Publications by authors named "Mike Gerards"

24 Publications

  • Page 1 of 1

Knockdown Results in Reduction of mtDNA Copy Number, OXPHOS Deficiency and Abnormalities in Zebrafish Embryos.

Front Cell Dev Biol 2020 12;8:381. Epub 2020 Jun 12.

Department of Genetics and Cell Biology, Maastricht University, Maastricht, Netherlands.

High mitochondrial DNA (mtDNA) copy numbers are essential for oogenesis and embryogenesis and correlate with fertility of oocytes and viability of embryos. To understand the pathology and mechanisms associated with low mtDNA copy numbers, we knocked down mitochondrial transcription factor A (), a regulator of mtDNA replication, during early zebrafish development. Reduction of using a splice-modifying morpholino (MO) resulted in a 42 ± 17% decrease in mtDNA copy number in embryos at 4 days post fertilization. Morphant embryos displayed abnormal development of the eye, brain, heart, and muscle, as well as a 50 ± 22% decrease in ATP production. Transcriptome analysis revealed a decrease in protein-encoding transcripts from the heavy strand of the mtDNA, and down-regulation of genes involved in haem production and the metabolism of metabolites, which appear to trigger increased rRNA and tRNA synthesis in the nucleoli. However, this stress or compensatory response appears to fall short as pathology emerges and expression of genes related to eye development are severely down-regulated. Taken together, this study highlights the importance of sufficient mtDNA copies for early zebrafish development. Zebrafish is an excellent model to manipulate the mtDNA bottleneck and study its effect on embryogenesis rapidly and in large numbers of offspring.
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http://dx.doi.org/10.3389/fcell.2020.00381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7303330PMC
June 2020

Manipulating mtDNA in vivo reprograms metabolism via novel response mechanisms.

PLoS Genet 2019 10 4;15(10):e1008410. Epub 2019 Oct 4.

Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.

Mitochondria have been increasingly recognized as a central regulatory nexus for multiple metabolic pathways, in addition to ATP production via oxidative phosphorylation (OXPHOS). Here we show that inducing mitochondrial DNA (mtDNA) stress in Drosophila using a mitochondrially-targeted Type I restriction endonuclease (mtEcoBI) results in unexpected metabolic reprogramming in adult flies, distinct from effects on OXPHOS. Carbohydrate utilization was repressed, with catabolism shifted towards lipid oxidation, accompanied by elevated serine synthesis. Cleavage and translocation, the two modes of mtEcoBI action, repressed carbohydrate rmetabolism via two different mechanisms. DNA cleavage activity induced a type II diabetes-like phenotype involving deactivation of Akt kinase and inhibition of pyruvate dehydrogenase, whilst translocation decreased post-translational protein acetylation by cytonuclear depletion of acetyl-CoA (AcCoA). The associated decrease in the concentrations of ketogenic amino acids also produced downstream effects on physiology and behavior, attributable to decreased neurotransmitter levels. We thus provide evidence for novel signaling pathways connecting mtDNA to metabolism, distinct from its role in supporting OXPHOS.
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http://dx.doi.org/10.1371/journal.pgen.1008410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6795474PMC
October 2019

A zebrafish model to study small-fiber neuropathy reveals a potential role for GDAP1.

Mitochondrion 2019 07 22;47:273-281. Epub 2019 Jan 22.

Department of Genetics and Cell Biology, Clinical Genomics Unit, Maastricht University, P.O. Box 616, 6200 MD, Maastricht, The Netherlands.; MHeNs School for Mental Health and Neuroscience, Maastricht University, Maastricht, The Netherlands.. Electronic address:

Mutations in genes involved in mitochondrial dynamics (fusion and fission) have been implicated in many peripheral neuropathies. We hypothesized that defects in these genes could result in a phenotype resembling features of small-fiber neuropathy (SFN). This was investigated in zebrafish by knocking down two genes involved in mitochondrial dynamics gdap1 (possibly fission and motility) and opa1 (fusion) using established morpholinos. Our read-outs were nerve density in the caudal fin and a behavioral response to temperature changes, both based on comparable hallmarks of SFN in patients. Knockdown of gdap1 resulted in zebrafish embryos with a reduced density of sensory neurites compared to control morpholino-injected embryos. Furthermore, these embryos demonstrated a decreased temperature-related activity. In contrast, a knockdown of opa1 did not affect the density of sensory neurites nor the temperature-related activity. However, only the opa1 morphants had an effect on mitochondrial network morphology. As we were not able to visualize the mitochondria in the neurons, it could well be that changes in the mitochondrial network remained undetected. Our data indicate that GDAP1 knockdown affects sensory neurite development, however, it is unclear if a problem in mitochondrial fission and network formation is the pathophysiological mechanism. Although we did not observe an effect of inhibiting mitochondrial fusion during development, we still propose that genes involved in mitochondrial dynamics should be screened for mutations in patients with SFN.
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http://dx.doi.org/10.1016/j.mito.2019.01.002DOI Listing
July 2019

RNase H1 promotes replication fork progression through oppositely transcribed regions of mitochondrial DNA.

