Genetic defects in patients with mitochondrial encephalomyopathies

Dit proefschrift is een bijdrage aan het snel groeiende kennisgebied gewijd aan de verbetering van de diagnostiek op DNA-niveau bij patiënten met mitochondriële encephalomyopathieën en is onder andere geïnspireerd door de hypothese van de communicatie over en weer tussen het kerngenoom (bijv. de 24 kDA subunit van complex I) en het mitochondriële genoom. Het presenteert de resultaten van klinische, biochemische en moleculair-genetische studies die uitgevoerd zijn op de Afdeling Humane Genetica, St.-Radboud Ziekenhuis Nijmegen; op de afdeling Genetica, Universiteit Maastricht; op de afdeling Neurologie, Erasmus MC Universitair Medisch Centrum Rotterdam, en in het Institute of Neurology, The National Hospital, Queen Square, Londen, Engeland (Introductie, hoofdstuk 1). In dit proefschrift worden verschillende strategieën beschreven om de oorzaak van een mitochondriële encephalomyopathie of in het mitochondriële of in het kerngenoom te localiseren (hoofdstukken 2-7). Eerst wordt een PCR-test beschreven om het spectrum van grote deleties van het mtDNA, zoals gevonden kan worden bij patiënten met een progressieve externe ophthalmoplegie of het Kearns-Sayre syndroom, aan te tonen. Het voordeel van deze, op een PCR gebaseerde test in vergelijking met de ‘Southern blotting’-techniek is de gevoeligheid van deze methode om deleties te detecteren. In veel gevallen is het DNA dat uit bloedleukocyten of uit haarwortels van de patiënt is verkregen, voldoende voor het stellen van de diagnose. Hierdoor kan de patiënt een spierbiopt worden bespaard (hoofdstuk 2). Met een ontwikkelde ‘single stranded conformation polymorfism’ (SSCP) analysemethode voor mitochondriële tRNA-mutaties is bij een patiënt met het klinische beeld van een mitochondriële encephalopathie met lactaatacidose en herseninfarctachtige episoden (MELAS-fenotype), een mutatie in het tRNAVal gen (G1642A) gevonden. Deze mutatie is eenmaal eerder gerapporteerd. Onze observatie is onafhankelijk van de eerdere rapportage gedaan. Het fenotype van onze patiënt is gelijk aan die van de eerdere, hetgeen de pathogeniciteit van deze mutatie waarschijnlijk maakt. Het fenotype van onze patiënt verschilt van andere MELAS-patiënten door de afsluiting van kleine cerebrale corticale arteriën. Betrokkenheid hiervan is niet eerder gerapporteerd (hoofdstuk 3). SAMENVATTING 191 Vervolgens wordt de analyse op mutaties in het enige mitochondrieel gecodeerde complex III-gen, het Cytochroom b-gen, bij vijf geïdentificeerde patiënten met een biochemisch fenotype van een complex III-defi-ciëntie weergegeven. Bij een patiënt wordt een vier base paar deletiemutatie gevonden op positie 14787 en een homoplasmisch polymorfisme. De mutatie is heteroplasmisch en is aanwezig in 95% in spier. Bij de klinisch gezonde moeder is geen mutatie vastgesteld. Deze ‘frameshift’-mutatie zal zeer waarschijnlijk zorgen voor een verstoring van de synthese van het cytochroom b-eiwit. Het fenotype van de patiënt is een overlappend beeld van Parkinsonisme en MELAS, wat niet eerder is beschreven noch geassocieerd met een complex III-deficiëntie, noch met een mutatie in het Cytochroom b-gen. Bij twee van de vier andere patiënten heeft de mutatieanalyse twee verschillende homoplasmische polymorfismen opgeleverd. Bij deze vier patiënten is geen mutatie gevonden. (hoofdstuk 4). Bij een patiënt met symptomen zoals gezien bij patiënten met een Leigh syndroom, wordt een de novo ontstane T8993C mitochondriële mutatie gerapporteerd. Hypothesen worden geformuleerd om deze de novogebeurtenis te verklaren en de stijging in mutatiepercentage van 0% bij het DNA in spier van de moeder naar 79% in spier van de patiënt. Kern gecodeerde, modificerende genen zijn waarschijnlijk nodig om het hoge percentage heteroplasmie van deze mutatie te begrijpen (hoofdstuk 5). Hierna wordt het gebruik van de ‘denaturing high performance liquid chromatography’ (DHPLC) technologie getoond als een antwoord op het toenemende aantal verschillende testen om alle mogelijke mtDNA-muta-ties uit te sluiten. De DHPLC-methode is een snelle, betrouwbare en gevoelige methode om heteroduplexen te detecteren die resulteren van heteroplasmische DNA-strengen. Deze methode is bij uitstek geschikt voor mutatiedetectie