Primary Hyperoxaluria Screening and Monitoring: Quantitative Measurement of Plasma Oxalate by Gas Chromatography-Mass Spectrometry With High Sensitivity.

Background: Plasma oxalate measurements can be used for the screening and therapeutic monitoring of primary hyperoxaluria. We developed a gas chromatography-mass spectrometry (GC-MS) assay for plasma oxalate measurements with high sensitivity and suitable testing volumes for pediatric populations. Methods: Plasma oxalate was extracted, derivatized, and analyzed by GC-MS. We measured the ion at m/z 261.10 to quantify oxalate and the 13C2-oxalate ion (m/z: 263.15) as the internal standard. Method validation included determination of the linear range, limit of blank, limit of detection, lower limit of quantification, precision, recovery, carryover, interference, and dilution effect. The cut-off value between primary and non-primary hyperoxaluria in a pediatric population was analyzed. Results: The detection limit was 0.78 µmol/L, and the linear range was up to 80.0 µmol/L. The between-day precision was 5.7% at 41.3 µmol/L and 13.1% at 1.6 µmol/L. The carryover was <0.2%. The recovery rate ranged from 90% to 110%. Interference analysis showed that Hb did not interfere with plasma oxalate quantification, whereas intralipids and bilirubin caused false elevation of oxalate concentrations. A cut-off of 13.9 µmol/L showed 63% specificity and 77% sensitivity, whereas a cut-off of 4.15 µmol/L showed 100% specificity and 20% sensitivity. The minimum required sample volume was 250 µL. The detected oxalate concentrations showed interference from instrument conditioning, sample preparation procedures, medications, and various clinical conditions. Conclusions: GC-MS is a sensitive assay for quantifying plasma oxalate and is suitable for pediatric patients. Plasma oxalate concentrations should be interpreted in a clinical context.

Biallelic pathogenic variants in the mitochondrial form of phosphoenolpyruvate carboxykinase cause peripheral neuropathy.

Phosphoenolpyruvate carboxykinase (PCK) plays a critical role in cytosolic gluconeogenesis, and defects in PCK1 cause a fasting-aggravated metabolic disease with hypoglycemia and lactic acidosis. However, there are two genes encoding PCK, and the role of the mitochondrial resident PCK (encoded by PCK2) is unclear, since gluconeogenesis is cytosolic. We identified three patients in two families with biallelic variants in PCK2. One has compound heterozygous variants (p.Ser23Ter/p.Pro170Leu), and the other two (siblings) have homozygous p.Arg193Ter variation. All three patients have weakness and abnormal gait, an absence of PCK2 protein, and profound reduction in PCK2 activity in fibroblasts, but no obvious metabolic phenotype. Nerve conduction studies showed reduced conduction velocities with temporal dispersion and conduction block compatible with a demyelinating peripheral neuropathy. To validate the association between PCK2 variants and clinical disease, we generated a mouse knockout model of PCK2 deficiency. The animals present abnormal nerve conduction studies and peripheral nerve pathology, corroborating the human phenotype. In total, we conclude that biallelic variants in PCK2 cause a neurogenetic disorder featuring abnormal gait and peripheral neuropathy.

Is impaired energy production a novel insight into the pathogenesis of pyridoxine-dependent epilepsy due to biallelic variants in ALDH7A1?

