Analysis of exome variant data for identifying causative SNVs of infantile mitochondrial disorders

This thesis presents a workflow for analysis of exome sequencing data aiming at identification of single nucleotide variants (SNVs) causing recessively inherited mitochondrial disease in children. Several variant selection criteria that are consistent with such group of genetic disorders are applied along the workflow in relation to mode of inheritance, allele frequency and the Finnish ancestry of the patients. These are combined with knowledge of nuclear-encoded mitochondrial proteins and prediction of pathogenic variants, narrowing down the total set of SNVs found in a patient to those most likely to be causative. Patient exomes are analysed individually (n=1 studies). The bioinformatic resources used for implementation include public and in-house databases of mitochondrial nuclear genes, human genetic variation and exome controls, as well as software tools for prediction of pathogenic SNVs and mitochondria-targeting proteins. Exome variant data from a cohort of 49 molecularly undiagnosed children were analysed through the workflow, leading to the identification of mitochondrial disease-causing SNVs located in nuclear genes for 10 of the patients. Therefore, a success rate of 20% was achieved. The workflow has been an important element in the use of exome sequencing as a new research tool at the Wartiovaara group of the Research Program for Molecular Neurology, Faculty of Medicine, University of Helsinki

Reply to : Proofreading deficiency in mitochondrial DNA polymerase does not affect total dNTP pools in mouse embryos

Non peer reviewe

Recessive PYROXD1 mutations cause adult-onset limb-girdle-type muscular dystrophy

Peer reviewe

Niacin Cures Systemic NAD(+) Deficiency and Improves Muscle Performance in Adult-Onset Mitochondrial Myopathy

NAD(+) is a redox-active metabolite, the depletion of which has been proposed to promote aging and degenerative diseases in rodents. However, whether NAD(+) depletion occurs in patients with degenerative disorders and whether NAD(+) repletion improves their symptoms has remained open. Here, we report systemic NAD(+) deficiency in adult-onset mitochondrial myopathy patients. We administered an increasing dose of NAD(+) booster niacin, a vitamin B3 form (to 750-1,000 mg/day; clinicaltrials.gov NCT03973203) for patients and their matched controls for 10 or 4 months, respectively. Blood NAD(+) increased in all subjects, up to 8-fold, and muscle-NAD(+) of patients reached the level of their controls. Some patients showed anemia tendency, while muscle strength and mitochondrial biogenesis increased in all subjects. In patients, muscle metabolome shifted toward controls and liver fat decreased even 50%. Our evidence indicates that blood analysis is useful in identifying NAD(+) deficiency and points niacin to be an efficient NAD(+) booster for treating mitochondrial myopathy.Peer reviewe

Recessive PYROXD1 mutations cause adult-onset limb-girdle-type muscular dystrophy

Objective: To describe adult-onset limb-girdle-type muscular dystrophy caused by biallelic variants in the PYROXD1 gene, which has been recently linked to early-onset congenital myofibrillar myopathy.Methods: Whole exome sequencing was performed for adult-onset neuromuscular disease patients with no molecular diagnosis. Patients with PYROXD1 variants underwent clinical characterization, lower limb muscle MRI, muscle biopsy and spirometry. A yeast complementation assay was used to determine the biochemical consequences of the genetic variants.Results: We identified four patients with biallelic PYROXD1 variants. Three patients, who had symptom onset in their 20s or 30s, were homozygous for the previously described p.Asn155Ser. The fourth patient, with symptom onset at age 49, was compound heterozygous for p.Asn155Ser variant and previously unknown p.Tyr354Cys. All patients presented with a LGMD-type phenotype of symmetric muscle weakness and wasting. Symptoms started in proximal muscles of the lower limbs, and progressed slowly to involve also upper limbs in a proximal-predominant fashion. All patients remained ambulant past the age of 60. They had restrictive lung disease but no cardiac impairment. Muscle MRI showed strong involvement of anterolateral thigh muscles. Muscle biopsy displayed chronic myopathic changes. Yeast complementation assay demonstrated the p.Tyr354Cys mutation to impair PYROXD1 oxidoreductase ability.Conclusion: PYROXD1 variants can cause an adult-onset slowly progressive LGMD-type phenotype.</p

Geneettisen tiedon lukutaidon kehittäminen kohderyhmänä terveydenhuollon AMK-opiskelijat