van mtDNA, daar de meeste mutaties in dit DNA heteroplasmisch zijn. Een mtDNA-DHPLC protocol dat het mogelijk maakt om binnen een dag een complete mtDNA-mutatieanalyse te doen, is ontwikkeld. Een minimaal heteroplasmieniveau van 0,5% voor de A8344G-mutatie is gedetecteerd. Bij drie van de eerste zes mitochondriële encephalomyopathiepatiënten die gescreend werden met deze methode, is een mutatie aangetoond. Het uitsluiten van betrokkenheid van mtDNAmutaties ondersteunt het doen van een vervolgonderzoek naar kerngenen en heeft belangrijke implicaties voor genetisch advies (hoofdstuk 6). Er zijn slechts enkele therapeutische mogelijkheden voor patiënten met mitochondriële encephalomyopathieën. In geval van een mtDNA-mutatie 192 SAMENVATTING als de oorzakelijke factor voor ziekte zijn de mogelijkheden voor prenatale diagnose beperkt vanwege de gecompliceerde manier waarop de mutatie wordt overgedragen. Voor de eerste keer wordt een prenatale diagnose en bevinding van een mutatieoverdracht gerapporteerd bij een familie met de T9176C (ATPase6-gen)-mutatie (hoofdstuk 7). Vervolgens wordt de karakterisering en mutatiescreening van de drie flavoproteïnen fractiegenen, de NDUFV1, NDUFV2, NDUFV3 genen, van complex I gegeven. Met behulp van chaotrope agentia kan complex I in drie fracties worden verdeeld. Een van de fracties, de flavoproteïnenfractie, bestaat uit drie subunits van 51, 24 en 10 kDa en is functioneel belangrijk voor complex I (hoofdstuk 8-11). Eerst is de klonering en genomische localisering van de kleinste van de drie subunits, de NDUFV3 of 10 kDa gen, beschreven. De menselijke cDNA-sequentie is opgehelderd met behulp van een menselijke nier cDNA-bibliotheek met runder 10kDa cDNA met bekende sequentie. Het 5' einde van het cDNA is verkregen met de snelle vermenigvuldigingsprocedure van cDNA-einden (RACE). Northern blot toonde aan dat het gen algemeen tot expressie komt. Het gen bevat drie exonen en bestrijkt ongeveer 20kb. Een Southern blot-procedure met een set humane/hamster somatische celhybriden toont dat het gen is gelocaliseerd op chromosoom 21. Een 10-kDa gen bevattende cosmide is verkregen van een chromosoom 21-specifieke cosmidenbibliotheek en gebruikt voor een fluorescentie in situ hybridisatie (Fish) procedure ter verfijning van de chromosoom 21-locatie naar een gebied beperkt tot 21q22.3 (hoofdstuk 8). Hierna wordt de klonering en karakterisering van het Fe-S cluster bevattende 24 kDa subunit gen beschreven. Het homologe runder 24 kDa cDNA is gebruikt om een humane cosmidenbibliotheek te screenen. De zoektocht is gecompliceerd geweest door de aanwezigheid van een pseudogen. De 24 kDa cDNA cosmiden zijn in kaart gebracht door het analyseren van de gebieden van hybridisatie met een Southern blot-panel met humane/ham-ster somatische celhybriden. Deze analyse verwijst naar twee genen. Een groot fragment is gelocaliseerd op chromosoom 19 en drie kleinere fragmenten op chromosoom 18. Verdere verfijning van de localisatie is verricht met chromosoomspecifieke somatische celhybriden voor chromosoom 18- en 19-fragmenten. Met deze procedure is het locus toegewezen aan 18p11.2-pter en aan chromosoom 19q13.3-qter. In een Fish-procedu-re zijn de loci verder verfijnd naar 18p11.2-11.31 en 19qter. De twee genen zijn gesequenced en onthullen dat de chromosoom 19 locus een SAMENVATTING 193 pseudogen representeert en de chromosoom 18 locus het actieve 24 kDa gen. De Northern blot toont een algemeen voorkomende genexpressie (hoofdstuk 9). Verder wordt de structuur van het NDUFV1-gen, coderend voor de 51 kDa flavoproteïne subunit van complex I, beschreven. De structuur van het gen is opgehelderd met behulp van de bekende runder 51kDa cDNA-sequen-tie. Met primers afgeleid van het cDNA, zijn PCR-fragmenten van genomisch DNA gegenereerd. Het gen blijkt 10 exonen te bevatten die coderen voor 464 aminozuren en strekt zich uit over 5kb. De Northern blot-analy-se toont algemene expressie die het hoogste is in de pancreas. Voor testis mRNA is een uniek mRNA lengtefragment aanwezig (hoofdstuk 10). Vervolgens wordt de mutatieanalyse voor de drie flavoproteïnen subunits, namelijk de genen NDUFV1, NDUFV2 en NDUFV3, beschreven. Voor deze mutatieanalyse zijn 20 patiënten met een mitochondriële encephalomyopathie en een geïsoleerde