CoQ deficiency causes disruption of mitochondrial sulfide oxidation, a new pathomechanism associated with this syndrome

Coenzyme Q (CoQ) is a key component of the mitochondrial respiratory chain, but it also has several other functions in the cellular metabolism. One of them is to function as an electron carrier in the reaction catalyzed by sulfide:quinone oxidoreductase (SQR), which catalyzes the first reaction in the hydrogen sulfide oxidation pathway. Therefore, SQR may be affected by CoQ deficiency. Using human skin fibroblasts and two mouse models with primary CoQ deficiency, we demonstrate that severe CoQ deficiency causes a reduction in SQR levels and activity, which leads to an alteration of mitochondrial sulfide metabolism. In cerebrum of Coq9R239X mice, the deficit in SQR induces an increase in thiosulfate sulfurtransferase and sulfite oxidase, as well as modifications in the levels of thiols. As a result, biosynthetic pathways of glutamate, serotonin, and catecholamines were altered in the cerebrum, and the blood pressure was reduced. Therefore, this study reveals the reduction in SQR activity as one of the pathomechanisms associated with CoQ deficiency syndrome

A spontaneous missense mutation in the chromodomain helicase DNA‐binding protein 8 (CHD8) gene: a novel association with congenital myasthenic syndrome

Aims: Congenital myasthenic syndromes (CMS) are characterized by muscle weakness, ptosis and episodic apnoea. Mutations affect integral protein components of the neuromuscular junction (NMJ). Here we searched for the genetic basis of CMS in female monozygotic twins. Methods: We employed whole-exome sequencing for mutation detection and Sanger sequencing for segregation analysis. Immunohistology was done with antibodies against CHD8, rapsyn, β-catenin (βCAT) and golgin on fi-bro-blasts, human and mouse muscle. We recorded superresolution images of the NMJ using 3D-structured illumination microscopy. Results: We discovered a spontaneous missense mutation in CHD8 [chr14:g.21,884,051G>A, GRCh37.p11 | c.1732C>T, NM_00117062 | p.(R578C)], the gene encoding chromodomain helicase DNA-binding protein 8. This is the first missense mutation affecting Duplin, the short 110 kDa isoform of CHD8. It is known that CHD8/Duplin negatively regulates βCAT signalling in the WNT pathway and plays a role in chromatin remodelling. Inactivating CHD8 mutations are associated with autism spectrum disorder and intellectual disability in combination with facial dysmorphism, overgrowth and macrocephalus. No muscle-specific phenotype has been reported to date. Co-immunostaining with rapsyn on human and mouse muscle revealed a strong presence of CHD8 at the NMJ being located towards the sarcoplasmic side of the rapsyn cluster, where it co-localizes with βCAT. Conclusion: We hypothesize CHD8 to have a role in the maintenance of the structural integrity and function of the NMJ. Both patients benefited from treatment with 3,4-diaminopyridine, a reversible blocker of voltage-gated potassium channels at the nerve terminal that prolongs the action potential and increases acetylcholine release

Systematic Comparison of Three Methods for Fragmentation of Long-Range PCR Products for Next Generation Sequencing

Next Generation Sequencing (NGS) technologies are gaining importance in the routine clinical diagnostic setting. It is thus desirable to simplify the workflow for high-throughput diagnostics. Fragmentation of DNA is a crucial step for preparation of template libraries and various methods are currently known. Here we evaluated the performance of nebulization, sonication and random enzymatic digestion of long-range PCR products on the results of NGS. All three methods produced high-quality sequencing libraries for the 454 platform. However, if long-range PCR products of different length were pooled equimolarly, sequence coverage drastically dropped for fragments below 3,000 bp. All three methods performed equally well with regard to overall sequence quality (PHRED) and read length. Enzymatic fragmentation showed highest consistency between three library preparations but performed slightly worse than sonication and nebulization with regard to insertions/deletions in the raw sequence reads. After filtering for homopolymer errors, enzymatic fragmentation performed best if compared to the results of classic Sanger sequencing. As the overall performance of all three methods was equal with only minor differences, a fragmentation method can be chosen solely according to lab facilities, feasibility and experimental design