J Biol Chem 2019 03 11;294(12):4331-4344. Epub 2019 Jan 11.

From the Faculty of Medicine and Health Technology and Tampere University Hospital, FI-33014 Tampere University, Finland.

Mitochondrial DNA (mtDNA) replication uses a simple core machinery similar to those of bacterial viruses and plasmids, but its components are challenging to unravel. Here, we found that, as in mammals, the single gene for RNase H1 () has alternative translational start sites, resulting in two polypeptides, targeted to either mitochondria or the nucleus. RNAi-mediated knockdown did not influence growth or viability of S2 cells, but compromised mtDNA integrity and copy number. knockdown in intact flies also produced a phenotype of impaired mitochondrial function, characterized by respiratory chain deficiency, locomotor dysfunction, and decreased lifespan. Its overexpression in S2 cells resulted in cell lethality after 5-9 days, attributable to the nuclearly localized isoform. knockdown and overexpression produced opposite effects on mtDNA replication intermediates. The most pronounced effects were seen in genome regions beyond the major replication pauses where the replication fork needs to progress through a gene cluster that is transcribed in the opposite direction. RNase H1 deficiency led to an accumulation of replication intermediates in these zones, abundant mtDNA molecules joined by four-way junctions, and species consistent with fork regression from the origin. These findings indicate replication stalling due to the presence of unprocessed RNA/DNA heteroduplexes, potentially leading to the degradation of collapsed forks or to replication restart by a mechanism involving strand invasion. Both mitochondrial RNA and DNA syntheses were affected by knockdown, suggesting that RNase H1 also plays a role in integrating or coregulating these processes in mitochondria.
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http://dx.doi.org/10.1074/jbc.RA118.007015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6433063PMC
March 2019

Whole Exome Sequencing Is the Preferred Strategy to Identify the Genetic Defect in Patients With a Probable or Possible Mitochondrial Cause.

Front Genet 2018 12;9:400. Epub 2018 Oct 12.

Department of Genetics and Cell Biology, Maastricht University Medical Centre, Maastricht, Netherlands.

Mitochondrial disorders, characterized by clinical symptoms and/or OXPHOS deficiencies, are caused by pathogenic variants in mitochondrial genes. However, pathogenic variants in some of these genes can lead to clinical manifestations which overlap with other neuromuscular diseases, which can be caused by pathogenic variants in non-mitochondrial genes as well. Mitochondrial pathogenic variants can be found in the mitochondrial DNA (mtDNA) or in any of the 1,500 nuclear genes with a mitochondrial function. We have performed a two-step next-generation sequencing approach in a cohort of 117 patients, mostly children, in whom a mitochondrial disease-cause could likely or possibly explain the phenotype. A total of 86 patients had a mitochondrial disorder, according to established clinical and biochemical criteria. The other 31 patients had neuromuscular symptoms, where in a minority a mitochondrial genetic cause is present, but a non-mitochondrial genetic cause is more likely. All patients were screened for pathogenic variants in the mtDNA and, if excluded, analyzed by whole exome sequencing (WES). Variants were filtered for being pathogenic and compatible with an autosomal or X-linked recessive mode of inheritance in families with multiple affected siblings and/or consanguineous parents. Non-consanguineous families with a single patient were additionally screened for autosomal and X-linked dominant mutations in a predefined gene-set. We identified causative pathogenic variants in the mtDNA in 20% of the patient-cohort, and in nuclear genes in 49%, implying an overall yield of 68%. We identified pathogenic variants in mitochondrial and non-mitochondrial genes in both groups with, obviously, a higher number of mitochondrial genes affected in mitochondrial disease patients. Furthermore, we show that 31% of the disease-causing genes in the mitochondrial patient group were not included in the MitoCarta database, and therefore would have been missed with MitoCarta based gene-panels. We conclude that WES is preferable to panel-based approaches for both groups of patients, as the mitochondrial gene-list is not complete and mitochondrial symptoms can be secondary. Also, clinically and genetically heterogeneous disorders would require sequential use of multiple different gene panels. We conclude that WES is a comprehensive and unbiased approach to establish a genetic diagnosis in these patients, able to resolve multi-genic disease-causes.
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http://dx.doi.org/10.3389/fgene.2018.00400DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6194163PMC
October 2018

Intracellular vesicle trafficking plays an essential role in mitochondrial quality control.

Mol Biol Cell 2018 Apr;29(7):809-819

Faculty of Medicine and Life Sciences and Tampere University Hospital, FI-33014 University of Tampere, Finland.