complex I-deficiëntie geselecteerd. Er zijn geen mutaties in deze groep patiënten gevonden. Drie polymorfismen zijn gevonden in het NDUFV2-gen. Deze studie ondersteunt het idee dat de flavoproteïnenfractie van complex I geen ‘hotspot’ is voor mutaties (hoofdstuk 11). Tot slot worden de nieuwe bevindingen, gepresenteerd in dit proefschrift, in perspectief gezet en aanwijzingen voor toekomstig onderzoek bediscussieerd (hoofdstuk 12).Summary This thesis is a contribution to the fast growing field devoted to the improvement of the diagnostics in patients with mitochondrial encephalomyopathies at the DNA level and is inspired amongst others by the hypothesis of intergenomic crosstalk between the nuclear genome (e.g. the 24 kDa subunit of complex I) and the mitochondrial genome. It presents the results of clinical, biochemical and molecular genetic studies that have been performed at the Department of Human Genetics, University Hospital Nijmegen, Nijmegen; at the Division of Genetics, University of Maastricht, Maastricht; at the Department of Neurology, Erasmus MC -University Medical Center Rotterdam, The Netherlands and at the Institute of Neurology, The National Hospital, Queen Square, London, UK (Introduction, Chapter 1). In this thesis, different strategies are described to discriminate the cause of a mitochondrial encephalomyopathy to be located either in the mitochondrial or in the nuclear genome (Chapter 2-7). First a PCR-based test is described to detect the whole spectrum of large deletions as can be found in Progressive External Ophthalmoplegia and in the Kearns-Sayre syndrome. The advantage of this PCR-based test compared to Southern blot analysis is the sensitivity of the method for detecting deletions. In many cases patient’s leukocyte DNA is sufficient for making a diagnosis saving the patient a muscle biopsy procedure (Chapter 2). A single stranded conformation analysis (SSCP) screening method for mitochondrial tRNA mutations revealed in a patient with a mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS-pheno-type), a mutation in the tRNAVal gene (G1642A) being the second report of this mutation. Our observation was done independently and our patient’s phenotype is the same as in the former report confirming its likely pathogenicity. The phenotype of this patient differed from other MELAS patients because of the involvement of small cerebral arteries in the disease process. This involvement has not been reported before (Chapter 3). Next the analysis of five patients, with a biochemical phenotype of a complex III deficiency, for mutations in the only mitochondrially encoded subunit of complex III, the cytochrome b gene is described. In one patient a four base pair deletion-mutation at position 14787 and a homoplasmic SUMMARY 187 polymorphism are found. The mutation is heteroplasmic and present in 95% in muscle. In the clinically unaffected mother no mutation is detected. This frame shift mutation is predicted to cause a severe disruption of the synthesis of the cytochrome b protein. The phenotype of the patient, a Parkinsonism-MELAS overlap syndrome, has not been described before, neither in association with a complex III deficiency nor with a mutation in the cytochrome b gene. In two out of the four other patients the mutation analysis revealed two different homoplasmic polymorphisms (Chapter 4). In a patient with symptoms as seen in patients with a Leigh syndrome is a de novo arisen T8993C mitochondrial mutation reported. Hypotheses are formulated to explain this de novo event and the rise in mutant rate from 0% in the mother’s muscle mtDNA to 79% in the patient. Nuclear encoded, modifier genes are very likely necessary to understand the high percentage of heteroplasmy for this mutation (Chapter 5). Hereafter is the use of denaturing high performance liquid chromatography (DHPLC) technology demonstrated as an answer to the increasing number of different tests to exclude all the possible mtDNA mutations. The DHPLC method is a fast, reliable and sensitive method to detect heteroduplexes that result from heteroplasmic strands. Therefore this method is particularly suited for mtDNA screening, because most mutations in the mtDNA are heteroplasmic. A mtDNA-DHPLC protocol was developed that enables a complete mtDNA