Most pathogenic mitochondrial DNA (mtDNA) variants occur in the 22 mtDNA-encoded tRNA (mt-tRNA) genes. However, despite more than 270 reported mt-tRNA gene mutations, only 5 reside within mt-tRNAGly (MT-TG). We report a rare MT-TG variant and evaluate this, in addition to all previously reported MT-TG variants, against the published criteria used to help determine the pathogenicity of the mt-tRNA variants

Bugiardini, E

Hanna, MG

Hardy, SA

Hargreaves, IP

Horga, A

Merve, A

Pitceathly, RDS

Poole, OV

Quinlivan, R

Taylor, RW

Woodward, CE

English

UCL Discovery

CLINICAL/SCIENTIFIC NOTES OPEN ACCESSMultisystem mitochondrial disease caused bya rare m.10038G>A mitochondrial tRNAGly(MT-TG) variantOlivia V. Poole, MD,* Alejandro Horga, MD, PhD,* Steven A. Hardy, PhD, Enrico Bugiardini, MD,Cathy E. Woodward, BSc, Iain P. Hargreaves, PhD, FRCPath, Ashirwad Merve, PhD, FRCPath,Rosaline Quinlivan, FRCP, Robert W. Taylor, PhD, FRCPath, Michael G. Hanna, FRCP, andRobert D.S. Pitceathly, PhD, MRCPNeurol Genet 2020;6:e413. doi:10.1212/NXG.0000000000000413CorrespondenceDr. Pitceathlyr.pitceathly@ucl.ac.ukMost pathogenic mitochondrial DNA (mtDNA) variants occur in the 22 mtDNA-encodedtRNA (mt-tRNA) genes. However, despite more than 270 reported mt-tRNA gene mutations,only 5 reside within mt-tRNAGly (MT-TG).1 We report a rareMT-TG variant and evaluate this,in addition to all previously reportedMT-TG variants, against the published criteria used to helpdetermine the pathogenicity of the mt-tRNA variants.2Case reportA 39 year old woman, born to nonconsanguineous parents, was reviewed in a specialistmitochondrial disorders clinic. She presented with hearing loss in her late teens followed byvisual impairment, with bilateral cataracts, retinal dystrophy, and subsequent bilateral retinaldetachments in her twenties; hypothyroidism in her thirties; and secondary amenorrhea.Clinical examination was otherwise normal, apart from short stature. There was no familyhistory of neuromuscular or neurologic disease. Blood tests, including creatine kinase,plasma amino acids, acylcarnitine profile, very long chain fatty acids, and white cell enzymes,were normal. Plasma lactate was elevated (3.70 mmol/L, reference range 0.5–2.2 mmol/L).Nerve conduction studies and EMG showed no evidence of generalized myopathy or largefiber neuropathy. Histochemical analyses of muscle tissue revealed ragged-red and cyto-chrome c oxidase (COX) deficient fibers (figure A). Spectrophotometric determination ofmitochondrial respiratory chain enzyme activities as a ratio to citrate synthase activity3confirmed decreased activities of complexes I (0.076, reference range 0.104–0.268) and IV(0.006, reference range 0.014–0.034). Analysis of the next generation sequencing data(Ilumina MiSeq) of the entire mitochondrial genome extracted from the muscle3 revealeda rare m.10038G>A variant (GenBank reference accession number: NC_012920.1) in MT-TG (figure B) that was present at variable heteroplasmy levels across tissue types: 15%blood, 40% urinary epithelial cells, and 92% skeletal muscle. Maternal transmission wasconfirmed: 3% mutant load was present in the mother’s urinary epithelial cells (method-ology detects heteroplasmy levels ≥1%). Heteroplasmy levels within individual laser-captured COX-positive and COX-deficient muscle fibers were quantified by pyrosequenc-ing.4 Single fiber segregation studies confirmed a higher mutation load in COX-deficient(mean 95.30% ± 0.50%, SD, n = 27) compared with COX-positive fibers (mean 78.92% ±4.43%, SD, n = 26; p = 0.0005, figure C).*These authors contributed equally to the manuscript.From the Department of Neuromuscular Diseases (O.V.P., A.H., E.B., R.Q., M.G.H., R.D.S.P.), UCL Queen Square Institute of Neurology and The National Hospital for Neurology andNeurosurgery, London, United Kingdom; Wellcome Centre for Mitochondrial Research (S.A.H., R.W.T.), Translational and Clinical Research Institute, Newcastle University, NewcastleUpon Tyne; Neurogenetics Unit (C.E.W.), and Neurometabolic Unit (I.P.H.), The National Hospital for Neurology and Neurosurgery; Division of Neuropathology (A.M.), UCL QueenSquare Institute of Neurology; Department of Histopathology (A.M.), Camelia Botnar Laboratory, Great Ormond Street Hospital; and Dubowitz Neuromuscular Centre (R.Q.), GreatOrmond Street Hospital, London, United Kingdom.Go to Neurology.org/NG for full disclosures. Funding information is provided at the end of the article.The Article Processing Charge was funded by the authors.This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (CC BY), which permits unrestricted use, distribution, and reproduction in anymedium, provided the original work is properly cited.Copyright © 2020 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology. 