BACKGROUND: Pyridoxine-dependent epilepsy (PDE) is due to biallelic variants in ALDH7A1 (PDE-ALDH7A1). ALDH7A1 encodes α-aminoadipic semialdehyde dehydrogenase in lysine catabolism. We investigated the gamma aminobutyric acid (GABA) metabolism and energy production pathways in human PDE-ALDH7A1 and its knock-out aldh7a1 zebrafish model. METHODS: We measured GABA pathway, and tricarboxylic acid cycle metabolites and electron transport chain activities in patients with PDE-ALDH7A1 and in knock-out aldh7a1 zebrafish. RESULTS: We report results of three patients with PDE-ALDH7A1: low paired complex I+II and complex II+III and individual complex IV activities in muscle biopsy in patient 1 (likely more severe phenotype); significantly elevated CSF glutamate in the GABA pathway and elevated CSF citrate, succinate, isocitrate and α-ketoglutarate in the TCA cycle in patient 3 (likely more severe phenotype); and normal CSF GABA pathway and TCA cycle metabolites on long-term pyridoxine therapy in patient 2 (likely milder phenotype). All GABA pathway metabolites (γ-hydroxybutyrate, glutamine, glutamate, total GABA, succinic semialdehyde) and TCA cycle metabolites (citrate, malate, fumarate, isocitrate, lactate) were significantly low in the homozygous knock-out aldh7a1 zebrafish compared to the wildtype zebrafish. Homozygous knock-out aldh7a1 zebrafish had decreased electron transport chain enzyme activities compared to wildtype zebrafish. DISCUSSION: We report impaired electron transport chain function, accumulation of glutamate in the central nervous system and TCA cycle dysfunction in human PDE-ALDH7A1 and abnormal GABA pathway, TCA cycle and electron transport chain in knock-out aldh7a1 zebrafish. Central nervous system glutamate toxicity and impaired energy production may play important roles in the disease neuropathogenesis and severity in human PDE-ALDH7A1.

PPA2-associated sudden cardiac death: extending the clinical and allelic spectrum in 20 new families.

PURPOSE: Biallelic hypomorphic variants in PPA2, encoding the mitochondrial inorganic pyrophosphatase 2 protein, have been recently identified in individuals presenting with sudden cardiac death, occasionally triggered by alcohol intake or a viral infection. Here we report 20 new families harboring PPA2 variants. METHODS: Synthesis of clinical and molecular data concerning 34 individuals harboring five previously reported PPA2 variants and 12 novel variants, 11 of which were functionally characterized. RESULTS: Among the 34 individuals, only 6 remain alive. Twenty-three died before the age of 2 years while five died between 14 and 16 years. Within these 28 cases, 15 died of sudden cardiac arrest and 13 of acute heart failure. One case was diagnosed prenatally with cardiomyopathy. Four teenagers drank alcohol before sudden cardiac arrest. Progressive neurological signs were observed in 2/6 surviving individuals. For 11 variants, recombinant PPA2 enzyme activities were significantly decreased and sensitive to temperature, compared to wild-type PPA2 enzyme activity. CONCLUSION: We expand the clinical and mutational spectrum associated with PPA2 dysfunction. Heart failure and sudden cardiac arrest occur at various ages with inter- and intrafamilial phenotypic variability, and presentation can include progressive neurological disease. Alcohol intake can trigger cardiac arrest and should be strictly avoided.

Effect of rapamycin on mitochondria and lysosomes in fibroblasts from patients with mtDNA mutations.

Maintaining mitochondrial function and dynamics is crucial for cellular health. In muscle, defects in mitochondria result in severe myopathies where accumulation of damaged mitochondria causes deterioration and dysfunction. Importantly, understanding the role of mitochondria in disease is a necessity to determine future therapeutics. One of the most common myopathies is mitochondrial encephalopathy lactic acidosis stroke-like episodes (MELAS), which has no current treatment. Recently, patients with MELAS treated with rapamycin exhibited improved clinical outcomes. However, the cellular mechanisms of rapamycin effects in patients with MELAS are currently unknown. In this study, we used cultured skin fibroblasts as a window into the mitochondrial dysfunction evident in MELAS cells, as well as to study the mechanisms of rapamycin action, compared with control, healthy individuals. We observed that mitochondria from patients were fragmented, had a threefold decline in the average speed of motility, a twofold reduced mitochondrial membrane potential, and a 1.5- to 2-fold decline in basal respiration. Despite the reduction in mitochondrial function, mitochondrial import protein Tim23 was elevated in patient cell lines. MELAS fibroblasts exhibited increased MnSOD levels and lysosomal function when compared with healthy controls. Treatment of MELAS fibroblasts with rapamycin for 24 h resulted in increased mitochondrial respiration compared with control cells, a higher lysosome content, and a greater localization of mitochondria to lysosomes. Our studies suggest that rapamycin has the potential to improve cellular health even in the presence of mtDNA defects, primarily via an increase in lysosomal content.

