Article

Molecular analysis of ANT1, TWINKLE and POLG in patients with multiple deletions or depletion of mitochondrial DNA by a dHPLC-based assay

Department of Medical Genetics, Archet 2 Hospital, CHU Nice, France.
European Journal of HumanGenetics (Impact Factor: 4.23). 09/2006; 14(8):917-22. DOI: 10.1038/sj.ejhg.5201627
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ABSTRACT ANT1, TWINKLE and POLG genes affect mtDNA stability and are involved in autosomal dominant PEO, while mutations in POLG are responsible for numerous clinical presentations, including autosomal recessive PEO, sensory ataxic neuropathy, dysarthria and ophthalmoparesis (SANDO), spino-cerebellar ataxia and epilepsy (SCAE) or Alpers syndrome. In this study, we report on the mutational analysis of ANT1, TWINKLE and POLG genes in 15 unrelated patients, using a dHPLC-based protocol. This series of patients illustrates the large array of clinical presentations associated with mtDNA stability defects, ranging from isolated benign PEO to fatal Alpers syndrome. A total of seven different mutations were identified in six of 15 patients (40%). Six different recessive mutations were found in POLG, one in TWINKLE while no mutation was identified in ANT1. Among the POLG mutations, three are novel and include two missense and one frameshift changes. Seventeen neutral changes and polymorphisms were also identified, including four novel neutral polymorphisms. Overall, this study illustrates the variability of phenotypes associated with mtDNA stability defects, increases the mutational spectrum of POLG variants and provides an efficient and reliable detection protocol for ANT1, TWINKLE and POLG mutational screening.

