Improved cardiac outcomes with combined atenolol and diazepam intervention in seizure.

OBJECTIVE: Altered autonomic activity has been implicated in the development of cardiac dysfunction during seizures. This study investigates whether intervening in seizure progression with diazepam will reduce seizure-induced cardiomyopathy. Second, this study examines the hypothesis that combining atenolol with diazepam, as an intervention after seizure onset, will combat cardiac injury during status epilepticus. METHODS: Male Sprague-Dawley rats were implanted with electroencephalographic/electrocardiographic electrodes to allow simultaneous recordings during seizures induced by intrahippocampal (2 nmol, 1 µL) kainic acid (KA). Subcutaneous saline, atenolol (5 mg·kg-1 ), diazepam (5 mg·kg-1 ), or atenolol and diazepam (n = 12/group) were administered at 60 minutes post-KA and daily for 7 days, at which point echocardiography, susceptibility to aconitine-induced arrhythmias, and histology were evaluated. RESULTS: Seizure activity was associated with immediately increased heart rate, QTc interval, and blood pressure (BP; 10%-30% across indices). Seven days postseizure, saline-treated animals were found to have reduced left ventricular function, increased fibrotic scarring, and an elevated risk of aconitine-induced arrhythmias. Diazepam treatment significantly reduced cumulative seizure behaviors by 79% compared to saline-treated animals but offered no cardiac protection. Diazepam significantly raised BP (35%) and increased the risk of bigeminal arrhythmias (36%) compared to saline-treated animals. Atenolol administration, either alone or with diazepam, reduced heart rate, QTc interval, and BP back to control levels. Atenolol also preserved cardiac morphology and reduced arrhythmia risk. SIGNIFICANCE: Attenuation of seizure with diazepam offered no cardiac protection; however, coadministration of atenolol with diazepam prevented the development of seizure-induced cardiac dysfunction. This study demonstrates that atenolol intervention should be strongly considered as an adjunct clinical treatment to reduce cardiomyopathy during seizures.

Enzymatic testing sensitivity, variability and practical diagnostic algorithm for pyruvate dehydrogenase complex (PDC) deficiency.

Pyruvate dehydrogenase complex (PDC) deficiency is a major cause of primary lactic acidemia in children. Prompt and correct diagnosis of PDC deficiency and differentiating between specific vs generalized, or secondary deficiencies has important implications for clinical management and therapeutic interventions. Both genetic and enzymatic testing approaches are being used in the diagnosis of PDC deficiency. However, the diagnostic efficacy of such testing approaches for individuals affected with PDC deficiency has not been systematically investigated in this disorder. We sought to evaluate the diagnostic sensitivity and variability of the various PDC enzyme assays in females and males at the Center for Inherited Disorders of Energy Metabolism (CIDEM). CIDEM data were filtered by lactic acidosis and functional PDC deficiency in at least one cell/tissue type (blood lymphocytes, cultured fibroblasts or skeletal muscle) identifying 186 subjects (51% male and 49% female), about half were genetically resolved with 78% of those determined to have a pathogenic PDHA1 mutation. Assaying PDC in cultured fibroblasts in cases where the underlying genetic etiology is PDHA1, was highly sensitive irrespective of gender; 97% (95% confidence interval [CI]: 90%-100%) and 91% (95% CI: 82%-100%) in females and males, respectively. In contrast to the fibroblast-based testing, the lymphocyte- and muscle-based testing were not sensitive (36% [95% CI: 11%-61%, p=0.0003] and 58% [95% CI: 30%-86%, p=0.014], respectively) for identifying known PDC deficient females with pathogenic PDHA1 mutations. In males with a known PDHA1 mutation, the sensitivity of the various cell/tissue assays (75% lymphocyte, 91% fibroblast and 88% muscle) were not statistically different, and the discordance frequency due to the specific cell/tissue used for assaying PDC was 0.15±0.11. Based on this data, a practical diagnostic algorithm is proposed accounting for current molecular approaches, enzyme testing sensitivity, and variability due to gender, cell/tissue type used for testing, and successive repeat testing.

Lethal neonatal case and review of primary short-chain enoyl-CoA hydratase (SCEH) deficiency associated with secondary lymphocyte pyruvate dehydrogenase complex (PDC) deficiency.