Structural Variant in Mitochondrial-Associated Gene (MRPL3) Induces Adult-Onset Neurodegeneration with Memory Impairment in the Mouse.

An impediment to the development of effective therapies for neurodegenerative disease is that available animal models do not reproduce important clinical features such as adult-onset and stereotypical patterns of progression. Using in vivo magnetic resonance imaging and behavioral testing to study male and female decrepit mice, we found a stereotypical neuroanatomical pattern of progression of the lesion along the limbic system network and an associated memory impairment. Using structural variant analysis, we identified an intronic mutation in a mitochondrial-associated gene (Mrpl3) that is responsible for the decrepit phenotype. While the function of this gene is unknown, embryonic lethality in Mrpl3 knock-out mice suggests it is critical for early development. The observation that a mutation linked to energy metabolism precipitates a pattern of neurodegeneration via cell death across disparate but linked brain regions may explain how stereotyped patterns of neurodegeneration arise in humans or define a not yet identified human disease.SIGNIFICANCE STATEMENT The development of novel therapies for adult-onset neurodegenerative disease has been impeded by the limitations of available animal models in reproducing many of the clinical features. Here, we present a novel spontaneous mutation in a mitochondrial-associated gene in a mouse (termed decrepit) that results in adult-onset neurodegeneration with a stereotypical neuroanatomical pattern of progression and an associated memory impairment. The decrepit mouse model may represent a heretofore undiagnosed human disease and could serve as a new animal model to study neurodegenerative disease.

Bi-allelic mutations of LONP1 encoding the mitochondrial LonP1 protease cause pyruvate dehydrogenase deficiency and profound neurodegeneration with progressive cerebellar atrophy.

LonP1 is crucial for maintaining mitochondrial proteostasis and mitigating cell stress. We identified a novel homozygous missense LONP1 variant, c.2282 C > T, (p.Pro761Leu), by whole-exome and Sanger sequencing in two siblings born to healthy consanguineous parents. Both siblings presented with stepwise regression during infancy, profound hypotonia and muscle weakness, severe intellectual disability and progressive cerebellar atrophy on brain imaging. Muscle biopsy revealed the absence of ragged-red fibers, however, scattered cytochrome c oxidase-negative staining and electron dense mitochondrial inclusions were observed. Primary cultured fibroblasts from the siblings showed normal levels of mtDNA and mitochondrial transcripts, and normal activities of oxidative phosphorylation complexes I through V. Interestingly, fibroblasts of both siblings showed glucose-repressed oxygen consumption compared to their mother, whereas galactose and palmitic acid utilization were similar. Notably, the siblings' fibroblasts had reduced pyruvate dehydrogenase (PDH) activity and elevated intracellular lactate:pyruvate ratios, whereas plasma ratios were normal. We demonstrated that in the siblings' fibroblasts, PDH dysfunction was caused by increased levels of the phosphorylated E1α subunit of PDH, which inhibits enzyme activity. Blocking E1α phosphorylation activated PDH and reduced intracellular lactate concentrations. In addition, overexpressing wild-type LonP1 in the siblings' fibroblasts down-regulated phosphoE1α. Furthermore, in vitro studies demonstrated that purified LonP1-P761L failed to degrade phosphorylated E1α, in contrast to wild-type LonP1. We propose a novel mechanism whereby homozygous expression of the LonP1-P761L variant leads to PDH deficiency and energy metabolism dysfunction, which promotes severe neurologic impairment and neurodegeneration.