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Available from: Claude Desnuelle, Aug 28, 2015
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    • "Depletion syndromes are associated with nuclear mutations of DGUOK [42], SUCLA2, SUCLG1, TK2, MPV17 [43], PEO1 [44], RRM2B, and POLG; the last is the case in two of our patients. Mutations in the POLG gene were previously reported in AHS by three groups [17-19,45-47] and most recently by Stewart et al. [15] who describe 9 patients with a COX mosaic in muscle, but none in liver. "
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    ABSTRACT: Thesis focuses on mutations of POLG1 gene encoding catalytic subunit polγ-α of mitochondrial DNA polymerase gamma holoenzyme (polG) and the association of mutations with different clinical phenotypes. In addition, particular defective mutant variants of the protein were characterized biochemically in vitro. PolG-holoenzyme is the sole DNA polymerase found in mitochondria. It is involved in replication and repair of the mitochondrial genome, mtDNA. Holoenzyme also includes the accessory subunit polγ-β, which is required for the enhanced processivity of polγ-α. Defective polγ-α causes accumulation of secondary mutations on mtDNA, which leads to a defective oxidative phosphorylation system. The clinical consequences of such mutations are variable, affecting nervous system, skeletal muscles, liver and other post-mitotic tissues. The aims of the studies included: 1) Determination of the role of POLG1 mutations in neurological syndromes with features of mitochondrial dysfunction and an unknown molecular cause. 2) Development and set up of diagnostic tests for routine clinical purposes. 3) Biochemical characterization of the functional consequences of the identified polγ-α variants. Studies describe new neurological phenotypes in addition to PEO caused by POLG1 mutations, including parkinsonism, premature amenorrhea, ataxia and Parkinson s disease (PD). POLG1 mutations and polymorphisms are both common and/or potential genetic risk factors at least among the Finnish population. The major findings and applications reported here are: 1) POLG1 mutations cause parkinsonism and premature menopause in PEO families in either a recessive or a dominant manner. 2) A common recessive POLG1 mutations (A467T and W748S) in the homozygous state causes severe adult or juvenile-onset ataxia without muscular symptoms or histological or mtDNA abnormalities in muscles. 3) A common recessive pathogenic change A467T can also cause a mild dominant disease in heterozygote carriers. 4) The A467T variant shows reduced polymerase activity due to defective template binding. 5) Rare polyglutamine tract length variants of POLG1 are significantly enriched in Finnish idiopathic Parkinson s disease patients. 6) Dominant mutations are clearly restricted to the highly conserved polymerase domain motifs, whereas recessive ones are more evenly distributed along the protein. The present results highlight and confirm the new role of mitochondria in parkinsonism/Parkinson s disease and describe a new mitochondrial ataxia. Based on these results, a POLG1 diagnostic routine has been set up in Helsinki University Central Hospital (HUSLAB). Ihmisen perintötekijät eli geenit sijaitsevat lähes yksinomaan solujen tumassa. Niiden määräksi on ihmisellä arvioitu noin 20-30 000. Vain 37 näistä geeneistä sijaitsee tuman ulkopuolella solujen mitokondrioissa, joita usein kuvataan solujen voimalaitoksiksi . Niiden tärkeimpänä tehtävänä on muuntaa ravintoaineiden kemiallinen energia yhdenmukaiseen muotoon, polttoaineeksi eli ATP:ksi, jota solujen rakentamiseen ja ylläpitoon osallistuvat entsyymit voivat yleisesti käyttää. Tässä muutostyössä hapella on tärkeä rooli, puhumme soluhengityksestä. Kun soluhengitys ei toimi kunnolla, lisääntyy soluissa myös hapen vahingolliset muodot aiheuttaen solurakenteiden vaurioitumista. Kuten tuman geeneistä, myös mitokondrioissa olevista geeneistä täytyy pitää huolta: monistaa ja korjata vaurioita. Tässä työssä keskeisimpiä entsyymejä on DNA polymeraasi gamma. Kuten tiedämme, voi yhden ainoan geenin yksi virhe tuottaa vakavan sairauden ihmisessä, näin on asian laita myös DNA polymeraasi gamman kohdalla. Erona kuitenkin useimpien muiden geenien mutaatioihin nähden on se, että DNA polymeraasi gamman mutaatiot saavat aikaan lisää mutaatioita niissä 37:ssä geenissä, jotka ovat mitokondrioiden sisällä. Koska kyseiset geenit ovat tärkeitä soluhengityksen kannalta, aiheuttaa niiden vaurioituminen ongelmia soluhengitykselle ja energia-aineenvaihdunnalle ja tästä koituu vakavia seurauksia ihmiselle. Tässä väitöstyössä kuvataan uusia DNA polymeraasi gamman mutaatioita ja niihin liittyviä sairauksia, joista tärkeimpinä ataksia sekä Parkinsonin tauti. Työssä mm. kuvataan uusi mutaatio, joka on osoittautunut yleisimmäksi ataksian aiheuttajaksi Suomessa. Sen lisäksi työssä osoitettiin DNA polymeraasi gammalla olevan yleisimmän päätyypin lisäksi ainakin 17 erilaista alatyyppiä suomalaisessa väestössä. Osan näistä harvinaisista DNA polymeraasi gamman alatyypeistä osoitettiin olevan taustalla huomattavalla määrällä suomalaisista Parkinsonin tautia sairastavista. DNA polymeraasi gamma saattaa osoittautua yleisimmäksi tautigeeniksi Parkinsonin taudin taustalla Suomessa. Tulokset vahvistavat osaltaan mitokondrioiden tärkeää roolia kyseisissä sairauksissa. Tärkeänä havaintona voidaan myös pitää sitä että sama mutaatio eri perheissä saattaa aiheuttaa joko vallitsevasti tai peittyvästi periytyvän taudin. Vallitsevasti periytyvässä taudissa yksi, esimerkiksi äidiltä periytyvässä geenissä oleva mutaatio aiheuttaa yksinään sairauden. Peittyvästi periytyvässä sairaudessa tarvitaan sekä isältä että äidiltä peritystä geenistä mutatoitunut muoto. Näiden tutkimustulosten pohjalta on saatu tärkeää tietoa neurologisten sairauksien molekulaarisista mekanismeista sekä luotu Helsingin yliopistolliseen keskussairaalaan uusia diagnostisia menetelmiä, sekä tietoa jota voidaan soveltaa perinnöllisyysneuvonnassa.
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