Mutations in ECHS1 result in short-chain enoyl-CoA hydratase (SCEH) deficiency which mainly affects the catabolism of various amino acids, particularly valine. We describe a case compound heterozygous for ECHS1 mutations c.836T>C (novel) and c.8C>A identified by whole exome sequencing of proband and parents. SCEH deficiency was confirmed with very low SCEH activity in fibroblasts and nearly absent immunoreactivity of SCEH. The patient had a severe neonatal course with elevated blood and cerebrospinal fluid lactate and pyruvate concentrations, high plasma alanine and slightly low plasma cystine. 2-Methyl-2,3-dihydroxybutyric acid was markedly elevated as were metabolites of the three branched-chain α-ketoacids on urine organic acids analysis. These urine metabolites notably decreased when lactic acidosis decreased in blood. Lymphocyte pyruvate dehydrogenase complex (PDC) activity was deficient, but PDC and α-ketoglutarate dehydrogenase complex activities in cultured fibroblasts were normal. Oxidative phosphorylation analysis on intact digitonin-permeabilized fibroblasts was suggestive of slightly reduced PDC activity relative to control range in mitochondria. We reviewed 16 other cases with mutations in ECHS1 where PDC activity was also assayed in order to determine how common and generalized secondary PDC deficiency is associated with primary SCEH deficiency. For reasons that remain unexplained, we find that about half of cases with primary SCEH deficiency also exhibit secondary PDC deficiency. The patient died on day-of-life 39, prior to establishing his diagnosis, highlighting the importance of early and rapid neonatal diagnosis because of possible adverse effects of certain therapeutic interventions, such as administration of ketogenic diet, in this disorder. There is a need for better understanding of the pathogenic mechanisms and phenotypic variability in this relatively recently discovered disorder.

Succinyl-CoA synthetase (SUCLA2) deficiency in two siblings with impaired activity of other mitochondrial oxidative enzymes in skeletal muscle without mitochondrial DNA depletion.

Mutations in SUCLA2 result in succinyl-CoA ligase (ATP-forming) or succinyl-CoA synthetase (ADP-forming) (A-SCS) deficiency, a mitochondrial tricarboxylic acid cycle disorder. The phenotype associated with this gene defect is largely encephalomyopathy. We describe two siblings compound heterozygous for SUCLA2 mutations, c.985A>G (p.M329V) and c.920C>T (p.A307V), with parents confirmed as carriers of each mutation. We developed a new LC-MS/MS based enzyme assay to demonstrate the decreased SCS activity in the siblings with this unique genotype. Both siblings shared bilateral progressive hearing loss, encephalopathy, global developmental delay, generalized myopathy, and dystonia with choreoathetosis. Prior to diagnosis and because of lactic acidosis and low activity of muscle pyruvate dehydrogenase complex (PDC), sibling 1 (S1) was placed on dichloroacetate, while sibling 2 (S2) was on a ketogenic diet. S1 developed severe cyclic vomiting refractory to therapy, while S2 developed Leigh syndrome, severe GI dysmotility, intermittent anemia, hypogammaglobulinemia and eventually succumbed to his disorder. The mitochondrial DNA contents in skeletal muscle (SM) were normal in both siblings. Pyruvate dehydrogenase complex, ketoglutarate dehydrogenase complex, and several mitochondrial electron transport chain (ETC) activities were low or at the low end of the reference range in frozen SM from S1 and/or S2. In contrast, activities of PDC, other mitochondrial enzymes of pyruvate metabolism, ETC and, integrated oxidative phosphorylation, in skin fibroblasts were not significantly impaired. Although we show that propionyl-CoA inhibits PDC, it does not appear to account for decreased PDC activity in SM. A better understanding of the mechanisms of phenotypic variability and the etiology for tissue-specific secondary deficiencies of mitochondrial enzymes of oxidative metabolism, and independently mitochondrial DNA depletion (common in other cases of A-SCS deficiency), is needed given the implications for control of lactic acidosis and possible clinical management.

Three rare diseases in one Sib pair: RAI1, PCK1, GRIN2B mutations associated with Smith-Magenis Syndrome, cytosolic PEPCK deficiency and NMDA receptor glutamate insensitivity.

The National Institutes of Health Undiagnosed Diseases Program evaluates patients for whom no diagnosis has been discovered despite a comprehensive diagnostic workup. Failure to diagnose a condition may arise from the mutation of genes previously unassociated with disease. However, we hypothesized that this could also co-occur with multiple genetic disorders. Demonstrating a complex syndrome caused by multiple disorders, we report two siblings manifesting both similar and disparate signs and symptoms. They shared a history of episodes of hypoglycemia and lactic acidosis, but had differing exam findings and developmental courses. Clinical acumen and exome sequencing combined with biochemical and functional studies identified three genetic conditions. One sibling had Smith-Magenis Syndrome and a nonsense mutation in the RAI1 gene. The second sibling had a de novo mutation in GRIN2B, which resulted in markedly reduced glutamate potency of the encoded receptor. Both siblings had a protein-destabilizing homozygous mutation in PCK1, which encodes the cytosolic isoform of phosphoenolpyruvate carboxykinase (PEPCK-C). In summary, we present the first clinically-characterized mutation of PCK1 and demonstrate that complex medical disorders can represent the co-occurrence of multiple diseases.