mutation analysis within one day. Levels of heteroplasmy as low as 0.5% for the A8344G mutation can be detected. The first six mitochondrial encephalomyopathy patients screened with this method showed a mutation in three out of six patients tested. Exclusion of mtDNA involvement supports a subsequent investigation of nuclear genes and has important implications for counselling (Chapter 6). There are only a few therapeutic possibilities for patients with mitochondrial encephalomyopathies. In case of a mtDNA mutation as the causative factor for disease also the possibilities for prenatal diagnosis are limited because of the complicated way the mutation is transmitted. For the first time a prenatal diagnosis and transmission findings are reported in a family with Leigh syndrome associated with the T9176C (ATPase6 gene) mutation (Chapter 7). In the second part of the thesis the characterization and mutation screening of the three flavoprotein fraction genes, the NDUFV1, NDUFV2, NDUFV3 genes, of the complex I are described. Using chaotropic agents 188 SUMMARY complex I can be divided in three fractions. One of the fractions, the flavoprotein fraction, consists of three subunits of 51, 24 and 10 kDa and is functionally important for complex I and the oxidative phosphorylation (Chapter 8-11). First the cloning and mapping of the gene coding for the smallest of the three subunits the NDUFV3 or 10 kDa-gene is described. The human cDNA sequence was elucidated by screening a human renal cDNA library with the known bovine 10-kDa cDNA. The 5' end of the cDNA was obtained with the rapid amplification of cDNA ends (RACE) procedure. Northern blot procedures showed the gene to be ubiquitously expressed. The gene contains three exons and spans about 20 kb. A Southern blot panel with human/hamster somatic cell hybrids showed that the gene is localized on chromosome 21. A 10 kDa gene containing cosmid was derived from a chromosome 21-specific cosmid library and used for a fluorescence in situ hybridization (Fish) procedure to refine the chromosome 21 location to 21q22.3 (Chapter 8). Next the cloning and characterization of the Fe-S cluster containing 24 kDa subunit gene is reported. The homologous bovine 24 kDa cDNA was used to screen a human cosmid library. The search was complicated by the presence of a pseudogene. The 24kDa cDNA cosmids were mapped by screening a Southern blot panel of human/hamster somatic cell hybrids to two different genes. One large fragment mapped to chromosome 19 and three smaller fragments to chromosome 18. Further refinement of the mapping was done with somatic cell hybrids containing either chromosome 18 or 19 fragments. With this procedure the locus could be assigned to 18p11.2-pter and to chromosome 19q13.3-qter. In a Fish procedure the loci were further refined to 18p11.2-11.31 and 19qter. The two genes were sequenced and revealed that the chromosome 19 locus represented a pseudogene and that the chromosome 18 locus represented the active 24 kDa gene. Northern blot analysis showed an ubiquitous gene expression (Chapter 9). Then the structure of the NDUFV1 gene, encoding the 51 kDa flavoprotein subunit of complex I, is described. The structure of the gene was clarified by using the known bovine 51 kDa cDNA sequence. With primers derived from the cDNA, PCR fragments from genomic DNA were generated. The gene appeared to contain 10 exons coding for 464 amino acids and spanned about 5 kb of the human genome. Northern blot analysis showed ubiquitous gene expression with the highest expression in pan- SUMMARY 189 creas. For testis mRNA a unique mRNA length fragment was present (Chapter 10). Following the characterisation of the three flavoprotein subunit genes NDUFV1, NDUFV2 and NDUFV3 the mutation analysis is described. For this comprehensive mutation analysis twenty patients with a mitochondrial encephalomyopathy and an isolated complex I deficiency were selected. No mutations in this group of patients were detected. Three polymorphisms were found in the NDUFV2 gene. This study supports the idea that the flavoprotein fraction of complex I is not a hotspot for mutations (Chapter 11). Finally the new findings presented in this thesis are put in perspective and directions for future research are discussed (Chapter 12)

