Disorders of mitochondrial DNA polymerase : a clinical, biochemical and structural study

Mitochondrial dysfunction is a common cause of hereditary neurodegenerative diseases, such as Parkinson s disease and inherited ataxia syndromes. Frequent causes of mitochondrial dysfunction are mutations in mitochondrial DNA (mtDNA) or mutations in the genes encoding the mtDNA replication machinery. Mutations in the gene POLG1, which encodes the catalytic subunit of mitochondrial DNA polymerase, POLG, cause a heterogeneous group of disorders with varying clinical phenotypes, such as mitochondrial recessive ataxia syndrome (MIRAS), progressive external ophthalmoplegia (PEO), PEO with parkinsonism (POLG1-PD) and Alpers syndrome. The cause of variability in the clinical manifestations is unknown and is most evident in POLG1 diseases. In this thesis, we set to elucidate this variability by examining the neuropathological changes in MIRAS, POLG1-PD, IOSCA and PEO. We found that the cell number in substantia nigra correlated well with parkinsonism in POLG1 defects. Respiratory chain complex I defect, however, is a general consequence of mtDNA maintenance defects, and does not explain manifestation of parkinsonism or degree of mesencephalic cell death in patients with mtDNA maintenance disorders. We studied the biochemical background underlying MIRAS by biochemically characterizing the recombinant POLG1, with the MIRAS causing amino acid change, p.Trp748Ser. The recombinant POLG1 does not exhibit a biochemical phenotype in DNA polymerase function. Our findings challenge previously published results and their implications. We examined the distribution of Alpers syndrome causing mutations on the protein structure of POLG1. We found that the mutations can be divided into five distinct groups, each with a different biochemical profile. Our findings were supported by the published biochemical characterizations of various mutant POLG1 enzymes. Based on the structural data, we predict that the p.Trp748Ser amino acid change affects the interaction with mitochondrial single stranded-DNA binding protein and not intrinsic DNA polymerase activity or DNA binding of POLG1. We also studied the clinical picture of MIRAS. The high carrier frequency of the MIRAS disease allele in Finland, Norway and Belgium may lead to multiple introductions of the disease allele and some MIRAS cases to be mistakenly recognised as dominantly inherited, thereby leading to the exclusion of MIRAS as the diagnosis. We described a family in which both mother and son had MIRAS-like disease. We found that the father was a carrier of the MIRAS disease allele. Thus, MIRAS should not be ruled out by an apparently dominant mode of inheritance in countries with high carrier frequency of the disease allele.Mitokondriot ovat soluelimiä, joiden toimintahäiriöt ovat yleinen syy perinnöllisille hermoston rappeumasairauksille, kuten ataksioille ja Parkinsonin taudille. Perinnöllisten mitokondrioiden toimintahäiriöiden taustalla on mutaatiot mitokondriaalisessa DNA:ssa tai mitokondrioiden toimintaan osallistuvissa geeneissä. Mitokondriaalisen DNA polymeraasin, polymeraasi gamman mutaatiot aiheuttavat Suomen yleisimmän perinnöllisen ataksian, mitokondriaalisen resessiivisen ataksiaoireyhtymän (MIRAS). Lisäksi polymeraasi gamman geenin mutaatiot voivat aiheuttaa etenevää silmänliikuttajalihasten heikkenemää ja Parkinsonin tautia. Väitöstutkimuksessa selvitimme syitä tämän monimuotoisuuden taustalla. Tutkimus tehtiin yhteistyössä Helsingin yliopiston ja Michigan State Universityn kanssa. Mitokondrioiden toimintahäiriöt, etenkin hengitysketjun häiriöt, ovat mahdollisesti tärkeässä roolissa Parkinsonin taudin synnyssä. Selvitimme parkinsonismin syntyä mitokondriotautipotilailla tutkimalla muutoksia heidän keskushermostossaan. Jokaisella tutkituilla potilailla oli hermosolujen merkittävää tuhoutumista niillä keskiaivojen alueilla, jotka liittyvät Parkinsonin taudin syntyyn. Kuolleiden hermosolujen määrä oli suurimmillaan potilailla, joilla oli parkinsonismia. Kuitenkin kaikilla tutkituilla potilailla oli yhtä hankala hengitysketjun toiminnan häiriö, joka ei ollut riippuvainen parkinsonismista. Tämä tulos on ristiriidassa aikaisemmin julkaistujen tulosten kanssa ja viittaa siihen, että hengitysketjun toiminnan häiriö ei ole ensisijainen syy parkinsonismin tai Parkinsonin taudin taustalla. MIRAS on Suomen yleisin perinnöllinen ataksia, jota esiintyy myös muualla maailmassa. Tutkimuksessa kuvasimme perheen, jossa sekä äidillä että pojalla oli MIRAS. MIRAS:ä aiheuttavan tautialleelin kantajatiheys on hyvin korkea Suomessa. Korkeasta kantajatiheydestä johtuen resessiivisesti periytyvä MIRAS pitäisi sulkea aina pois perinnöllistä ataksiaa epäiltäessä, vaikka periytyminen vaikuttaisi dominantilta. MIRAS on tärkeä erottaa muista ataksioista, jotta vältyttäisiin epilepsialääke valproaatin aiheuttamilta maksavaurioilta MIRAS-potilailla. MIRAS kliiniset oireet ja ennuste ovat hyvin vaihtelevia ja niin alkamisikä, eliniänodote ja oirekuvan hankaluus vaihtelevat merkittävästi. Biokemiallinen ja rakenteellinen selvitys polymeraasi gamma-entsyymin toiminnasta MIRAS-potilailla ei selittänyt tätä monimuotoisuutta. MIRAS muutos häiritsee mahdollisesti polymeraasi gamma-entsyymin vuorovaikutusta muiden mitokondriaalisen DNA:n kahdentamiseen osallistuvien entsyymien kanssa. Selvitimme lisäksi peittyvästi perityvien muutosten jakautumista polymeraasi gamma-entsyymin kolmiulotteiselle rakenteelle. Peittyvästi periytyvät polymeraasi gamman mutaatiot muodostivat viisi erilaista rypästä, joilla jokaisella oli erityinen biokemiallinen profiili. Tämä tulos antaa mitokondriotautipotilaita hoitaville lääkärille uuden työkalun, jonka avulla he pystyvät mahdollisesti arvioimaan uusien polymeraasi gamma-geenistä löytyvien mutaatioiden tautia aiheuttavaa vaikutusta