Infection-triggered familial or recurrent cases of acute necrotizing encephalopathy caused by mutations in a component of the nuclear pore, RANBP2.

Acute necrotizing encephalopathy (ANE) is a rapidly progressive encephalopathy that can occur in otherwise healthy children after common viral infections such as influenza and parainfluenza. Most ANE is sporadic and nonrecurrent (isolated ANE). However, we identified a 7 Mb interval containing a susceptibility locus (ANE1) in a family segregating recurrent ANE as an incompletely penetrant, autosomal-dominant trait. We now report that all affected individuals and obligate carriers in this family are heterozygous for a missense mutation (c.1880C-->T, p.Thr585Met) in the gene encoding the nuclear pore protein Ran Binding Protein 2 (RANBP2). To determine whether this mutation is the susceptibility allele, we screened controls and other patients with ANE who are unrelated to the index family. Patients from 9 of 15 additional kindreds with familial or recurrent ANE had the identical mutation. It arose de novo in two families and independently in several other families. Two other patients with familial ANE had different RANBP2 missense mutations that altered conserved residues. None of the three RANBP2 missense mutations were found in 19 patients with isolated ANE or in unaffected controls. We conclude that missense mutations in RANBP2 are susceptibility alleles for familial and recurrent cases of ANE.

High prevalence of overweight and obesity in females with phenylketonuria.

The primary treatment for phenylketonuria (PKU) is a low phenylalanine diet together with an amino acid-based, phenylalanine-free formula. Thus, PKU patients tend to consume a diet enriched in carbohydrates which could predispose to obesity. Studies in the 1980s and 1990s demonstrated that school-age phenylketonuria (PKU) patients have a higher mean body weight compared to a control population. However, no recent studies in the United States PKU population have examined whether this trend has persisted or whether adolescents are also affected. To investigate whether pediatric PKU populations (ages 2-20 years) in two major metropolitan areas of the United States (Cleveland, OH and Houston, TX) have a higher than expected percentage of overweight (BMI≥85th percentile) relative to the general population in the United States (NHANES), a retrospective chart review of PKU patients born between 1990 and 2008 and followed in Cleveland, OH (Rainbow Babies and Children's Hospital/University Hospitals Case Medical Center) and in Houston, TX (Texas Children's Hospital) was performed. Based on data from the U.S., 40% of pediatric PKU patients were overweight or obese. However, the percentage of overweight females (55%) and obese females (33%) is 1.8× and 2.1× higher respectively than expected based on comparison data from U.S. children. Further studies are necessary to identify potential strategies for prevention of excessive weight gain in children with PKU, especially in females.

Spectrum of neurological and survival outcomes in pyruvate dehydrogenase complex (PDC) deficiency: lack of correlation with genotype.