1DiscussionThe m.10038G nucleotide is highly conserved across species(figure D, figure e-1, links.lww.com/NXG/A248) and the G>Atransition at this location disrupts a C-G Watson-Crick basepair in the TΨC stem of the tRNA molecule. Them.10038G>A variant is rare (absent from 3,450 in-house and48,882 GenBank1 sequences) and detectable at variable het-eroplasmy levels across different tissues, with the highest levelsin the postmitotic muscle. Histochemical and biochemical ev-idence of reduced complex I and IV activities in the muscle issupportive of impaired mitochondrial protein synthesis,whereas single fiber studies confirmed segregation of the COXdefect with higher mutant levels. Consequently, the mutationwould be considered “definitely pathogenic” based on the ac-cepted criteria for assigning pathogenicity to tRNA mutations(table e-1, links.lww.com/NXG/A249).2Review of the 5 previously reportedMT-TG variants accordingto these criteria (table e-1, links.lww.com/NXG/A249) revealsthat only 2 variants; m.9997T>C and m.10010T>C, should beconsidered “definitely pathogenic.” Although the m.9997T>Cvariant has only been reported in one family, trans-mitochondrial cybrid studies support its pathogenic effects.The m.10010T>C variant has been reported a number oftimes, and its pathogenicity has been confirmed using singlefiber segregation studies. The m.10006A>G variant has onlybeen reported in individuals harboring additional mtDNAvariants, and thus may represent a benign polymorphism or beinsufficient to cause disease in isolation. Current evidencesuggests that the m.10014A>G variant is a benign poly-morphism. Finally, although the scoring system indicates thatthe m.10044A>G variant is “possibly pathogenic,” it has beendetected in healthy controls, and thus may represent a hap-logroup specific polymorphism.5It remains unclear why pathogenic variants occur more fre-quently in specific mt-tRNA genes. Possible explanations in-clude intensive investigation of mt-tRNAs in which pathogenicvariants have previously been identified, and survivor bias andFigure Characterization of a rare pathogenic MT-TG variant(A) Staining of themuscle sections with haematoxylin and eosin demonstrated ragged-red fibers (*) which were confirmed in the Gomori trichrome preparation(*)with the inset showing highmagnificationof left top fiber. Anumber of fibers, oftenwith ragged-redmorphology, showedamild increase in thenumber of lipiddroplets with Sudan black (*). Sequential COX and SDH histochemistry demonstrated frequent COX-deficient fibers, some of which had a ragged red–likeappearance (*). The bar represents 100 μm for all stains with the inset 50 μm. (B) Two-dimensional cloverleaf structure of mitochondrial DNA-encoded tRNAGly.Black arrows indicate previously reported pathogenic variants. Red arrow indicates the rare variant identified in our patient. (C) Single muscle fiber segregationstudies. COX-deficient fibers, (blue circles) harbor a higher mutational load compared with COX-positive fibers (red circles). (D) Evolutionary conservation of them.10038G residue across species. COX = cytochrome c oxidase; SDH = succinate dehydrogenase.2 Neurology: Genetics | Volume 6, Number 2 | April 2020 Neurology.org/NGthe absence of maternal transmission of mutations in mt-tRNAgenes linked with severe biochemical deficiencies, with isolatedcases dissuading clinicians from further investigation of anunderlying mitochondrial disorder.6 Variants in mt-tRNAGlyaffecting its canonical or noncanonical functions may also po-tentially be better tolerated than other mt-tRNAs or, con-versely, be severely deleterious and embryonic lethal.The detection of the rare mt-tRNA gene variants in suspectedmitochondrial disease has become more commonplace, giventhe widespread availability of whole mtDNA high throughputsequencing. Furthermore, whole genome sequencing, whichincludes