Yksilöllistetyn lääketieteen jalkautuessa ennakoidaan, että geneettisen tiedon käyttö tulee vähitellen yleistymään terveydenhuollossa. Hoitotyön ammattikorkeakouluopinnoista löytyy kuitenkin vain vähäistä sisältöä genetiikasta, sen merkityksestä terveyteen ja siitä, kuinka geneettisen sairastumisriskin huomioiminen edistää yksilöllisempää terveysneuvontaa. Tämä motivoi opinnäytetyön, jonka tarkoitus oli tutustuttaa AMK-opiskelijaryhmä geneettisen tiedon hyödyntämiseen terveydenhuollossa. Opinnäytetyössä suunniteltiin ja toteutettiin työpaja, jossa valmistuvat terveydenhoitajaopiskelijat rakensivat asiakaskohtaista terveysneuvontaa, jossa ovat huomioitu asiakkaiden arvioitu perinnöllinen sekä kokonaisriski sairastua tyypin 2 diabetekseen. Opiskelijat vastasivat myös kyselyihin genetiikkaan ja geneettisen tiedon käyttöön liittyvistä näkemyksistään sekä aihepiiriin liittyvistä aiemmista opinnoistaan tai kokemuksistaan. Työpajan toteutuksessa käytettiin Flipped Learning -menetelmää, jonka mukaisesti opiskelijoille tuotettiin kirjallinen perehtymismateriaali itsenäiseen valmistautumiseen työpajassa tapahtuvaan työskentelyyn. Materiaalissa esiteltiin lyhyesti ihmisgenetiikan perusteet sekä kerrattiin tyypin 2 diabetekseen liittyvä osaaminen. Varsinaisessa työpajassa itsenäinen opiskelu vaihtui vuorovaikutukseen ja tiedon jakamiseen opiskelijoiden kesken. Opiskelijoiden vastaukset kyselyjen avoimiin kysymyksiin analysoitiin teemoittelun avulla. Numeerisilla arvoilla annetut vastaukset laskettiin yhteen yksinkertaisesti vastaajien lukumääriksi tai prosenttiosuuksiksi. Opiskelijoiden tekemät terveysneuvonnan suunnitelmat sekä tulokset kyselyistä yleisesti ottaen viittaavat siihen, että opiskelijat omaksuivat ja osasivat soveltaa vasta kohdattuja genetiikan aihepiirin käsitteitä. Työpajan lopputuloksena oli se, että opiskelijoiden valmius antaa geneettiseen riskiin pohjautuvaa, kansantautien ennaltaehkäisevää terveysneuvontaa kehittyi. Osallistujat myönsivät kuitenkin, että työpajan johdosta kehittynyttä laajempi ja syvempi osaaminen vaatisi pitkäjänteisempää koulutusta ja kokemusta. Jo ennen työpajaa opiskelijoilla oli melko vahva näkemys siihen, että geneettisen tiedon lukutaito tulee olemaan tärkeä osaaminen terveydenhuollon työssä. Työpajaan osallistumisen jälkeen opiskelijoiden mielipide geneettisen tiedon osaamisen merkityksestä hoitotyössä vahvistui.As personalised medicine gains ground, the use of genetic information is expected to gradually become commonplace in health care. Yet, health care degree programmes at the undergraduate level only include a minimal amount of content about genetics, its relevance to health, and how taking into account information on genetic risks promotes more individualised health advice. This was the motivation for this work, whilst its purpose was to introduce a group of undergraduate students to utilising genetic information in health care. A workshop was planned and carried out where final-semester Public Health Care students outlined health advice plans for described client cases, taking into consideration their estimated genetic and overall risk of developing type 2 diabetes. The students also answered questionnaires that briefly surveyed their views and prior studies or experience related to genetics and the use of genetic information. The Flipped Learning model was applied, whereby written material concisely introducing basic concepts in human genetics and recapitulating type 2 diabetes was provided to be independently used by the students in preparation for the workshop. Independent learning transitioned in the workshop event itself to students interacting and sharing knowledge. The students’ answers to the open-ended questions in the survey were analysed by theme elicitation. Answers given in numeric scales were simply quantified by number or proportion of respondents. The outlined health advice plans as well as the results from the survey in general indicate that the students grasped and were able to apply newly met concepts from the field of genetics. The overall outcome from the workshop was the students’ improved ability to provide preventive health advice with a basis on an individual’s genetic risk for common disease. The participants, however, recognised that widening and deepening such competence would require longer-term training and experience. Already before the workshop, most students tended to agree, to some or full extent, that genetic information literacy is to become an important competence in health care work. After participating in the workshop, the students’ view of genetic information literacy being relevant to healthcare work had strengthened

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

A complex genomic locus drives mtDNA replicase POLG expression to its disease-related nervous system regions

DNA polymerase gamma (POLG), the mtDNA replicase, is a common cause of mitochondrial neurodegeneration. Why POLG defects especially cause central nervous system (CNS) diseases is unknown. We discovered a complex genomic regulatory locus for POLG, containing three functional CNS-specific enhancers that drive expression specifically in oculomotor complex and sensory interneurons of the spinal cord, completely overlapping with the regions showing neuronal death in POLG patients. The regulatory locus also expresses two functional RNAs, LINC00925-RNA and MIR9-3, which are coexpressed with POLG. The MIR9-3 targets include NR2E1, a transcription factor maintaining neural stem cells in undifferentiated state, and MTHFD2, the regulatory enzyme of mitochondrial folate cycle, linking POLG expression to stem cell differentiation and folate metabolism. Our evidence suggests that distant genomic non-coding regions contribute to regulation of genes encoding mitochondrial proteins. Such genomic arrangement of POLG locus, driving expression to CNS regions affected in POLG patients, presents a potential mechanism for CNS-specific manifestations in POLG disease.Peer reviewe