Mechanisms and effects of mitochondrial DNA instability and copy number manipulation

Defects in mitochondrial DNA (mtDNA) maintenance cause a range of human diseases, including autosomal dominant progressive external ophthalmoplegia (adPEO). This study aimed to clarify the molecular background of adPEO. We discovered that deoxynucleoside triphosphate (dNTP) metabolism plays a crucial in mtDNA maintenance and were thus prompted to search for therapeutic strategies based on the modulation of cellular dNTP pools or mtDNA copy number. Human mtDNA is a 16.6 kb circular molecule present in hundreds to thousands of copies per cell. mtDNA is compacted into nucleoprotein clusters called nucleoids. mtDNA maintenance diseases result from defects in nuclear encoded proteins that maintain the mtDNA. These syndromes typically afflict highly differentiated, post-mitotic tissues such as muscle and nerve, but virtually any organ can be affected. adPEO is a disease where mtDNA molecules with large-scale deletions accumulate in patients tissues, particularly in skeletal muscle. Mutations in five nuclear genes, encoding the proteins ANT1, Twinkle, POLG, POLG2 and OPA1, have previously been shown to cause adPEO. Here, we studied a large North American pedigree with adPEO, and identified a novel heterozygous mutation in the gene RRM2B, which encodes the p53R2 subunit of the enzyme ribonucleotide reductase (RNR). RNR is the rate-limiting enzyme in dNTP biosynthesis, and is required both for nuclear and mitochondrial DNA replication. The mutation results in the expression of a truncated form of p53R2, which is likely to compete with the wild-type allele. A change in enzyme function leads to defective mtDNA replication due to altered dNTP pools. Therefore, RRM2B is a novel adPEO disease gene. The importance of adequate dNTP pools and RNR function for mtDNA maintenance has been established in many organisms. In yeast, induction of RNR has previously been shown to increase mtDNA copy number, and to rescue the phenotype caused by mutations in the yeast mtDNA polymerase. To further study the role of RNR in mammalian mtDNA maintenance, we used mice that broadly overexpress the RNR subunits Rrm1, Rrm2 or p53R2. Active RNR is a heterotetramer consisting of two large subunits (Rrm1) and two small subunits (either Rrm2 or p53R2). We also created bitransgenic mice that overexpress Rrm1 together with either Rrm2 or p53R2. In contrast to the previous findings in yeast, bitransgenic RNR overexpression led to mtDNA depletion in mouse skeletal muscle, without mtDNA deletions or point mutations. The mtDNA depletion was associated with imbalanced dNTP pools. Furthermore, the mRNA expression levels of Rrm1 and p53R2 were found to correlate with mtDNA copy number in two independent mouse models, suggesting nuclear-mitochondrial cross talk with regard to mtDNA copy number. We conclude that tight regulation of RNR is needed to prevent harmful alterations in the dNTP pool balance, which can lead to disordered mtDNA maintenance. Increasing the copy number of wild-type mtDNA has been suggested as a strategy for treating PEO and other mitochondrial diseases. Only two proteins are known to cause a robust increase in mtDNA copy number when overexpressed in mice; the mitochondrial transcription factor A (TFAM), and the mitochondrial replicative helicase Twinkle. We studied the mechanisms by which Twinkle and TFAM elevate mtDNA levels, and showed that Twinkle specifically implements mtDNA synthesis. Furthermore, both Twinkle and TFAM were found to increase mtDNA content per nucleoid. Increased mtDNA content in mouse tissues correlated with an age-related accumulation of mtDNA deletions, depletion of mitochondrial transcripts, and progressive respiratory dysfunction. Simultaneous overexpression of Twinkle and TFAM led to a further increase in the mtDNA content of nucleoids, and aggravated the respiratory deficiency. These results suggested that high mtDNA levels have detrimental long-term effects in mice. These data have to be considered when developing and evaluating treatment strategies for elevating mtDNA copy number.Genetiska sjukdomar orsakas av mutationer i cellens DNA. De mitokondriella sjukdomarna beror på fel i mitokondrierna, som är cellens energifabriker, och som också har eget mitokondriellt DNA (mtDNA). I denna studie hittade vi ett nytt genfel, som påvisade ett viktigt samband mellan syntesen av DNA s byggstenar och uppehället av mtDNA. Som ett potentiellt botemedel för mitokondriella sjukdomar undersökte vi möjligheten att manipulera mängden mtDNA. Överraskande nog fann vi att mitokondrierna inte tycks kunna fungera normalt om de innehåller för mycket DNA. Studiens resultat ökar vår förståelse av de mekanismer som ligger till grund för mitokondriernas normala funktion, en förutsättning för ett normalt liv. Största delen av vårt DNA är packat i kromosomerna i cellens kärna, men ca 1 % utgörs av mtDNA, som innehåller 37 livsviktiga gener. De mitokondriella sjukdomarna är en stor grupp allvarliga genetiska sjukdomar. Mitokondriernas funktion kan störas som en följd av mutationer i mtDNA eller i någon av de av kärnans gener som kodar mitokondriella proteiner. Nära nog samtliga av kroppens organ kan ta skada om mitokondrierna inte fungerar normalt, men särskilt ömtåliga är organ med stort energibehov, t.ex. hjärnan och skelettmuskulaturen. Vi undersökte en familj med en mitokondriell sjukdom, kallad autosomal dominant progressiv extern oftalmoplegi (adPEO). I adPEO sker en långsam ansamling av skadade mtDNA-molekyler i patientens celler. Sjukdomen drabbar i synnerhet de ytterst aktiva ögonmusklerna. Vi lyckades identifiera en mutation i genen RRM2B, hos de drabbade personerna. Denna gen finns i kärnan, och den kodar en enhet i enzymet ribonukleotid reduktas (RNR), som deltar i syntesen av deoxynukleotider, byggstenar för både kärnans DNA och mtDNA. Skadorna i mtDNA uppstod sannolikt som en följd av en obalans av deoxynukleotiderna. Vi fördjupade vår studie av RNR genom att undersöka möss som har en kraftigt ökad produktion av enzymet. Dessa möss mådde i övrigt bra, men deoxynukleotiderna i deras skelettmuskulatur visade sig vara i obalans, vilket ledde till att mängden mtDNA minskade. Såldes drog vi slutsatsen att korrekt uppehälle av mtDNA är beroende av en mycket noggrann balansering av dess byggstenar. En ökning av mängden mtDNA har tänkts kunna ha fördelaktiga effekter i mitokondriella sjukdomar. Genom att korsa två tidigare beskrivna muslinjer, skapade vi en linje möss med den största mängden mtDNA som hittills rapporterats. Övarraskande fann vi att denna mycket stora mängd mtDNA verkade ha ofördelaktiga konsekvenser: nukleoiderna, de proteinpaket som mtDNA packas i, var kraftigt förstorade, och mössens musklers mtDNA uppvisade defekter i likhet med patienter som har PEO. Därmed är vår studie den första att ge bevis för att en för stor mängd mtDNA kan vara skadlig