The gene products Bet1, Slh, and CG10144, predicted to function in intracellular vesicle trafficking, were previously found to be essential for mitochondrial nucleoid maintenance. Here we show that Slh and Bet1 cooperate to maintain mitochondrial functions. In their absence, mitochondrial content, membrane potential, and respiration became abnormal, accompanied by mitochondrial proteotoxic stress, but without direct effects on mtDNA. Immunocytochemistry showed that both Slh and Bet1 are localized at the Golgi, together with a proportion of Rab5-positive vesicles. Some Bet1, as well as a tiny amount of Slh, cofractionated with highly purified mitochondria, while live-cell imaging showed coincidence of fluorescently tagged Bet1 with most Lysotracker-positive and a small proportion of Mitotracker-positive structures. This three-way association was disrupted in cells knocked down for Slh, although colocalized lysosomal and mitochondrial signals were still seen. Neither Slh nor Bet1 was required for global mitophagy or endocytosis, but prolonged Slh knockdown resulted in G2 growth arrest, with increased cell diameter. These effects were shared with knockdown of betaCOP but not of CG1044, Snap24, or Syntaxin6. Our findings implicate vesicle sorting at the -Golgi in mitochondrial quality control.
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http://dx.doi.org/10.1091/mbc.E17-10-0619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5905294PMC
April 2018

Elevated mitochondrial DNA copy numbers in pediatric acute lymphoblastic leukemia: A potential biomarker for predicting inferior survival.

Pediatr Blood Cancer 2018 03 14;65(3). Epub 2017 Nov 14.

Department of Biochemistry, All India Institute of Medical Sciences (AIIMS), New Delhi, India.

Background: Studies on mitochondrial DNA copy number reveal an increase or decrease in copy number that appears to be cancer specific, but data on acute lymphoblastic leukemia have been inconsistent regarding the significance of changes in mitochondrial DNA copies. The purpose of this pilot study was to analyze mitochondrial DNA copy number and mitochondrial DNA integrity.

Procedure: Copy number and mitochondrial deletion ratios were estimated in the bone marrow of 51 patients and peripheral blood of 30 healthy controls using quantitative real-time PCR. The copy number values were correlated with prognostic markers in patients.

Results: Significantly increased mitochondrial DNA copy number (P-value < 0.0001) and increased mitochondrial deletion ratios (P-value = 0.0018) were observed in patients compared with controls. The copy numbers were significantly decreased in patients after chemotherapy (P-value = 0.0232). Patients with higher copy numbers exhibited significantly inferior survival than patients with lower copy numbers (for event-free survival, P-value = 0.04 and overall survival, P-value = 0.1175).

Conclusions: Significant decreases in mitochondrial DNA copy number with therapy indicates that copy number could be evaluated as a potential marker for therapeutic efficacy and a higher mitochondrial DNA copy number could be a poor prognostic marker.
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http://dx.doi.org/10.1002/pbc.26874DOI Listing
March 2018

Selection and Characterization of Palmitic Acid Responsive Patients with an OXPHOS Complex I Defect.

Front Mol Neurosci 2017 18;10:336. Epub 2017 Oct 18.

Department of Clinical Genetics, Maastricht University Medical Centre, Maastricht, Netherlands.

Mitochondrial disorders are genetically and clinically heterogeneous, mainly affecting high energy-demanding organs due to impaired oxidative phosphorylation (OXPHOS). Currently, effective treatments for OXPHOS defects, with complex I deficiency being the most prevalent, are not available. Yet, clinical practice has shown that some complex I deficient patients benefit from a high-fat or ketogenic diet, but it is unclear how these therapeutic diets influence mitochondrial function and more importantly, which complex I patients could benefit from such treatment. Dietary studies in a complex I deficient patient with exercise intolerance showed increased muscle endurance on a high-fat diet compared to a high-carbohydrate diet. We performed whole-exome sequencing to characterize the genetic defect. A pathogenic homozygous p.G212V missense mutation was identified in the gene, encoding an early assembly factor of complex I. A complementation study in fibroblasts confirmed that the p.G212V mutation caused the complex I deficiency. The mechanism turned out to be an incomplete assembly of the peripheral arm of complex I, leading to a decrease in the amount of mature complex I. The patient clinically improved on a high-fat diet, which was supported by the 25% increase in maximal OXPHOS capacity in TMEM126B defective fibroblast by the saturated fatty acid palmitic acid, whereas oleic acid did not have any effect in those fibroblasts. Fibroblasts of other patients with a characterized complex I gene defect were tested in the same way. Patient fibroblasts with complex I defects in NDUFS7 and NDUFAF5 responded to palmitic acid, whereas ACAD9, NDUFA12, and NDUFV2 defects were non-responding. Although the data are too limited to draw a definite conclusion on the mechanism, there is a tendency that protein defects involved in early assembly complexes, improve with palmitic acid, whereas proteins defects involved in late assembly, do not. Our data show at a clinical and biochemical level that a high fat diet can be beneficial for complex I patients and that our cell line assay will be an easy tool for the selection of patients, who might potentially benefit from this therapeutic diet.
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http://dx.doi.org/10.3389/fnmol.2017.00336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5651253PMC
October 2017

Thiamine deficiency in childhood with attention to genetic causes: Survival and outcome predictors.