Mitochondrial neurogastrointestinal encephalomyopathy caused by thymidine phosphorylase enzyme deficiency: From pathogenesis to emerging therapeutic options

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a progressive metabolic disorder caused by thymidine phosphorylase (TP) enzyme deficiency. The lack of TP results in systemic accumulation of deoxyribonucleosides thymidine (dThd) and deoxyuridine (dUrd). In these patients, clinical features include mental regression, ophthalmoplegia, and fatal gastrointestinal complications. The accumulation of nucleosides also causes imbalances in mitochondrial DNA (mtDNA) deoxyribonucleoside triphosphates (dNTPs), which may play a direct or indirect role in the mtDNA depletion/deletion abnormalities, although the exact underlying mechanism remains unknown. The available therapeutic approaches include dialysis and enzyme replacement therapy, both can only transiently reverse the biochemical imbalance. Allogeneic hematopoietic stem cell transplantation is shown to be able to restore normal enzyme activity and improve clinical manifestations in MNGIE patients. However, transplant related complications and disease progression result in a high mortality rate. New therapeutic approaches, such as adeno-associated viral vector and hematopoietic stem cell gene therapy have been tested in Tymp−/− Upp1−/− mice, a murine model for MNGIE. This review provides background information on disease manifestations of MNGIE with a focus on current management and treatment options. It also outlines the pre-clinical approaches toward future treatment of the disease