Reply : A novel MCM3AP mutation in a Lebanese family with recessive Charcot-Marie-Tooth neuropathy

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Perinnöllinen neuropatia ja spastinen parapareesi

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Motoneuronitautien lääkehoito - uutuuksia näköpiirissä

Vertaisarvioitu.Motoneuronitaudit ovat ryhmä harvinaisia tauteja, jotka johtavat usein kuolemaan ja joihin on toistaiseksi ollut tarjolla vain oireenmukaista hoitoa. Geenivirheiden osuus näiden tautien etiologiassa vaihtelee tuntemattomasta myötävaikuttavan kautta täydelliseen. Karttuva tieto sairauksien ja niille altistavien tekijöiden geneettisestä taustasta on mahdollistanut täsmälääketieteen esiinmarssin eli lisännyt räätälöityjen hoitojen tarjontaa. Yhtenä ongelmana on kustannus-hyötysuhde, koska uudet hoidot ovat kalliita. Esittelemme yleisimpien motoneuronitautien, amyotrofisen lateraaliskleroosin (ALS) ja spinaalisen lihasatrofian (SMA) hoitojen nykytilaa ja lähitulevaisuuden näkymiä.Peer reviewe

ABCD1-geenin mutaatiosta johtuva adrenomyeloneuropatia spastisen parapareesin taustatekijänä

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Absence of NEFL in patient-specific neurons in early-onset Charcot-Marie-Tooth neuropathy