Ann Neurol 2017 Sep 30;82(3):317-330. Epub 2017 Aug 30.

Division of Child Neurology, Sant Joan de Déu Hospital, University of Barcelona, Barcelona, Spain.

Primary and secondary conditions leading to thiamine deficiency have overlapping features in children, presenting with acute episodes of encephalopathy, bilateral symmetric brain lesions, and high excretion of organic acids that are specific of thiamine-dependent mitochondrial enzymes, mainly lactate, alpha-ketoglutarate, and branched chain keto-acids. Undiagnosed and untreated thiamine deficiencies are often fatal or lead to severe sequelae. Herein, we describe the clinical and genetic characterization of 79 patients with inherited thiamine defects causing encephalopathy in childhood, identifying outcome predictors in patients with pathogenic SLC19A3 variants, the most common genetic etiology. We propose diagnostic criteria that will aid clinicians to establish a faster and accurate diagnosis so that early vitamin supplementation is considered. Ann Neurol 2017;82:317-330.
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http://dx.doi.org/10.1002/ana.24998DOI Listing
September 2017

Specific MRI Abnormalities Reveal Severe Perrault Syndrome due to CLPP Defects.

Front Neurol 2016 16;7:203. Epub 2016 Nov 16.

Department of Clinical Genetics, Maastricht University Medical Centre, Maastricht, Netherlands; Department of Genetics and Cell Biology, School for Oncology and Developmental Biology, Maastricht University Medical Centre, Maastricht, Netherlands; Maastricht Centre for Systems Biology (MaCSBio), Maastricht, Netherlands.

In establishing a genetic diagnosis in heterogeneous neurological disease, clinical characterization and whole exome sequencing (WES) go hand-in-hand. Clinical data are essential, not only to guide WES variant selection and define the clinical severity of a genetic defect but also to identify other patients with defects in the same gene. In an infant patient with sensorineural hearing loss, psychomotor retardation, and epilepsy, WES resulted in identification of a novel homozygous frameshift mutation (c.21delA). Based on the gene defect and clinical symptoms, the diagnosis Perrault syndrome type 3 (PRLTS3) was established. The patient's brain-MRI revealed specific abnormalities of the subcortical and deep cerebral white matter and the middle blade of the corpus callosum, which was used to identify similar patients in the Amsterdam brain-MRI database, containing over 3000 unclassified leukoencephalopathy cases. In three unrelated patients with similar MRI abnormalities the gene was sequenced, and in two of them novel missense mutations were identified together with a large deletion that covered part of the gene on the other allele. The severe neurological and MRI abnormalities in these young patients were due to the drastic impact of the mutations, correlating with the variation in clinical manifestations among previously reported patients. Our data show that similarity in brain-MRI patterns can be used to identify novel PRLTS3 patients, especially during early disease stages, when only part of the disease manifestations are present. This seems especially applicable to the severely affected cases in which CLPP function is drastically affected and MRI abnormalities are pronounced.
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http://dx.doi.org/10.3389/fneur.2016.00203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5110515PMC
November 2016

Replication Errors Made During Oogenesis Lead to Detectable De Novo mtDNA Mutations in Zebrafish Oocytes with a Low mtDNA Copy Number.

Genetics 2016 Dec 21;204(4):1423-1431. Epub 2016 Oct 21.

Department of Genetics and Cell Biology, Clinical Genomics Unit, School for Oncology and Developmental Biology, Maastricht University, 6200 MD, The Netherlands

Of all pathogenic mitochondrial DNA (mtDNA) mutations in humans, ∼25% is de novo, although the occurrence in oocytes has never been directly assessed. We used next-generation sequencing to detect point mutations directly in the mtDNA of 3-15 individual mature oocytes and three somatic tissues from eight zebrafish females. Various statistical and biological filters allowed reliable detection of de novo variants with heteroplasmy ≥1.5%. In total, we detected 38 de novo base substitutions, but no insertions or deletions. These 38 de novo mutations were present in 19 of 103 mature oocytes, indicating that ∼20% of the mature oocytes carry at least one de novo mutation with heteroplasmy ≥1.5%. This frequency of de novo mutations is close to that deducted from the reported error rate of polymerase gamma, the mitochondrial replication enzyme, implying that mtDNA replication errors made during oogenesis are a likely explanation. Substantial variation in the mutation prevalence among mature oocytes can be explained by the highly variable mtDNA copy number, since we previously reported that ∼20% of the primordial germ cells have a mtDNA copy number of ≤73 and would lead to detectable mutation loads. In conclusion, replication errors made during oogenesis are an important source of de novo mtDNA base substitutions and their location and heteroplasmy level determine their significance.
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http://dx.doi.org/10.1534/genetics.116.194035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5161276PMC
December 2016

Differences in Strength and Timing of the mtDNA Bottleneck between Zebrafish Germline and Non-germline Cells.