Clustering of Alpers disease mutations and catalytic defects in biochemical variants reveal new features of molecular mechanism of hte human mitochondrial replicase, Pol gamma

Peer reviewe

The Variant p.(Arg183Trp) in SPTLC2 Causes Late-Onset Hereditary Sensory Neuropathy

Hereditary sensory and autonomic neuropathy 1 (HSAN1) is an autosomal dominant disorder that can be caused by variants in SPTLC1 or SPTLC2, encoding subunits of serine palmitoyl-CoA transferase. Disease variants alter the enzyme's substrate specificity and lead to accumulation of neurotoxic 1-deoxysphingolipids. We describe two families with autosomal dominant HSAN1C caused by a new variant in SPTLC2, c.547C > T, p.(Arg183Trp). The variant changed a conserved amino acid and was not found in public variant databases. All patients had a relatively mild progressive distal sensory impairment, with onset after age 50. Small fibers were affected early, leading to abnormalities on quantitative sensory testing. Sural biopsy revealed a severe chronic axonal neuropathy with subtotal loss of myelinated axons, relatively preserved number of non-myelinated fibers and no signs for regeneration. Skin biopsy with PGP9.5 labeling showed lack of intraepidermal nerve endings early in the disease. Motor manifestations developed later in the disease course, but there was no evidence of autonomic involvement. Patients had elevated serum 1-deoxysphingolipids, and the variant protein produced elevated amounts of 1-deoxysphingolipids in vitro, which proved the pathogenicity of the variant. Our results expand the genetic spectrum of HSAN1C and provide further detail about the clinical characteristics. Sequencing of SPTLC2 should be considered in all patients presenting with mild late-onset sensory-predominant small or large fiber neuropathy.Peer reviewe

SNCA mutation p.Ala53Glu is derived from a common founder in the Finnish population

Peer reviewe

Genetic background of ataxia in children younger than 5 years in Finland

Objective To characterize the genetic background of molecularly undefined childhood-onset ataxias in Finland. Methods This study examined a cohort of patients from 50 families with onset of an ataxia syndrome before the age of 5 years collected from a single tertiary center, drawing on the advantages offered by next generation sequencing. A genome-wide genotyping array (Illumina Infinium Global Screening Array MD-24 v.2.0) was used to search for copy number variation undetectable by exome sequencing. Results Exome sequencing led to a molecular diagnosis for 20 probands (40%). In the 23 patients examined with a genome-wide genotyping array, 2 additional diagnoses were made. A considerable proportion of probands with a molecular diagnosis had de novo pathogenic variants (45%). In addition, the study identified a de novo variant in a gene not previously linked to ataxia: MED23. Patients in the cohort had medically actionable findings. Conclusions There is a high heterogeneity of causative mutations in this cohort despite the defined age at onset, phenotypical overlap between patients, the founder effect, and genetic isolation in the Finnish population. The findings reflect the heterogeneous genetic background of ataxia seen worldwide and the substantial contribution of de novo variants underlying childhood ataxia.Peer reviewe

Genetic Basis of Severe Childhood-Onset Cardiomyopathies.

BACKGROUND: Childhood cardiomyopathies are progressive and often lethal disorders, forming the most common cause of heart failure in children. Despite severe outcomes, their genetic background is still poorly characterized. OBJECTIVES: The purpose of this study was to characterize the genetics of severe childhood cardiomyopathies in a countrywide cohort. METHODS: The authors collected a countrywide cohort, KidCMP, of 66 severe childhood cardiomyopathies from the sole center in Finland performing cardiac transplantation. For genetic diagnosis, next-generation sequencing and subsequent validation using genetic, cell biology, and computational approaches were used. RESULTS: The KidCMP cohort presents remarkable early-onset and severe disorders: the median age of diagnosis was 0.33 years, and 17 patients underwent cardiac transplantation. The authors identified the pathogenic variants in 39% of patients: 46% de novo, 34% recessive, and 20% dominantly-inherited. The authors report NRAP underlying childhood dilated cardiomyopathy, as well as novel phenotypes for known heart disease genes. Some genetic diagnoses have immediate implications for treatment: CALM1 with life-threatening arrhythmias, and TAZ with good cardiac prognosis. The disease genes converge on metabolic causes (PRKAG2, MRPL44, AARS2, HADHB, DNAJC19, PPA2, TAZ, BAG3), MAPK pathways (HRAS, PTPN11, RAF1, TAB2), development (NEK8 and TBX20), calcium signaling (JPH2, CALM1, CACNA1C), and the sarcomeric contraction cycle (TNNC1, TNNI3, ACTC1, MYH7, NRAP). CONCLUSIONS: Childhood cardiomyopathies are typically caused by rare, family-specific mutations, most commonly de novo, indicating that next-generation sequencing of trios is the approach of choice in their diagnosis. Genetic diagnoses may suggest intervention strategies and predict prognosis, offering valuable tools for prioritization of patients for transplantation versus conservative treatment