Variability of Creatine Metabolism Genes in Children with Autism Spectrum Disorder.

Creatine deficiency syndrome (CDS) comprises three separate enzyme deficiencies with overlapping clinical presentations: arginine:glycine amidinotransferase (GATM gene, glycine amidinotransferase), guanidinoacetate methyltransferase (GAMT gene), and creatine transporter deficiency (SLC6A8 gene, solute carrier family 6 member 8). CDS presents with developmental delays/regression, intellectual disability, speech and language impairment, autistic behaviour, epileptic seizures, treatment-refractory epilepsy, and extrapyramidal movement disorders; symptoms that are also evident in children with autism. The objective of the study was to test the hypothesis that genetic variability in creatine metabolism genes is associated with autism. We sequenced GATM, GAMT and SLC6A8 genes in 166 patients with autism (coding sequence, introns and adjacent untranslated regions). A total of 29, 16 and 25 variants were identified in each gene, respectively. Four variants were novel in GATM, and 5 in SLC6A8 (not present in the 1000 Genomes, Exome Sequencing Project (ESP) or Exome Aggregation Consortium (ExAC) databases). A single variant in each gene was identified as non-synonymous, and computationally predicted to be potentially damaging. Nine variants in GATM were shown to have a lower minor allele frequency (MAF) in the autism population than in the 1000 Genomes database, specifically in the East Asian population (Fisher's exact test). Two variants also had lower MAFs in the European population. In summary, there were no apparent associations of variants in GAMT and SLC6A8 genes with autism. The data implying there could be a lower association of some specific GATM gene variants with autism is an observation that would need to be corroborated in a larger group of autism patients, and with sub-populations of Asian ethnicities. Overall, our findings suggest that the genetic variability of creatine synthesis/transport is unlikely to play a part in the pathogenesis of autism spectrum disorder (ASD) in children.

Novel recessive mutations in COQ4 cause severe infantile cardiomyopathy and encephalopathy associated with CoQ10 deficiency.

Coenzyme Q10 (CoQ10) or ubiquinone is one of the two electron carriers in the mitochondrial respiratory chain which has an essential role in the process of oxidative phosphorylation. Defects in CoQ10 synthesis are usually associated with the impaired function of CoQ10-dependent complexes I, II and III. The recessively transmitted CoQ10 deficiency has been associated with a number of phenotypically and genetically heterogeneous groups of disorders manifesting at variable age of onset. The infantile, multisystemic presentation is usually caused by mutations in genes directly involved in CoQ10 biosynthesis. To date, mutations in COQ1 (PDSS1 and PDSS2), COQ2, COQ4, COQ6, COQ7, COQ8A/ADCK3, COQ8B/ADCK4, and COQ9 genes have been identified in patients with primary form of CoQ10 deficiency. Here we report novel mutations in the COQ4 gene, which were identified in an infant with profound mitochondrial disease presenting with perinatal seizures, hypertrophic cardiomyopathy and severe muscle CoQ10 deficiency.

Novel homozygous PCK1 mutation causing cytosolic phosphoenolpyruvate carboxykinase deficiency presenting as childhood hypoglycemia, an abnormal pattern of urine metabolites and liver dysfunction.

Clinical and laboratory data were collected from three Finnish patients including a sibling pair and another unrelated child with unexplained childhood hypoglycemia. Transient elevation of alanine transaminase, lactate and tricarboxylic acid cycle intermediates, especially fumarate, were noticed in urine organic acid analysis. Exome sequencing was performed for the patients and their parents. A novel homozygous PCK1 c.925G>A (p.G309R) mutation was detected in all affected individuals. COS-1 cells transfected with mutant PCK1 transcripts were used to study the pathogenic nature of the detected variant. The COS-1 transfected cells showed the mutant gene to be incapable of producing a normally functioning cytosolic phosphoenolpyruvate carboxykinase (PEPCK) enzyme. This report further delineates the clinical phenotype of isolated cytosolic PEPCK deficiency and offers a metabolic pattern helping to recognize these patients. Cytosolic PEPCK deficiency should be considered in the differential diagnosis of children presenting with hypoglycemia, hepatic dysfunction and elevated tricarboxylic acid intermediates in urinary organic acid analysis.