Cell Rep 2016 07 30;16(3):622-30. Epub 2016 Jun 30.

Department of Genetics and Cell Biology, Clinical Genomics Unit, School for Oncology and Developmental Biology (GROW), Maastricht University Medical Centre, 6200MD Maastricht, the Netherlands; Maastricht Centre for Systems Biology (MaCSBio), Maastricht University Medical Centre, 6200MD, the Netherlands. Electronic address:

We studied the mtDNA bottleneck in zebrafish to elucidate size, timing, and variation in germline and non-germline cells. Mature zebrafish oocytes contain, on average, 19.0 × 10(6) mtDNA molecules with high variation between oocytes. During embryogenesis, the mtDNA copy number decreases to ∼170 mtDNA molecules per primordial germ cell (PGC), a number similar to that in mammals, and to ∼50 per non-PGC. These occur at the same developmental stage, implying considerable variation in mtDNA copy number in (non-)PGCs of the same female, dictated by variation in the mature oocyte. The presence of oocytes with low mtDNA numbers, if similar in humans, could explain how (de novo) mutations can reach high mutation loads within a single generation. High mtDNA copy numbers in mature oocytes are established by mtDNA replication during oocyte development. Bottleneck differences between germline and non-germline cells, due to early differentiation of PGCs, may account for different distribution patterns of familial mutations.
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http://dx.doi.org/10.1016/j.celrep.2016.06.023DOI Listing
July 2016

Leigh syndrome: Resolving the clinical and genetic heterogeneity paves the way for treatment options.

Mol Genet Metab 2016 Mar 19;117(3):300-12. Epub 2015 Dec 19.

Department of Clinical Genetics, Research School GROW, Maastricht University Medical Centre, Maastricht, The Netherlands.

Leigh syndrome is a progressive neurodegenerative disorder, affecting 1 in 40,000 live births. Most patients present with symptoms between the ages of three and twelve months, but adult onset Leigh syndrome has also been described. The disease course is characterized by a rapid deterioration of cognitive and motor functions, in most cases resulting in death due to respiratory failure. Despite the high genetic heterogeneity of Leigh syndrome, patients present with identical, symmetrical lesions in the basal ganglia or brainstem on MRI, while additional clinical manifestations and age of onset varies from case to case. To date, mutations in over 60 genes, both nuclear and mitochondrial DNA encoded, have been shown to cause Leigh syndrome, still explaining only half of all cases. In most patients, these mutations directly or indirectly affect the activity of the mitochondrial respiratory chain or pyruvate dehydrogenase complex. Exome sequencing has accelerated the discovery of new genes and pathways involved in Leigh syndrome, providing novel insights into the pathophysiological mechanisms. This is particularly important as no general curative treatment is available for this devastating disorder, although several recent studies imply that early treatment might be beneficial for some patients depending on the gene or process affected. Timely, gene-based personalized treatment may become an important strategy in rare, genetically heterogeneous disorders like Leigh syndrome, stressing the importance of early genetic diagnosis and identification of new genes/pathways. In this review, we provide a comprehensive overview of the most important clinical manifestations and genes/pathways involved in Leigh syndrome, and discuss the current state of therapeutic interventions in patients.
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http://dx.doi.org/10.1016/j.ymgme.2015.12.004DOI Listing
March 2016

Pathogenic CWF19L1 variants as a novel cause of autosomal recessive cerebellar ataxia and atrophy.

Eur J Hum Genet 2016 Apr 22;24(4):619-22. Epub 2015 Jul 22.

Department of Clinical Genetics, Unit Clinical Genomics, Maastricht University Medical Centre, Maastricht, The Netherlands.