Changes in globus pallidus with (pre)term kernicterus

OBJECTIVE: We report serial magnetic resonance (MR) and sonographic behavior of globus pallidus in 5 preterm and 3 term infants with kernicterus and describe the clinical context in very low birth weight preterm infants. On the basis of this information, we suggest means of diagnosis and prevention. METHODS: Charts and MR and ultrasound images of 5 preterm infants and 3 term infants with suspected bilirubin-associated brain damage were reviewed. Included were preterm infants with severe hearing loss, quadriplegic hypertonia, and abnormal hypersignal of globus pallidus on T2-weighted MR imaging (MRI). In 1 infant who died on day 150, the diagnosis was confirmed during the neonatal period. The others were picked up as outpatients and scanned at 12 or 22 months' corrected age. Three instances of term kernicterus were included for comparison of serial MRI in the neonatal period and early infancy: they were caused by glucose-6-phosphate dehydrogenase deficiency, urosepsis, and dehydration plus fructose 1-6 biphosphatase deficiency. RESULTS: Five preterm infants of 25 to 29 weeks' gestational age presented with total serum bilirubin (TSB) levels below exchange transfusion thresholds commonly advised. Mixed acidosis was present in 3 infants around the TSB peak. The bilirubin/albumin molar ratio was >0.5 in all, in the absence of displacing drugs. All failed to pass bedside hearing screen tests and had severe hearing loss on auditory brain response testing. Symmetrical homogeneous hyperechogenicity of globus pallidus was the alerting feature in 1 infant. Globus pallidus was hyperintense on T1-weighted MR images in this child. The other infants presented with severe developmental delay as a result of dyskinetic quadriplegia and hearing loss. Globus pallidus was normal on T1- but hyperintense on T2-weighted MR images at 12

How do changes in the mtDNA and mitochondrial dysfunction influence cancer and cancer therapy? Challenges, opportunities and models

Several mutations in nuclear genes encoding for mitochondrial components have been associated with an increased cancer risk or are even causative, e.g. succinate dehydrogenase (SDHB, SDHC and SDHD genes) and iso-citrate dehydrogenase (IDH1 and IDH2 genes). Recently, studies have suggested an eminent role for mitochondrial DNA (mtDNA) mutations in the development of a wide variety of cancers. Various studies associated mtDNA abnormalities, including mutations, deletions, inversions and copy number alterations, with mitochondrial dysfunction. This might, explain the hampered cellular bioenergetics in many cancer cell types. Germline (e.g. m.10398A>G; m.6253T>C) and somatic mtDNA mutations as well as differences in mtDNA copy number seem to be associated with cancer risk. It seems that mtDNA can contribute as driver or as complementary gene mutation according to the multiple-hit model. This can enhance the mutagenic/clonogenic potential of the cell as observed for m.8993T>G or influences the metastatic potential in later stages of cancer progression. Alternatively, other mtDNA variations will be innocent passenger mutations in a tumor and therefore do not contribute to the tumorigenic or metastatic potential. In this review, we discuss how reported mtDNA variations interfere with cancer treatment and what implications this has on current successful pharmaceutical interventions. Mutations in MT-ND4 and mtDNA depletion have been reported to be involved in cisplatin resistance. Pharmaceutical impairment of OXPHOS by metformin can increase the efficiency of radiotherapy. To study mitochondrial dysfunction in cancer, different cellular models (like ρ0 cells or cybrids), in vivo murine models (xenografts and specific mtDNA mouse models in combination with a spontaneous cancer mouse model) and small animal models (e.g. Danio rerio) could be potentially interesting to use. For future research, we foresee that unraveling mtDNA variations can contribute to personalized therapy for specific cancer types and improve the outcome of the disease

Anatomic & metabolic brain markers of the m.3243A>G mutation: A multi-parametric 7T MRI study