CoQ deficiency causes disruption of mitochondrial sulfide oxidation, a new pathomechanism associated with this syndrome

Coenzyme Q (CoQ) is a key component of the mitochondrial respiratory chain, but it also has several other functions in the cellular metabolism. One of them is to function as an electron carrier in the reaction catalyzed by sulfide:quinone oxidoreductase (SQR), which catalyzes the first reaction in the hydrogen sulfide oxidation pathway. Therefore, SQR may be affected by CoQ deficiency. Using human skin fibroblasts and two mouse models with primary CoQ deficiency, we demonstrate that severe CoQ deficiency causes a reduction in SQR levels and activity, which leads to an alteration of mitochondrial sulfide metabolism. In cerebrum of Coq9R239X mice, the deficit in SQR induces an increase in thiosulfate sulfurtransferase and sulfite oxidase, as well as modifications in the levels of thiols. As a result, biosynthetic pathways of glutamate, serotonin, and catecholamines were altered in the cerebrum, and the blood pressure was reduced. Therefore, this study reveals the reduction in SQR activity as one of the pathomechanisms associated with CoQ deficiency syndrome.Ministerio de Economía y CompetitividadERDF/SAF2013-47761-RERDF/SAF2014-55523-RERDF/RD12/0042/0011ERDF/SAF2015-65786-RJunta de AndalucíaNIH/P01HD08064

Habitual Physical Activity in Mitochondrial Disease

Mitochondrial disease is the most common neuromuscular disease and has a profound impact upon daily life, disease and longevity. Exercise therapy has been shown to improve mitochondrial function in patients with mitochondrial disease. However, no information exists about the level of habitual physical activity of people with mitochondrial disease and its relationship with clinical phenotype.Habitual physical activity, genotype and clinical presentations were assessed in 100 patients with mitochondrial disease. Comparisons were made with a control group individually matched by age, gender and BMI. = −0.49; 95% CI −0.33, −0.63, P<0.01). There were no systematic differences in physical activity between different genotypes mitochondrial disease.These results demonstrate for the first time that low levels of physical activity are prominent in mitochondrial disease. Combined with a high prevalence of obesity, physical activity may constitute a significant and potentially modifiable risk factor in mitochondrial disease

Statistical Guidance for Experimental Design and Data Analysis of Mutation Detection in Rare Monogenic Mendelian Diseases by Exome Sequencing

Recently, whole-genome sequencing, especially exome sequencing, has successfully led to the identification of causal mutations for rare monogenic Mendelian diseases. However, it is unclear whether this approach can be generalized and effectively applied to other Mendelian diseases with high locus heterogeneity. Moreover, the current exome sequencing approach has limitations such as false positive and false negative rates of mutation detection due to sequencing errors and other artifacts, but the impact of these limitations on experimental design has not been systematically analyzed. To address these questions, we present a statistical modeling framework to calculate the power, the probability of identifying truly disease-causing genes, under various inheritance models and experimental conditions, providing guidance for both proper experimental design and data analysis. Based on our model, we found that the exome sequencing approach is well-powered for mutation detection in recessive, but not dominant, Mendelian diseases with high locus heterogeneity. A disease gene responsible for as low as 5% of the disease population can be readily identified by sequencing just 200 unrelated patients. Based on these results, for identifying rare Mendelian disease genes, we propose that a viable approach is to combine, sequence, and analyze patients with the same disease together, leveraging the statistical framework presented in this work

Regionalized Pathology Correlates with Augmentation of mtDNA Copy Numbers in a Patient with Myoclonic Epilepsy with Ragged-Red Fibers (MERRF-Syndrome)