Autosomal recessive cerebellar ataxia (ARCA) is a group of neurological disorders characterized by degeneration or abnormal development of the cerebellum and spinal cord. ARCA is clinically and genetically highly heterogeneous, with over 20 genes involved. Exome sequencing of a girl with ARCA from non-consanguineous Dutch parents revealed two pathogenic variants c.37G>C; p.D13H and c.946A>T; p.K316* in CWF19L1, a gene with an unknown function, recently reported to cause ARCA in a Turkish family. Sanger sequencing showed that the c.37G>C variant was inherited from the father and the c.946A>T variant from the mother. Pathogenicity was based on the damaging effect on protein function as the c.37G>C variant changed the highly conserved, negatively charged aspartic acid to the positively charged histidine and the c.946A>T variant introduced a premature stop codon. In addition, 27 patients with ARCA were tested for pathogenic variants in CWF19L1, however, no pathogenic variants were identified. Our data confirm CWF19L1 as a novel but rare gene causing ARCA.
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http://dx.doi.org/10.1038/ejhg.2015.158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4929877PMC
April 2016

Leigh syndrome: the genetic heterogeneity story continues.

Authors:
Mike Gerards

Brain 2014 Nov 24;137(Pt 11):2872-3. Epub 2014 Oct 24.

Clinical Genomics - Maastricht University Medical Centre, The Netherlands E-mail:

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http://dx.doi.org/10.1093/brain/awu264DOI Listing
November 2014

Ciona intestinalis NADH dehydrogenase NDX confers stress-resistance and extended lifespan on Drosophila.

Biochim Biophys Acta 2014 Nov 12;1837(11):1861-1869. Epub 2014 Aug 12.

BioMediTech and Tampere University Hospital, University of Tampere, FI-33014, Finland; Research Program of Molecular Neurology, University of Helsinki, FI-00014, Finland. Electronic address:

An assembled cDNA coding for the putative single-subunit NADH dehydrogenase (NDX) of Ciona intestinalis was introduced into Drosophila melanogaster. The encoded protein was found to localize to mitochondria and to confer rotenone-insensitive substrate oxidation in organello. Transgenic flies exhibited increased resistance to menadione, starvation and temperature stress, and manifested a sex and diet-dependent increase in mean lifespan of 20-50%. However, NDX was able only weakly to complement the phenotypes produced by the knockdown of complex I subunits.
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http://dx.doi.org/10.1016/j.bbabio.2014.08.001DOI Listing
November 2014

Screen for mitochondrial DNA copy number maintenance genes reveals essential role for ATP synthase.

Mol Syst Biol 2014 Jun 21;10:734. Epub 2014 Jun 21.

BioMediTech and Tampere University Hospital, University of Tampere, Tampere, Finland Research Program of Molecular Neurology, University of Helsinki, Helsinki, Finland

The machinery of mitochondrial DNA (mtDNA) maintenance is only partially characterized and is of wide interest due to its involvement in disease. To identify novel components of this machinery, plus other cellular pathways required for mtDNA viability, we implemented a genome-wide RNAi screen in Drosophila S2 cells, assaying for loss of fluorescence of mtDNA nucleoids stained with the DNA-intercalating agent PicoGreen. In addition to previously characterized components of the mtDNA replication and transcription machineries, positives included many proteins of the cytosolic proteasome and ribosome (but not the mitoribosome), three proteins involved in vesicle transport, some other factors involved in mitochondrial biogenesis or nuclear gene expression, > 30 mainly uncharacterized proteins and most subunits of ATP synthase (but no other OXPHOS complex). ATP synthase knockdown precipitated a burst of mitochondrial ROS production, followed by copy number depletion involving increased mitochondrial turnover, not dependent on the canonical autophagy machinery. Our findings will inform future studies of the apparatus and regulation of mtDNA maintenance, and the role of mitochondrial bioenergetics and signaling in modulating mtDNA copy number.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4265055PMC
http://dx.doi.org/10.15252/msb.20145117DOI Listing
June 2014

Reply: Infantile Leigh-like syndrome caused by SLC19A3 mutations is a treatable disease.

Brain 2014 Sep 30;137(Pt 9):e296. Epub 2014 May 30.

1 Department of Clinical Genetics, Unit Clinical Genomics, Maastricht University Medical Centre, Maastricht, The Netherlands2 School for Oncology and Developmental Biology, Maastricht University Medical Centre, Maastricht, The Netherlands.

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http://dx.doi.org/10.1093/brain/awu129DOI Listing
September 2014

Exome sequencing reveals a novel Moroccan founder mutation in SLC19A3 as a new cause of early-childhood fatal Leigh syndrome.

Brain 2013 Mar 18;136(Pt 3):882-90. Epub 2013 Feb 18.

Department of Genetics and Cell Biology, P.O. Box 616, 6200 MD Maastricht, the Netherlands.