One of the most common mitochondrial DNA (mtDNA) mutations, the A to G transition at base pair 3243, has been linked to changes in the brain, in addition to commonly observed hearing problems, diabetes and myopathy. However, a detailed quantitative description of m.3243A>G patients' brains has not been provided so far. In this study, ultra-high field MRI at 7T and volume- and surface-based data analyses approaches were used to highlight morphology (i.e. atrophy)-, microstructure (i.e. myelin and iron concentration)- and metabolism (i.e. cerebral blood flow)-related differences between patients (N = 22) and healthy controls (N = 15). The use of quantitative MRI at 7T allowed us to detect subtle changes of biophysical processes in the brain with high accuracy and sensitivity, in addition to typically assessed lesions and atrophy. Furthermore, the effect of m.3243A>G mutation load in blood and urine epithelial cells on these MRI measures was assessed within the patient population and revealed that blood levels were most indicative of the brain's state and disease severity, based on MRI as well as on neuropsychological data. Morphometry MRI data showed a wide-spread reduction of cortical, subcortical and cerebellar gray matter volume, in addition to significantly enlarged ventricles. Moreover, surface-based analyses revealed brain area-specific changes in cortical thickness (e.g. of the auditory cortex), and in T1, T2* and cerebral blood flow as a function of mutation load, which can be linked to typically m.3243A>G-related clinical symptoms (e.g. hearing impairment). In addition, several regions linked to attentional control (e.g. middle frontal gyrus), the sensorimotor network (e.g. banks of central sulcus) and the default mode network (e.g. precuneus) were characterized by alterations in cortical thickness, T1, T2* and/or cerebral blood flow, which has not been described in previous MRI studies. Finally, several hypotheses, based either on vascular, metabolic or astroglial implications of the m.3243A>G mutation, are discussed that potentially explain the underlying pathobiology. To conclude, this is the first 7T and also the largest MRI study on this patient population that provides macroscopic brain correlates of the m.3243A>G mutation indicating potential MRI biomarkers of mitochondrial diseases and might guide future (longitudinal) studies to extensively track neuropathological and clinical changes

Healthy, mtDNA-mutation free mesoangioblasts from mtDNA patients qualify for autologous therapy

BACKGROUND: Myopathy and exercise intolerance are prominent clinical features in carriers of a point-mutation or large-scale deletion in the mitochondrial DNA (mtDNA). In the majority of patients, the mtDNA mutation is heteroplasmic with varying mutation loads between tissues of an individual. Exercise-induced muscle regeneration has been shown to be beneficial in some mtDNA mutation carriers, but is often not feasible for this patient group. In this study, we performed in vitro analysis of mesoangioblasts from mtDNA mutation carriers to assess their potential to be used as source for autologous myogenic cell therapy. METHODS: We assessed the heteroplasmy level of patient-derived mesoangioblasts, isolated from skeletal muscle of multiple carriers of different mtDNA point-mutations (n = 25). Mesoangioblast cultures with < 10% mtDNA mutation were further analyzed with respect to immunophenotype, proliferation capacity, in vitro myogenic differentiation potential, mitochondrial function, and mtDNA quantity. RESULTS: This study demonstrated that mesoangioblasts in half of the patients contained no or a very low mutation load (< 10%), despite a much higher mutation load in their skeletal muscle. Moreover, none of the large-scale mtDNA deletion carriers displayed the deletion in mesoangioblasts, despite high percentages in skeletal muscle. The mesoangioblasts with no or a very low mutation load (< 10%) displayed normal mitochondrial function, proliferative capacity, and myogenic differentiation capacity. CONCLUSIONS: Our data demonstrates that in half of the mtDNA mutation carriers, their mesoangioblasts are (nearly) mutation free and can potentially be used as source for autologous cell therapy for generation of new muscle fibers without mtDNA mutation and normal mitochondrial function

Benign familial infantile convulsions: A clinical study of seven Dutch families

Benign familial infantile convulsions (BFIC) is a recently identified partial epilepsy syndrome with onset between 3 and 12 months of age. We describe the clinical characteristics and outcome of 43 patients with BFIC from six Dutch families and one Dutch-Canadian family and the encountered difficulties in classifying the syndrome. Four families had a pure BFIC phenotype; in two families BFIC was accompanied by paroxysmal kinesigenic dyskinesias; in one family BFIC was associated with later onset focal epilepsy in older generations. Onset of seizures was between 6 weeks and 10 months, and seizures remitted before the age of 3 years in all patients with BFIC. In all, 29 (67%) of the 43 patients had been treated with anti-epileptic drugs for a certain period of time. BFIC is often not recognized as (hereditary) epilepsy by the treating physician. Seizures often remit shortly after the start of anti-epileptic drugs but, because of the benign course of the syndrome and the spontaneous remission of seizures, patients with low seizure fr