capture and deep sequencing of the mitochondrialgenome, is identifying mtDNA variants in those in whom mi-tochondrial disease may not previously have been considered.However, despite advances in the DNA sequencing technol-ogy, the challenge of assigning pathogenicity to the raremtDNA variants remains. It is therefore crucial that pathogenicvariants are reported after confirmation using “gold-standard”techniques, for example, single fiber or transmitochondrialcybrid studies, given the implications to genetic counseling andthe available reproductive options for mtDNA mutations, in-cluding mitochondrial donation. Moreover, generating a com-prehensive data set for “definitely pathogenic” mt-tRNAvariants would potentially advance the understanding of themolecular mechanisms underpinning the susceptibility of in-dividual genes to deleterious variants and facilitate the de-velopment of targeted therapies to treat this group of disorders.AcknowledgmentThe authors would like to sincerely thank the family thatparticipated in the study.Study fundingPart of this work was undertaken in the University CollegeLondon Hospitals/University College London Queen SquareInstitute of Neurology sequencing facility, which receiveda proportion of funding from the Department of Health’sNational Institute for Health Research Biomedical ResearchCentres funding scheme. This research was supported by theNational Institute for Health Research University CollegeLondon Hospitals Biomedical Research Centre. O.V. Poolehas received funding from the Lily Foundation. R.W. Taylor issupported by the Wellcome Centre for Mitochondrial Re-search (203105/Z/16/Z), the Medical Research Council(MRC) International Centre for Genomic Medicine inNeuromuscular Disease, Mitochondrial Disease Patient Co-hort (UK) (G0800674), the UK NIHR Biomedical ResearchCentre for Ageing and Age-related disease award to theNewcastle upon Tyne Foundation Hospitals NHS Trust, theMRC/EPSRC Molecular Pathology Node and The LilyFoundation. R.D.S. Pitceathly is supported by a Medical Re-search Council Clinician Scientist Fellowship (MR/S002065/1). The clinical and diagnostic mitochondrial services inLondon and Newcastle upon Tyne are funded by the UKNHS Highly Specialised Commissioners to provide the “RareMitochondrial Disorders” Service.DisclosureThe present study is not industry sponsored. O.V. Poole,A. Horga, S.A. Hardy, E. Bugiardini, C. Woodward, I.P.Hargreaves, A. Merve, R. Quinlivan, R.W. Taylor, M.G.Hanna, and R.D.S. Pitceathly report no disclosures. Go toNeurology.org/NG for full disclosures.Publication historyReceived by Neurology: Genetics July 7, 2019. Accepted in final formJanuary 23, 2020.Appendix AuthorsName Location ContributionOlivia V.Poole, MDUCL Queen SquareInstitute of Neurology andThe National Hospital forNeurology andNeurosurgery, London,United KingdomStudy concept and design,Major role in acquisition ofdata, Analysis/interpretation of data,Revising the manuscriptfor intellectual contentAlejandroHorga, MDUCL Queen Square Instituteof Neurology and TheNational Hospital forNeurology andNeurosurgery, London,United KingdomStudy concept and design,Major role in acquisition ofdata, Analysis/interpretation of data,Revising the manuscriptfor intellectual contentSteven A.Hardy, PhDWellcome Centre forMitochondrial Research,Translational and ClinicalResearch Institute,NewcastleUniversity, Newcastle UponTyne, United KingdomMajor role in acquisition ofdata, Analysis/interpretation of data,Revising the manuscriptfor intellectual contentEnricoBugiardini,MDUCL Queen Square Instituteof Neurology and TheNational Hospital forNeurology andNeurosurgery, London,United KingdomMajor role in acquisition ofdata, Analysis/interpretation of data,Drafting/revising themanuscript for intellectualcontentCathy E.Woodward,BScThe National Hospital forNeurology andNeurosurgery, London,United KingdomMajor role in acquisition ofdata, Analysis/interpretation of data,Revising the manuscriptfor intellectual contentIain PHargreaves,PhD,FRCPathThe National Hospital forNeurology andNeurosurgery, London,United KingdomMajor role in acquisition ofdata, Analysis/interpretation of data,Drafting/revising themanuscript for intellectualcontentAshirwadMerve, PhD,FRCPathUCL Queen SquareInstitute of Neurology,London, United KingdomMajor role in acquisition