Objective: We used patient-specific neuronal cultures to characterize the molecular genetic mechanism of recessive nonsense mutations in neurofilament light (NEFL) underlying early-onset Charcot-Marie-Tooth (CMT) disease. Methods: Motor neurons were differentiated from induced pluripotent stem cells of a patient with early-onset CMT carrying a novel homozygous nonsense mutation in NEFL. Quantitative PCR, protein analytics, immunocytochemistry, electron microscopy, and single-cell transcriptomics were used to investigate patient and control neurons. Results: We show that the recessive nonsense mutation causes a nearly total loss of NEFL messenger RNA (mRNA), leading to the complete absence of NEFL protein in patient's cultured neurons. Yet the cultured neurons were able to differentiate and form neuronal networks and neurofilaments. Single-neuron gene expression fingerprinting pinpointed NEFL as the most downregulated gene in the patient neurons and provided data of intermediate filament transcript abundancy and dynamics in cultured neurons. Blocking of nonsense-mediated decay partially rescued the loss of NEFL mRNA. Conclusions: The strict neuronal specificity of neurofilament has hindered the mechanistic studies of recessive NEFL nonsense mutations. Here, we show that such mutation leads to the absence of NEFL, causing childhood-onset neuropathy through a loss-of-function mechanism. We propose that the neurofilament accumulation, a common feature of many neurodegenerative diseases, mimics the absence of NEFL seen in recessive CMT if aggregation prevents the proper localization of wild-type NEFL in neurons. Our results suggest that the removal of NEFL as a proposed treatment option is harmful in humans.Peer reviewe

Effectiveness of clinical exome sequencing in adult patients with difficult-to-diagnose neurological disorders

Objectives Clinical diagnostics in adults with hereditary neurological diseases is complicated by clinical and genetic heterogeneity, as well as lifestyle effects. Here, we evaluate the effectiveness of exome sequencing and clinical costs in our difficult-to-diagnose adult patient cohort. Additionally, we expand the phenotypic and genetic spectrum of hereditary neurological disorders in Finland. Methods We performed clinical exome sequencing (CES) to 100 adult patients from Finland with neurological symptoms of suspected genetic cause. The patients were classified as myopathy (n = 57), peripheral neuropathy (n = 16), ataxia (n = 15), spastic paraplegia (n = 4), Parkinsonism (n = 3), and mixed (n = 5). In addition, we gathered the costs of prior diagnostic work-up to retrospectively assess the cost-effectiveness of CES as a first-line diagnostic tool. Results The overall diagnostic yield of CES was 27%. Pathogenic variants were found for 14 patients (in genes ANO5, CHCHD10, CLCN1, DES, DOK7, FKBP14, POLG, PYROXD1, SCN4A, TUBB3, and TTN) and likely pathogenic previously undescribed variants for 13 patients (in genes ABCD1, AFG3L2, ATL1, CACNA1A, COL6A1, DYSF, IRF2BPL, KCNA1, MT-ATP6, SAMD9L, SGCB, and TPM2). Age of onset below 40 years increased the probability of finding a genetic cause. Our cost evaluation of prior diagnostic work-up suggested that early CES would be cost-effective in this patient group, in which diagnostic costs increase linearly with prolonged investigations. Conclusions Based on our results, CES is a cost-effective, powerful first-line diagnostic tool in establishing the molecular diagnosis in adult neurological patients with variable symptoms. Importantly, CES can markedly shorten the diagnostic odysseys of about one third of patients.Peer reviewe

Attitudes towards genetic testing and information : does parenthood shape the views?

This study examines how parents of pediatric patients might differ in their views and attitudes towards genetic technology and information when compared to adult patients. There is surprisingly little evidence on how parents compare to other parts of population in their attitudes. Previous empirical studies often relate health-related preferences and attitudes to factors such as age, education, and income instead of parental status, thus evading comparison of parents to others as health-related decision makers. Findings related to the parental status can be useful when implementing genetic technology in clinical practice. We conducted a survey of views on genetic technology and information for groups of adult neurology patients (n = 68) and parents of pediatric neurology patients (n = 31) to shed some light on this issue. In addition to our own survey instrument, we conducted other surveys to gain insight on psychosocial factors that might affect these attitudes. The results suggest that parents are more concerned about their children's genetic risk factors when compared to the attitudes of adult patients about their own risk. For both groups, negative emotional state was associated with more concerns towards genetic information. Our study provides insights on how parental views might affect the acceptance of genetic technology and information.Peer reviewe

Truncated HSPB1 causes axonal neuropathy and impairs tolerance to unfolded protein stress

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CHCHD10 mutations and motor neuron disease: the distribution in Finnish patients.

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