Non-Random mtDNA Segregation Patterns Indicate a Metastable Heteroplasmic Segregation Unit in m.3243A>G Cybrid Cells

Many pathogenic mitochondrial DNA mutations are heteroplasmic, with a mixture of mutated and wild-type mtDNA present within individual cells. The severity and extent of the clinical phenotype is largely due to the distribution of mutated molecules between cells in different tissues, but mechanisms underpinning segregation are not fully understood. To facilitate mtDNA segregation studies we developed assays that measure m.3243A>G point mutation loads directly in hundreds of individual cells to determine the mechanisms of segregation over time. In the first study of this size, we observed a number of discrete shifts in cellular heteroplasmy between periods of stable heteroplasmy. The observed patterns could not be parsimoniously explained by random mitotic drift of individual mtDNAs. Instead, a genetically metastable, heteroplasmic mtDNA segregation unit provides the likely explanation, where stable heteroplasmy is maintained through the faithful replication of segregating units with a fixed wild-type/m.3243A>G mutant ratio, and shifts occur through the temporary disruption and re-organization of the segregation units. While the nature of the physical equivalent of the segregation unit remains uncertain, the factors regulating its organization are of major importance for the pathogenesis of mtDNA diseases

A multicenter study on Leigh syndrome: Disease course and predictors of survival

Background: Leigh syndrome is a progressive neurodegenerative disorder, associated with primary or secondary dysfunction of the mitochondrial oxidative phosphorylation. Despite the fact that Leigh syndrome is the most common phenotype of mitochondrial disorders in children, longitudinal natural history data is missing. This study was undertaken to assess the phenotypic and genotypic spectrum of patients with Leigh syndrome, characterise the clinical course and identify predictors of survival in a large cohort of patients. Methods. This is a retrospective study of patients with Leigh syndrome that have been followed at eight centers specialising in mitochondrial diseases in Europe; Gothenburg, Rotterdam, Helsinki, Copenhagen, Stockholm, Brussels, Bergen and Oulu. Results: A total of 130 patients were included (78 males; 52 females), of whom 77 patients had identified pathogenic mutations. The median age of disease onset was 7 months, w

Transplantation, gene therapy and intestinal pathology in MNGIE patients and mice

Background: Gastrointestinal complications are the main cause of death in patients with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). Available treatments often restore biochemical homeostasis, but fail to cure gastrointestinal symptoms. Methods: We evaluated the small intestine neuromuscular pathology of an untreated MNGIE patient and two recipients of hematopoietic stem cells, focusing on enteric neurons and glia. Additionally, we evaluated the intestinal neuromuscular pathology in a mouse model of MNGIE treated with hematopoietic stem cell gene therapy. Quantification of muscle wall thickness and ganglion cell density was performed blind to the genotype with ImageJ. Significance of differences between groups was determined by two-tailed Mann-Whitney U test (P < 0.05). Results: Our data confirm that MNGIE presents with muscle atrophy and loss of Cajal cells and CD117/c-kit immunoreactivity in the small intestine. We also show that hematopoietic stem cell transplantation does not benefit human intestinal pathology at least on short-term. Conclusions: We suggest that hematopoietic stem cell transplantation may be insufficient to restore intestinal neuropathology, especially at later stages of MNGIE. As interstitial Cajal cells and their networks play a key role in development of gastrointestinal dysmotility, alternative therapeutic approaches taking absence of these cells into account could be required