ofdata, Analysis/interpretationof data, Drafting/revising themanuscript for intellectualcontentRosalineQuinlivan,FRCPUCL Queen SquareInstitute of Neurology andThe National Hospital forNeurology andNeurosurgery, London,United KingdomAnalysis/interpretation ofdata, Drafting/revising themanuscript for intellectualcontentRobert W.Taylor, PhD,FRCPathWellcome Centre forMitochondrial Research,Translational and ClinicalResearch Institute,Newcastle University,Newcastle Upon Tyne,United KingdomMajor role in acquisition ofdata, Analysis/interpretation of data,Drafting/revising themanuscript for intellectualcontentContinuedNeurology.org/NG Neurology: Genetics | Volume 6, Number 2 | April 2020 3References1. Lott MT, Leipzig JN, Derbeneva O, et al. mtDNA variation and analysis usingmitomap and mitomaster. Curr Protoc Bioinformatics 2013;44:1–23.2. Yarham JW, Al-Dosary M, Blakely EL, et al. A comparative analysis approach to de-termining the pathogenicity of mitochondrial tRNA mutations. Hum Mutat 2011;32:1319–1325.3. Poole OV, Everett CM, Gandhi S, et al Adult-onset Leigh syndrome linked to thenovel stop codon mutation m. 6579G> A in MT-CO1. Mitochondrion 2019;47:294–297.4. Blakely EL, Yarham JW, Alston CL, et al. Pathogenic mitochondrial tRNA pointmutations: nine novel mutations affirm their importance as a cause of mitochondrialdisease. Hum Mutat 2013;34:1260–1268.5. Lehtonen MS, Meinilä M, Hassinen IE, Majamaa KJ. Haplotype-matched controls asa tool to discriminate polymorphisms from pathogenic mutations in mtDNA. HumGenet 1999;105:513–514.6. McFarland R, Elson JL, Taylor RW, Howell N, Turnbull DM. Assigning pathogenicityto mitochondrial tRNA mutations: when “definitely maybe”is not good enough.Trends Genet 2004;20:591–596.Appendix (continued)Name Location ContributionMichael G.Hanna, FRCPUCL Queen Square Instituteof Neurology and TheNational Hospital forNeurologyandNeurosurgery,London, United KingdomStudy concept and design,Analysis/interpretation ofdata, Revising themanuscript for intellectualcontentRobert D.S.Pitceathly,PhDUCL Queen SquareInstitute of Neurology andThe National Hospital forNeurology andNeurosurgery, London,United KingdomStudy concept and design,Analysis/interpretation ofdata, Revising themanuscript for intellectualcontent4 Neurology: Genetics | Volume 6, Number 2 | April 2020 Neurology.org/NGDOI 10.1212/NXG.00000000000004132020;6; Neurol Genet Olivia V. Poole, Alejandro Horga, Steven A. Hardy, et al. ) variantMT-TG (GlyMultisystem mitochondrial disease caused by a rare m.10038G>A mitochondrial tRNAThis information is current as of March 18, 2020reserved. Online ISSN: 2376-7839.Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.. All rightsan open-access, online-only, continuous publication journal. Copyright Copyright © 2020 The Author(s). is an official journal of the American Academy of Neurology. Published since April 2015, it isNeurol Genet ServicesUpdated Information & http://ng.neurology.org/content/6/2/e413.full.htmlincluding high resolution figures, can be found at:References http://ng.neurology.org/content/6/2/e413.full.html##ref-list-1This article cites 6 articles, 0 of which you can access for free at: Subspecialty Collections http://ng.neurology.org//cgi/collection/visual_lossVisual loss http://ng.neurology.org//cgi/collection/mitochondrial_disordersMitochondrial disorders http://ng.neurology.org//cgi/collection/metabolic_disease_inheritedMetabolic disease (inherited) http://ng.neurology.org//cgi/collection/endocrineEndocrine http://ng.neurology.org//cgi/collection/all_geneticsAll Geneticsfollowing collection(s): This article, along with others on similar topics, appears in the  Permissions & Licensing http://ng.neurology.org/misc/about.xhtml#permissionsits entirety can be found online at:Information about reproducing this article in parts (figures,tables) or in  Reprints http://ng.neurology.org/misc/addir.xhtml#reprintsusInformation about ordering reprints can be found online:reserved. 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Multisystem mitochondrial disease caused by a rare m.10038G>A mitochondrial tRNAGly (MT-TG) variant

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Multisystem mitochondrial disease caused by a rare m.10038G>A mitochondrial tRNAGly (MT-TG) variant

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