Exome Sequencing and the Management of Neurometabolic Disorders.

BACKGROUND: Whole-exome sequencing has transformed gene discovery and diagnosis in rare diseases. Translation into disease-modifying treatments is challenging, particularly for intellectual developmental disorder. However, the exception is inborn errors of metabolism, since many of these disorders are responsive to therapy that targets pathophysiological features at the molecular or cellular level. METHODS: To uncover the genetic basis of potentially treatable inborn errors of metabolism, we combined deep clinical phenotyping (the comprehensive characterization of the discrete components of a patient's clinical and biochemical phenotype) with whole-exome sequencing analysis through a semiautomated bioinformatics pipeline in consecutively enrolled patients with intellectual developmental disorder and unexplained metabolic phenotypes. RESULTS: We performed whole-exome sequencing on samples obtained from 47 probands. Of these patients, 6 were excluded, including 1 who withdrew from the study. The remaining 41 probands had been born to predominantly nonconsanguineous parents of European descent. In 37 probands, we identified variants in 2 genes newly implicated in disease, 9 candidate genes, 22 known genes with newly identified phenotypes, and 9 genes with expected phenotypes; in most of the genes, the variants were classified as either pathogenic or probably pathogenic. Complex phenotypes of patients in five families were explained by coexisting monogenic conditions. We obtained a diagnosis in 28 of 41 probands (68%) who were evaluated. A test of a targeted intervention was performed in 18 patients (44%). CONCLUSIONS: Deep phenotyping and whole-exome sequencing in 41 probands with intellectual developmental disorder and unexplained metabolic abnormalities led to a diagnosis in 68%, the identification of 11 candidate genes newly implicated in neurometabolic disease, and a change in treatment beyond genetic counseling in 44%. (Funded by BC Children's Hospital Foundation and others.).

Cytosolic phosphoenolpyruvate carboxykinase deficiency presenting with acute liver failure following gastroenteritis.

We report a patient from a consanguineous family who presented with transient acute liver failure and biochemical patterns suggestive of disturbed urea cycle and mitochondrial function, for whom conventional genetic and metabolic investigations for acute liver failure failed to yield a diagnosis. Whole exome sequencing revealed a homozygous 12-bp deletion in PCK1 (MIM 614168) encoding cytosolic phosphoenolpyruvate carboxykinase (PEPCK); enzymatic studies subsequently confirmed its pathogenic nature. We propose that PEPCK deficiency should be considered in the young child with unexplained liver failure, especially where there are marked, accumulations of TCA cycle metabolites on urine organic acid analysis and/or an amino acid profile with hyperammonaemia suggestive of a proximal urea cycle defect during the acute episode. If suspected, intravenous administration of dextrose should be initiated. Long-term management comprising avoidance of fasting with the provision of a glucose polymer emergency regimen for illness management may be sufficient to prevent future episodes of liver failure. This case report provides further insights into the (patho-)physiology of energy metabolism, confirming the power of genomic analysis of unexplained biochemical phenotypes.

Prevalence of Creatine Deficiency Syndromes in Children With Nonsyndromic Autism.