Leigh syndrome is an early onset, often fatal progressive neurodegenerative disorder caused by mutations in the mitochondrial or nuclear DNA. Until now, mutations in more than 35 genes have been reported to cause Leigh syndrome, indicating an extreme genetic heterogeneity for this disorder, but still only explaining part of the cases. The possibility of whole exome sequencing enables not only mutation detection in known candidate genes, but also the identification of new genes associated with Leigh syndrome in small families and isolated cases. Exome sequencing was combined with homozygosity mapping to identify the genetic defect in a Moroccan family with fatal Leigh syndrome in early childhood and specific magnetic resonance imaging abnormalities in the brain. We detected a homozygous nonsense mutation (c.20C>A; p.Ser7Ter) in the thiamine transporter SLC19A3. In vivo overexpression of wild-type SLC19A3 showed an increased thiamine uptake, whereas overexpression of mutant SLC19A3 did not, confirming that the mutation results in an absent or non-functional protein. Seventeen additional patients with Leigh syndrome were screened for mutations in SLC19A3 using conventional Sanger sequencing. Two unrelated patients, both from Moroccan origin and one from consanguineous parents, were homozygous for the same p.Ser7Ter mutation. One of these patients showed the same MRI abnormalities as the patients from the first family. Strikingly, patients receiving thiamine had an improved life-expectancy. One patient in the third family deteriorated upon interruption of the thiamine treatment and recovered after reinitiating. Although unrelated, all patients came from the province Al Hoceima in Northern Morocco. Based on the recombination events the mutation was estimated to have occurred 1250-1750 years ago. Our data shows that SLC19A3 is a new candidate for mutation screening in patients with Leigh syndrome, who might benefit from high doses of thiamine and/or biotin. Especially, Moroccan patients with Leigh syndrome should be tested for the c.20C>A founder mutation in SLC19A3.
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http://dx.doi.org/10.1093/brain/awt013DOI Listing
March 2013

Riboflavin-responsive oxidative phosphorylation complex I deficiency caused by defective ACAD9: new function for an old gene.

Brain 2011 Jan 7;134(Pt 1):210-9. Epub 2010 Oct 7.

Department of Genetics and Cell Biology, Clinical Genomics Unit, Maastricht University, 6200 MD Maastricht, The Netherlands.

Mitochondrial complex I deficiency is the most common oxidative phosphorylation defect. Mutations have been detected in mitochondrial and nuclear genes, but the genetics of many patients remain unresolved and new genes are probably involved. In a consanguineous family, patients presented easy fatigability, exercise intolerance and lactic acidosis in blood from early childhood. In muscle, subsarcolemmal mitochondrial proliferation and a severe complex I deficiency were observed. Exercise intolerance and complex I activity was improved by a supplement of riboflavin at high dosage. Homozygosity mapping revealed a candidate region on chromosome three containing six mitochondria-related genes. Four genes were screened for mutations and a homozygous substitution was identified in ACAD9 (c.1594 C>T), changing the highly conserved arginine-532 into tryptophan. This mutation was absent in 188 ethnically matched controls. Protein modelling suggested a functional effect due to the loss of a stabilizing hydrogen bond in an α-helix and a local flexibility change. To test whether the ACAD9 mutation caused the complex I deficiency, we transduced fibroblasts of patients with wild-type and mutant ACAD9. Wild-type, but not mutant, ACAD9 restored complex I activity. An unrelated patient with the same phenotype was compound heterozygous for c.380 G>A and c.1405 C>T, changing arginine-127 into glutamine and arginine-469 into tryptophan, respectively. These amino acids were highly conserved and the substitutions were not present in controls, making them very probably pathogenic. Our data support a new function for ACAD9 in complex I function, making this gene an important new candidate for patients with complex I deficiency, which could be improved by riboflavin treatment.
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http://dx.doi.org/10.1093/brain/awq273DOI Listing
January 2011

Nonsense mutations in CABC1/ADCK3 cause progressive cerebellar ataxia and atrophy.

Mitochondrion 2010 Aug 23;10(5):510-5. Epub 2010 May 23.

Department of Genetics and Cell Biology, Unit Clinical Genomics Maastricht University, Maastricht, The Netherlands.

Hereditary ataxias are genetic disorders characterized by uncoordinated gait and often poor coordination of hands, speech, and eye movements. Frequently, atrophy of the cerebellum occurs. Many ataxias are autosomal dominant, but autosomal recessive (AR) disease occurs as well. Homozygosity mapping in a consanguineous family with three affected children with progressive cerebellar ataxia and atrophy revealed a candidate locus on chromosome 1, containing the CABC1/ADCK3 (the chaperone, ABC1 activity of bc1 complex homologue) gene. CABC1/ADCK3 is the homologue of the yeast Coq8 gene, which is involved in the ubiquinone biosynthesis pathway. Mutation analysis of this gene showed a homozygous nonsense mutation (c.1042C>T, p.R348X). Eight additional patients with AR cerebellar ataxia and atrophy were screened for mutations in the CABC1/ADCK3 gene. One patient was compound heterozygous for the same c.1042C>T mutation and a second nonsense mutation (c.1136T>A, p.L379X). Both mutations created a premature stop codon, triggering nonsense mediated mRNA decay as the pathogenic mechanism. We found no evidence of a Dutch founder for the c.1042C>T mutation in AR ataxia. We report here the first nonsense mutations in CABC1 that most likely lead to complete absence of a functional CABC1 protein. Our results indicate that CABC1 is an important candidate for mutation analysis in progressive cerebellar ataxia and atrophy on MRI to identify those patients, who may benefit from CoQ10 treatment.
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http://dx.doi.org/10.1016/j.mito.2010.05.008DOI Listing
August 2010

Clinical expression of Leber hereditary optic neuropathy is affected by the mitochondrial DNA-haplogroup background.

