymatic testing was normal. We underscore that this constellation of features, which may be misdiagnosed as periventricular leukomalacia, illustrates a pattern highly suggestive of a deficiency of pyruvate dehydrogenase E1 alpha in female patients and should trigger appropriate diagnostic investigations.

Somatic mosaicism for PDHA1 mutation in a male with pyruvate dehydrogenase complex deficiency.

Pyruvate dehydrogenase complex deficiency is a clinically heterogeneous disorder. Most cases are due to mutations in an X-linked PDHA1 gene encoding the E1alpha subunit of the multienzyme complex. Females with mutations in the PDHA1 gene may be asymptomatic or have a milder phenotype as a result of skewed X-inactivation, while males are typically more severely affected. We report a case of PDHA1 mosaicism in a male patient who had a milder phenotype.

A technical surgery for refractory gastroparesis in a patient with a mitochondrial disorder.

Gastroparesis is a symptomatic chronic disorder of the stomach characterized by delayed gastric emptying in the absence of mechanical obstruction. Gastroparesis has been associated with various diseases and may occur as part of a mitochondrial disorder. In a patient with pyruvate dehydrogenase complex deficiency, which is associated with abnormal mitochondrial metabolism, intragastric liquids were retained massively and gastroparesis was diagnosed by demonstrating delayed gastric emptying. For an atonic stomach with impaired fundic distention, partial gastric plication, modified stomach-partitioning gastrojejunostomy using the stapled gastrectomy with Braun's anastomosis, antireflux gastroplasty with stapled wedge, and re-gastrostomy were performed. After operation, intragastric liquids were reduced remarkably and delayed gastric emptying was improved. She was doing well and discharged uneventfully. Our technical surgery reported improvement in delayed gastric emptying and we confirm the benefits of this operation.

PDH E1ß deficiency with novel mutations in two patients with Leigh syndrome.

Most cases of pyruvate dehydrogenase complex (PDHc) deficiency are attributable to mutations in the PDHA1 gene which encodes the E(1)α subunit, with few cases of mutations in the genes for E(3), E3BP (E(3) binding protein), E(2) and E(1)-phosphatase being reported. Only seven patients with deficiency of the E(1)ß subunit have been described, with mutations in the PDHB gene in six of them. Clinically they presented with a non-specific encephalomyopathy. We report two patients with new mutations in PDHB and Leigh syndrome. Patient 1 was a boy with neonatal onset of hyperlactataemia, corpus callosum hypoplasia and a convulsive encephalopathy. After neurological deterioration, he died at age 5 months. Autopsy revealed the characteristic features of Leigh syndrome. Patient 2, also a boy, presented a milder clinical course. First symptoms were noticed at age 16 months with muscular hypotonia, lactic acidosis and recurrent episodes of somnolence and transient tetraparesis. MRI revealed bilateral signal hyperintensities in the globus pallidus, midbrain and crura cerebri. PDHc and E(1) activities were deficient in fibroblasts in patient 1; in patient 2 PDHc deficiency was found in skeletal muscle. Mutations in PDHA1 were excluded. Sequencing of PDHB revealed a homozygous point mutation (c.302T>C), causing a predicted amino acid change (p.M101T) in patient 1. Patient 2 is compound heterozygote for mutations c.301A>G (p.M101V) and c.313G>A (p.R105Q). All three mutations appear to destabilize the E(1) enzyme with a decrease of both E(1)α and E(1)ß subunits in immunoblot analysis. To our knowledge, these patients with novel PDHB mutations are the first reported with Leigh syndrome.

Four novel PDHA1 mutations in pyruvate dehydrogenase deficiency.

The pyruvate dehydrogenase (PDH) complex is a mitochondrial multienzyme that catalyses the irreversible oxidative decarboxylation of pyruvate to acetyl-CoA. We report four novel PDHA1 mutations in patients with pyruvate dehydrogenase deficiency. Analysis of PDH activity showed decreased activity in fibroblasts from all four patients, around 16-52% of mean control, similar to what has been found in previous studies. Two of the mutations were missense mutations: c.616G>A (p.Glu206Lys) and c.457A>G (p.Met153Val), one was a 3 bp in-frame deletion: c.429_431delAGG (p.Gly143del), and one was a 65 bp duplication: c.900-6_958dup65. cDNA analysis of the 65 bp duplication showed a small amount of normal transcript in addition to the transcript corresponding to the duplication. The small amount of normal transcript likely explains the survival of the patient, who was a boy. The duplication and one of the missense mutations were associated with decreased amounts of E(1)α And E(1)ß protein on western blot analysis, whereas the other two mutations were associated with normal amounts. This study adds four novel mutations to the around 90 reported mutations in PDHA1 (HGMD PDHA1 mutation database). The phenotypes of patients with PDH deficiency have been divided into three groups: a neonatal form with severe lactic acidosis, a form observed only in males and characterized by episodes of ataxia with relapses associated with hyperlactataemia, and an infantile form with hypotonia, lethargy, onset of seizures or dystonia, psychomotor retardation, in some cases Leigh-like lesions and mild to moderate hyperlactataemia. The four patients reported here all belong to the latter group, which is the largest.