BACKGROUND AND OBJECTIVE: Creatine deficiency may play a role in the neurobiology of autism and may represent a treatable cause of autism. The goal of the study was to ascertain the prevalence of creatine deficiency syndromes (CDSs) in children with autism spectrum disorder (ASD). METHODS: In a prospective multicenter study, 443 children were investigated after a confirmed diagnosis of ASD. Random spot urine screening for creatine metabolites (creatine, guanidinoacetate, creatinine, and arginine) with liquid chromatography-tandem mass spectrometry and second-tier testing with high-performance liquid chromatography methodology was followed by recall testing in 24-hour urines and confirmatory testing by Sanger-based DNA sequencing of GAMT, GATM, and SLC6A8 genes. Additional diagnostic tests included plasma creatine metabolites and in vivo brain proton magnetic resonance spectroscopy. The creatine metabolites in spot urine in the autism group were compared with 128 healthy controls controlled for age. RESULTS: In 443 subjects with ASD investigated for CDS, we had 0 events (event: 0, 95% confidence interval 0-0.0068), therefore with 95% confidence the prevalence of CDS is <7 in 1000 children with ASD. The autism and control groups did not vary in terms of creatine metabolites (P > .0125) in urine. CONCLUSION: Our study revealed a very low prevalence of CDS in children with nonsyndromic ASD and no obvious association between creatine metabolites and autism. Unlike our study population, we expect more frequent CDS among children with severe developmental delay, speech impairment, seizures, and movement disorders in addition to impairments in social communication, restricted interests, and repetitive behaviors.

Exome sequencing identifies complex I NDUFV2 mutations as a novel cause of Leigh syndrome.

BACKGROUND: Two siblings with hypertrophic cardiomyopathy and brain atrophy were diagnosed with Complex I deficiency based on low enzyme activity in muscle and high lactate/pyruvate ratio in fibroblasts. METHODS: Whole exome sequencing results of fibroblast gDNA from one sibling was narrowed down to 190 SNPs or In/Dels in 185 candidate genes by selecting non-synonymous coding sequence base pair changes that were not present in the SNP database. RESULTS: Two compound heterozygous mutations were identified in both siblings in NDUFV2, encoding the 24 kDa subunit of Complex I. The intronic mutation (c.IVS2 + 1delGTAA) is disease causing and has been reported before. The other mutation is novel (c.669_670insG, p.Ser224Valfs*3) and predicted to cause a pathogenic frameshift in the protein. Subsequent investigation of 10 probands with complex I deficiency from different families revealed homozygosity for the intronic c.IVS2 + 1delGTAA mutation in a second, consanguineous family. In this family three of five siblings were affected. Interestingly, they presented with Leigh syndrome but no cardiac involvement. The same genotype had been reported previously in a two families but presenting with hypertrophic cardiomyopathy, trunk hypotonia and encephalopathy. CONCLUSION: We have identified NDUFV2 mutations in two families with Complex I deficiency, including a novel mutation. The diagnosis of Leigh syndrome expands the clinical phenotypes associated with the c.IVS2 + 1delGTAA mutation in this gene.

Thiamine Deficiency-Mediated Brain Mitochondrial Pathology in Alaskan Huskies with Mutation in SLC19A3.1.

Alaskan Husky encephalopathy (AHE(1) ) is a fatal brain disease associated with a mutation in SLC19A3.1 (c.624insTTGC, c.625C>A). This gene encodes for a thiamine transporter 2 with a predominately (CNS) central nervous system distribution. Considering that brain is particularly vulnerable to thiamine deficiency because of its reliance on thiamine pyrophosphate (TPP)-dependent metabolic pathways involved in energy metabolism and neurotransmitter synthesis, we characterized the impact of this mutation on thiamine status, brain bioenergetics and the contribution of oxidative stress to this phenotype. In silico modeling of the mutated transporter indicated a significant loss of alpha-helices resulting in a more open protein structure suggesting an impaired thiamine transport ability. The cerebral cortex and thalamus of affected dogs were severely deficient in TPP-dependent enzymes accompanied by decreases in mitochondrial mass and oxidative phosphorylation (OXPHOS) capacity, and increases in oxidative stress. These results along with the behavioral and pathological findings indicate that the phenotype associated with AHE is consistent with a brain-specific thiamine deficiency, leading to brain mitochondrial dysfunction and increased oxidative stress. While some of the biochemical deficits, neurobehavior and affected brain areas in AHE were shared by Wernicke's and Korsakoff's syndromes, several differences were noted likely arising from a tissue-specific vs. that from a whole-body thiamine deficiency.