Human patients with myoclonic epilepsy with ragged-red fibers (MERRF) suffer from regionalized pathology caused by a mutation in the mitochondrial DNA (m.8344A→G). In MERRF-syndrome brain and skeletal muscles are predominantly affected, despite mtDNA being present in any tissue. In the past such tissue-specificity could not be explained by varying mtDNA mutation loads. In search for a region-specific pathology in human individuals we determined the mtDNA/nDNA ratios along with the mutation loads in 43 different post mortem tissue samples of a 16-year-old female MERRF patient and in four previously healthy victims of motor vehicle accidents. In brain and muscle we further determined the quantity of mitochondrial proteins (COX subunits II and IV), transcription factors (NRF1 and TFAM), and VDAC1 (Porin) as a marker for the mitochondrial mass. In the patient the mutation loads varied merely between 89–100%. However, mtDNA copy numbers were increased 3–7 fold in predominantly affected brain areas (e.g. hippocampus, cortex and putamen) and in skeletal muscle. Similar increases were absent in unaffected tissues (e.g. heart, lung, kidney, liver, and gastrointestinal organs). Such mtDNA copy number increase was not paralleled by an augmentation of mitochondrial mass in some investigated tissues, predominantly in the most affected tissue regions of the brain. We thus conclude that “futile” stimulation of mtDNA replication per se or a secondary failure to increase the mitochondrial mass may contribute to the regionalized pathology seen in MERRF-syndrome

Region-Specific Expression of Mitochondrial Complex I Genes during Murine Brain Development

Mutations in the nuclear encoded subunits of mitochondrial complex I (NADH:ubiquinone oxidoreductase) may cause circumscribed cerebral lesions ranging from degeneration of the striatal and brainstem gray matter (Leigh syndrome) to leukodystrophy. We hypothesized that such pattern of regional pathology might be due to local differences in the dependence on complex I function. Using in situ hybridization we investigated the relative expression of 33 nuclear encoded complex I subunits in different brain regions of the mouse at E11.5, E17.5, P1, P11, P28 and adult (12 weeks). With respect to timing and relative intensity of complex I gene expression we found a highly variant pattern in different regions during development. High average expression levels were detected in periods of intense neurogenesis. In cerebellar Purkinje and in hippocampal CA1/CA3 pyramidal neurons we found a second even higher peak during the period of synaptogenesis and maturation. The extraordinary dependence of these structures on complex I gene expression during synaptogenesis is in accord with our recent findings that gamma oscillations – known to be associated with higher cognitive functions of the mammalian brain – strongly depend on the complex I activity. However, with the exception of the mesencephalon, we detected only average complex I expression levels in the striatum and basal ganglia, which does not explain the exquisite vulnerability of these structures in mitochondrial disorders

Patterns of Retinal Damage Facilitate Differential Diagnosis between Susac Syndrome and MS

Susac syndrome, a rare but probably underdiagnosed combination of encephalopathy, hearing loss, and visual deficits due to branch retinal artery occlusion of unknown aetiology has to be considered as differential diagnosis in various conditions. Particularly, differentiation from multiple sclerosis is often challenging since both clinical presentation and diagnostic findings may overlap. Optical coherence tomography is a powerful and easy to perform diagnostic tool to analyse the morphological integrity of retinal structures and is increasingly established to depict characteristic patterns of retinal pathology in multiple sclerosis. Against this background we hypothesised that differential patterns of retinal pathology facilitate a reliable differentiation between Susac syndrome and multiple sclerosis. In this multicenter cross-sectional observational study optical coherence tomography was performed in nine patients with a definite diagnosis of Susac syndrome. Data were compared with age-, sex-, and disease duration-matched relapsing remitting multiple sclerosis patients with and without a history of optic neuritis, and with healthy controls. Using generalised estimating equation models, Susac patients showed a significant reduction in either or both retinal nerve fibre layer thickness and total macular volume in comparison to both healthy controls and relapsing remitting multiple sclerosis patients. However, in contrast to the multiple sclerosis patients this reduction was not distributed over the entire scanning area but showed a distinct sectorial loss especially in the macular measurements. We therefore conclude that patients with Susac syndrome show distinct abnormalities in optical coherence tomography in comparison to multiple sclerosis patients. These findings recommend optical coherence tomography as a promising tool for differentiating Susac syndrome from MS