Pyruvate dehydrogenase E1alpha subunit deficiency in a female patient: evidence of antenatal origin of brain damage and possible etiology of infantile spasms.

Enlargement of the lateral ventricles and atrophy of the brain were documented ultrasonographically in utero at as early as 28th week of gestation in a female patient with lactic acidosis due to deficiency of the pyruvate dehydrogenase E1alpha subunit, demonstrating that the changes characteristic of this disease can occur antenatally. The mechanism of infantile spasms in this disease may be linked to mosaicism of the brain cells involving the normal enzyme and the mutant enzyme.

Aerobic growth deficient Haemophilus influenzae mutants are non-virulent: implications on metabolism.

We investigated aerobic metabolism in Haemophilus influenzae to better understand its essential physiological growth pathways. We describe the isolation and characterization of transposon insertions leading to knockout mutations in lpdA, encoding dihydrolipoamide dehydrogenase. H. influenzae Rd lpdA::Tn10d-cat mutants were unable to grow aerobically and an H. influenzae type b lpdA::Tn10d-cat mutant was significantly attenuated in an infant rat infection model. Since LpdA is a functional subunit of both pyruvate dehydrogenase (aceEF) and alpha-ketoglutarate dehydrogenase (sucAB) the phenotype of the lpdA mutant was further explored by creating separate knockout mutants in the sucAB and aceEF loci. DeltaaceEF and deltasucAB mutants were both significantly attenuated in virulence in the infant rat, but only the sucAB mutant was able to grow aerobically. We therefore conclude that the ability for aerobic growth is critical for invasive disease, and furthermore that a TCA cycle enzyme, alpha-ketoglutarate dehydrogenase, appears to contribute a key metabolic function in vivo, but is not required for growth under laboratory conditions.

A novel Y243S mutation in the pyruvate dehydrogenase El alpha gene subunit: correlation with thiamine pyrophosphate interaction.

We identified a new Y243S mutation in the X-linked E1 alpha-PDH gene in a patient with pyruvate dehydrogenase complex (PDHc) deficiency. The activity in cultured fibroblasts was very low even in the presence of high thiamine pyrophosphate (TPP) concentrations, indicating that the defect could be due to decreased affinity of PDHc for TPP.

Gene therapy for pyruvate dehydrogenase E1alpha deficiency using recombinant adeno-associated virus 2 (rAAV2) vectors.

To determine the feasibility of gene transfer to correct defects in the E1alpha subunit of the pyruvate dehydrogenase (PDH) complex (PDC), we constructed rAAV vectors that expressed PDH E1alpha, either alone or with a green fluorescent protein tag, from a hybrid cytomegalovirus (CMV) enhancer/chicken beta-actin (CB) promoter. These vectors were functional in vitro, as judged by increased expression of mRNA in vector-transduced deficient cell lines and correction of the biochemical defect in PDH activity in these cells. Approximately 30% of wild-type levels of PDH activity were restored under conditions with which only about 15% of cells were transduced. These same vectors were then used in vivo to transduce neurons within the rat striatum. Gene transfer, expression, and translocation into mitochondria were observed, without any obvious untoward effects. In vivo vector-mediated PDH expression persisted for at least 1 year after injection, indicating the stability of gene transfer. These studies provide the basis for future efforts to develop a recombinant AAV (rAAV)-based gene therapy approach for the correction of PDC deficiency.

Cerebral white matter involvement in children with mitochondrial encephalopathies.

In childhood mitochondrial encephalopathies the common MRI features are bilateral symmetric abnormalities in basal nuclei and brainstem. The presence of diffuse white matter abnormality has been described only in a few cases. Among a series of 110 children with mitochondrial encephalopathies, 8 patients with MR imaging consistent with a leukoencephalopathy were retrospectively evaluated. Diagnosis was based on the recognition of the biochemical defect in muscle homogenate. H-MR spectroscopic imaging was performed in six of them. Biochemical analysis demonstrated a defect of respiratory chain complexes in six patients: complex I in two cases, complex II in two, complex IV in one, multiple complexes defect in one. Pyruvate dehydrogenase deficiency was demonstrated in two patients. MRI showed severe involvement of the brain white matter without significant basal nuclei or brainstem abnormalities. Two patients developed large cystic areas since onset; in two others progressive vacuolisation of affected white matter was seen later in the course of the disease. One patient with pyruvate dehydrogenase deficiency also presented with a diffuse cortical polymicrogyria. H-MR spectroscopic imaging showed a decrease of N-acetylaspartate, choline and creatine with lactate accumulation in five patients, and was normal in one. These findings suggest that mitochondrial disorders should be included in the differential diagnosis of white matter disorders.