Variant non ketotic hyperglycinemia is caused by mutations in LIAS, BOLA3 and the novel gene GLRX5.

Patients with nonketotic hyperglycinemia and deficient glycine cleavage enzyme activity, but without mutations in AMT, GLDC or GCSH, the genes encoding its constituent proteins, constitute a clinical group which we call 'variant nonketotic hyperglycinemia'. We hypothesize that in some patients the aetiology involves genetic mutations that result in a deficiency of the cofactor lipoate, and sequenced genes involved in lipoate synthesis and iron-sulphur cluster biogenesis. Of 11 individuals identified with variant nonketotic hyperglycinemia, we were able to determine the genetic aetiology in eight patients and delineate the clinical and biochemical phenotypes. Mutations were identified in the genes for lipoate synthase (LIAS), BolA type 3 (BOLA3), and a novel gene glutaredoxin 5 (GLRX5). Patients with GLRX5-associated variant nonketotic hyperglycinemia had normal development with childhood-onset spastic paraplegia, spinal lesion, and optic atrophy. Clinical features of BOLA3-associated variant nonketotic hyperglycinemia include severe neurodegeneration after a period of normal development. Additional features include leukodystrophy, cardiomyopathy and optic atrophy. Patients with lipoate synthase-deficient variant nonketotic hyperglycinemia varied in severity from mild static encephalopathy to Leigh disease and cortical involvement. All patients had high serum and borderline elevated cerebrospinal fluid glycine and cerebrospinal fluid:plasma glycine ratio, and deficient glycine cleavage enzyme activity. They had low pyruvate dehydrogenase enzyme activity but most did not have lactic acidosis. Patients were deficient in lipoylation of mitochondrial proteins. There were minimal and inconsistent changes in cellular iron handling, and respiratory chain activity was unaffected. Identified mutations were phylogenetically conserved, and transfection with native genes corrected the biochemical deficiency proving pathogenicity. Treatments of cells with lipoate and with mitochondrially-targeted lipoate were unsuccessful at correcting the deficiency. The recognition of variant nonketotic hyperglycinemia is important for physicians evaluating patients with abnormalities in glycine as this will affect the genetic causation and genetic counselling, and provide prognostic information on the expected phenotypic course.

Genome-wide association analysis identifies a mutation in the thiamine transporter 2 (SLC19A3) gene associated with Alaskan Husky encephalopathy.

Alaskan Husky Encephalopathy (AHE) has been previously proposed as a mitochondrial encephalopathy based on neuropathological similarities with human Leigh Syndrome (LS). We studied 11 Alaskan Husky dogs with AHE, but found no abnormalities in respiratory chain enzyme activities in muscle and liver, or mutations in mitochondrial or nuclear genes that cause LS in people. A genome wide association study was performed using eight of the affected dogs and 20 related but unaffected control AHs using the Illumina canine HD array. SLC19A3 was identified as a positional candidate gene. This gene controls the uptake of thiamine in the CNS via expression of the thiamine transporter protein THTR2. Dogs have two copies of this gene located within the candidate interval (SLC19A3.2 - 43.36-43.38 Mb and SLC19A3.1 - 43.411-43.419 Mb) on chromosome 25. Expression analysis in a normal dog revealed that one of the paralogs, SLC19A3.1, was expressed in the brain and spinal cord while the other was not. Subsequent exon sequencing of SLC19A3.1 revealed a 4bp insertion and SNP in the second exon that is predicted to result in a functional protein truncation of 279 amino acids (c.624 insTTGC, c.625 C>A). All dogs with AHE were homozygous for this mutation, 15/41 healthy AH control dogs were heterozygous carriers while 26/41 normal healthy AH dogs were wild type. Furthermore, this mutation was not detected in another 187 dogs of different breeds. These results suggest that this mutation in SLC19A3.1, encoding a thiamine transporter protein, plays a critical role in the pathogenesis of AHE.