Am J Hum Genet 2007 Aug 4;81(2):228-33. Epub 2007 Jun 4.

Mitochondrial Research Group, Department of Ophthalmology and Institute of Human Genetics, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK.

Leber hereditary optic neuropathy (LHON) is due primarily to one of three common point mutations of mitochondrial DNA (mtDNA), but the incomplete penetrance implicates additional genetic or environmental factors in the pathophysiology of the disorder. Both the 11778G-->A and 14484T-->C LHON mutations are preferentially found on a specific mtDNA genetic background, but 3460G-->A is not. However, there is no clear evidence that any background influences clinical penetrance in any of these mutations. By studying 3,613 subjects from 159 LHON-affected pedigrees, we show that the risk of visual failure is greater when the 11778G-->A or 14484T-->C mutations are present in specific subgroups of haplogroup J (J2 for 11778G-->A and J1 for 14484T-->C) and when the 3460G-->A mutation is present in haplogroup K. By contrast, the risk of visual failure is significantly less when 11778G-->A occurs in haplogroup H. Substitutions on MTCYB provide an explanation for these findings, which demonstrate that common genetic variants have a marked effect on the expression of an ostensibly monogenic mtDNA disorder.
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http://dx.doi.org/10.1086/519394DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1950812PMC
August 2007

mtDNA point mutations are present at various levels of heteroplasmy in human oocytes.

Mol Hum Reprod 2007 Mar 26;13(3):149-54. Epub 2007 Jan 26.

Department of Genetics and Cell Biology, University of Maastricht, The Netherlands.

Little is known about the load of mutations and polymorphisms in the mitochondrial DNA (mtDNA) of human oocytes and the possible effect these mutations may have during life. To investigate this, we optimised at the single cell level the recently developed method to screen the entire mtDNA for mainly heteroplasmic mutations by denaturing high performance liquid chromatography analysis. This method is sensitive (approximately 1% heteroplasmy detectable), specific and rapid. The entire mtDNA of 26 oocytes of 13 women was screened by this method. Ten different heteroplasmic mutations, of which only one was located in the D-loop and two were observed twice, were detected in seven oocytes with mutation loads ranging from <5% to 50%. From eight women >1 oocyte was received and in four of them heteroplasmic differences between oocytes of the same woman were observed. In one of these four, two homoplasmic D-loop variants were also detected. Additionally, four oocytes of a single woman were sequenced using the MitoChip (which lacks the D-loop region), but all sequences were identical. It is concluded that heteroplasmic mtDNA mutations are common in oocytes and that, depending on the position and mutation load, they might increase the risk of developing OXPHOS disease early or later in life.
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http://dx.doi.org/10.1093/molehr/gal112DOI Listing
March 2007

Chip-based mtDNA mutation screening enables fast and reliable genetic diagnosis of OXPHOS patients.

Genet Med 2006 Oct;8(10):620-7

Department of Clinical Genetics, Maastricht University, Maastricht, The Netherlands.

Purpose: Oxidative phosphorylation is under dual genetic control of the nuclear and the mitochondrial DNA (mtDNA). Oxidative phosphorylation disorders are clinically and genetically heterogeneous, which makes it difficult to determine the genetic defect, and symptom-based protocols which link clinical symptoms directly to a specific gene or mtDNA mutation are falling short. Moreover, approximately 25% of the pediatric patients with oxidative phosphorylation disorders is estimated to have mutations in the mtDNA and a standard screening approach for common mutations and deletions will only explain part of these cases. Therefore, we tested a new CHIP-based screening method for the mtDNA.

Methods: MitoChip (Affymetrix) resequencing was performed on three test samples and on 28 patient samples.

Results: Call rates were 94% on average and heteroplasmy detection levels varied from 5-50%. A genetic diagnosis can be made in almost one-quarter of the patients at a potential output of 8 complete mtDNA sequences every 4 days. Moreover, a number of potentially pathogenic unclassified variants (UV) were detected.

Conclusions: The availability of long-range PCR protocols and the predominance of single nucleotide substitutions in the mtDNA make the resequencing CHIP a very fast and reliable method to screen the complete mtDNA for mutations.
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http://dx.doi.org/10.1097/01.gim.0000237782.94878.05DOI Listing
October 2006