Pyruvate dehydrogenase complex (PDC) deficiency is a relatively common mitochondrial disorder that primarily presents with neurological manifestations and lactic acidemia. We analyzed the clinical outcomes and neurological features of 59 consented symptomatic subjects (27 M, 32 F), who were confirmed to have PDC deficiency with defined mutations in one of the genes of PDC (PDHA1, n = 53; PDHB, n = 4; DLAT, n = 2), including 47 different mutations, of which 22 were novel, and for whom clinical records and/or structured interviews were obtained. 39% of these subjects (23/59) have died. Of these, 91% (21/23) died before age 4 years, 61% (14/23) before 1 year, and 43% (10/23) before 3 months. 56% of males died compared with 25% of females. Causes of death included severe lactic acidosis, respiratory failure, and infection. In subjects surviving past 6 months, a broad range of intellectual outcomes was observed. Of 42 subjects whose intellectual abilities were professionally evaluated, 19% had normal or borderline intellectual ability (CQ/IQ ≥ 70), 10% had mild intellectual disability (ID) (CQ/IQ 55-69), 17% had moderate ID (CQ/IQ 40-54), 24% had severe ID (CQ/IQ 25-39) and 33% had profound ID (CQ/IQ<25). Assessment by parents was comparable. Of 10 subjects who reached age 12 years, 9 had had professional IQ assessments, and only 4 had IQs ≥ 70 (only 2 of these 4 had assessments after age 12 years). The average outcome for females was severe-to-profound ID, whereas that of males was mild-to-moderate ID. Of subjects for whom specific neurological data were available, the majority had hypotonia (89%), and hypertonia or mixed hyper-/hypotonia (49%) were common. Seizures (57%), microcephaly (49%), and structural brain abnormalities including ventriculomegaly (67%) and agenesis, dysgenesis, or hypoplasia of the corpus callosum (55%) were common. Leigh syndrome was found in only 35%. Structural brain abnormalities were more common in females, and Leigh syndrome was more common in males. In a subgroup of 16 ambulatory subjects >3.5 years in whom balance was evaluated, ataxia was found in 13. Peripheral neuropathy was documented in 2 cases but not objectively evaluated in most subjects. Outcomes of this population with genetically confirmed PDC deficiency are heterogeneous and not distinctive. Correlations between specific genotypes and outcomes were not established. Although more females survive, related to the prevalence of X-linked PDHA1 mutations, symptomatic surviving females are generally more severely impaired cognitively and have a different pattern of neurological impairment compared to males. Neonatal or infant onset of symptoms was associated with poor outcomes. Males with PDHA1 mutations and low fibroblast PDC activity were less likely to survive beyond infancy. Recurrence rate in siblings of subjects with PDHA1 mutation was less than 5%. Paradoxically, in this retrospective review, potential factors considered possibly relevant to development, such as in vitro PDC activity, specific mutations, use of ketogenic diets, supplements, or medications, were generally not confirmed to be significantly correlated with objective outcomes of survival or neuro-cognitive function. Therefore, the basis of variability of these outcomes remains largely undetermined.

Treatment of mitochondrial electron transport chain disorders: a review of clinical trials over the past decade.

While many treatments for mitochondrial electron transport (respiratory) chain disorders have been suggested, relatively few have undergone controlled clinical trials. This review focuses on the recent history of clinical trials of dichloroacetate (DCA), arginine, coenzyme Q(10), idebenone, and exercise in both primary (congenital) disorders and secondary (degenerative) disorders. Despite prior clinical impressions that DCA had a positive effect on mitochondrial disorders, two trials of diverse subjects failed to demonstrate a clinically significant benefit, and a trial of DCA in MELAS found a major negative effect of neuropathy. Arginine also has been used to treat MELAS with promising effects, although a controlled trial is still needed for this potentially toxic agent. The anti-oxidant coenzyme Q(10) is very widely used for primary mitochondrial disorders but has not yet undergone a controlled clinical trial; such a trial is now underway, as well as trials of the co-Q analogue idebenone for MELAS and LHON. Greater experience has accumulated with multi-center trials of coenzyme Q(10) treatment to prevent the progression of Parkinson disease. Although initial smaller trials indicated a benefit, this has not yet been confirmed in subsequent trials with higher doses; a larger Phase III trial is now underway. Similarly, a series of trials of idebenone for Friedreich ataxia have shown some benefit in slowing the progression of cardiomyopathy, and controlled clinical trials are now underway to determine if there is significant neurological protection. Uncontrolled trials of exercise showed an increase of exercise tolerance in patients with disorders of mitochondrial DNA, but did not selectively increase the percentage of normal mtDNA; a larger partially controlled trial is now underway to evaluate this possible benefit. In summary, none of the controlled trials so far has conclusively shown a benefit of treatment with the agents tested, but some promising therapies are currently being evaluated in a controlled manner. These experiences underscore the importance of controlled clinical trials for evaluation of benefits and risks of recommended therapies. Application of such clinical trials to future more effective therapies for mitochondrial disorders will require multi-center collaboration, organization, leadership, and financial and advocacy support.

Deletion at chromosomal band Xp22.12-Xp22.13 involving PDHA1 in a patient with congenital lactic acidosis.

We present a patient with congenital lactic acidosis, agenesis of the corpus callosum, and profound developmental delay. Assays of pyruvate dehydrogenase complex function were normal in lymphocytes, but decreased in fibroblasts. Sequencing of the PDHA1 gene did not reveal deleterious mutations, and BAC based microarray analysis did not reveal any chromosomal abnormality. However, gene dosage analysis with oligonucleotide-based chromosomal microarray revealed a deletion of Xp22.12-Xp22.13 involving complete deletion of PDHA1. This is the first report of a whole gene deletion of PDHA1 detected by oligonucleotide-based microarray.