Complex II deficiency--a case report and review of the literature.

Complex II deficiency is a rare cause of mitochondrial respiratory chain defects with a prevalence of 2-23%. It is exclusively nuclear encoded and functions in the citric acid cycle by oxidizing succinate to fumarate and in the mitochondrial electron transport chain (ETC) by transferring electrons to ubiquinone. Of the four subunits, SDHA and SDHB are catalytic and SDHC and SDHD are anchoring. Mutations in SDHA and SDHAF1 (assembly factor) have been found in patients with CII deficiency and a mitochondrial phenotype. We present a patient with CII deficiency with a previously undescribed phenotype of dilated cardiomyopathy, left ventricular noncompaction, failure to thrive, hypotonia, and developmental delay. Also, a comprehensive review of 36 cases published in the literature was undertaken. The results show that CII deficiency has a variable phenotype with no correlation with residual complex activity in muscle although the phenotype and enzyme activities are comparable within a family. For some, the condition was fatal in infancy, others had multisystem involvement and some had onset in adulthood with mild symptoms and normal cognition. Neurological involvement is most commonly observed and brain imaging commonly shows leukoencephalopathy, Leigh syndrome, or cerebellar atrophy. Mutations in SDHAF1 are associated with leukoencephalopathy. Other organ systems like heart, muscle, and eyes are only involved in about 50% of the cases but cardiomyopathy is associated with high mortality and morbidity. In some patients, riboflavin has provided clinical improvement.

Mitochondrial citrate synthase crystals: novel finding in Sengers syndrome caused by acylglycerol kinase (AGK) mutations.

We report on two families with Sengers syndrome and mutations in the acylglycerol kinase gene (AGK). In the first family, two brothers presented with vascular strokes, lactic acidosis, cardiomyopathy and cataracts, abnormal muscle cell histopathology and mitochondrial function. One proband had very abnormal mitochondria with citrate synthase crystals visible in electron micrographs, associated with markedly high citrate synthase activity. Exome sequencing was used to identify mutations in the AGK gene in the index patient. Targeted sequencing confirmed the same homozygous mutation (c.3G>A, p.M1I) in the brother. The second family had four affected members, of which we examined two. They also presented with similar clinical symptoms, but no strokes. Postmortem heart and skeletal muscle tissues showed low complex I, III and IV activities in the heart, but normal in the muscle. Skin fibroblasts showed elevated lactate/pyruvate ratios and low complex I+III activity. Targeted sequencing led to identification of a homozygous c.979A>T, p.K327* mutation. AGK is located in the mitochondria and phosphorylates monoacylglycerol and diacylglycerol to lysophosphatidic acid and phosphatidic acid. Disruption of these signaling molecules affects the mitochondria's response to superoxide radicals, resulting in oxidative damage to mitochondrial DNA, lipids and proteins, and stimulation of cellular detoxification pathways. High levels of manganese superoxide dismutase protein were detected in all four affected individuals, consistent with increased free radical damage. Phosphatidic acid is also involved in the synthesis of phospholipids and its loss will result in changes to the lipid composition of the inner mitochondrial membrane. These effects manifest as cataract formation in the eye, respiratory chain dysfunction and cardiac hypertrophy in heart tissue. These two pedigrees confirm that mutation of AGK is responsible for the severe neonatal presentation of Sengers syndrome. The identification of citrate synthase precipitates by electron microscopy and the presence of vascular strokes in two siblings may expand the cellular and clinical phenotype of this disease.