Dominant Optic Atrophy: How to Determine the Pathogenicity of Novel Variants?

Perspective Dominant Optic Atrophy: How to Determine the Pathogenicity of Novel Variants? Jason A. Zehden, PharmD, Subahari Raviskanthan, MBBS, Peter W. Mortensen, MD, Marc Ferré, MEng, PhD, Pascal Reynier, MD, PhD, Dan Milea, MD, PhD, Andrew G. Lee, MD A utosomal dominant optic atrophy (DOA, MIM #165500) is the most common inherited optic neuropathy (1). ADOA is typically characterized by progressive bilateral visual loss and color vision defects, classically beginning in early childhood (2). OPA1 is the most commonly mutated gene in DOA, and novel mutations are regularly described in addition to the already 450 (http://www.lovd. nl/OPA1; data as of March 10, 2021) distinct pathogenic reported OPA1 variants (2,3). Nonpathogenic variants of the OPA1 gene are common, (4) and therefore, establishing the responsibility of a newly discovered variant in a patient with otherwise unexplained optic neuropathy requires careful evaluation. The aim of this study is to describe the process of establishing true pathogenicity of a novel mutation, taking as an example the case of a patient with an unexplained optic neuropathy in a context of absent family history of a similar condition. A 15-year-old Asian adolescent girl presented to optometry at the age of 4, her parents reporting painless Baylor College of Medicine (JZ), Houston, Texas, US; Department of Ophthalmology (SR, PWM, AGL), Blanton Eye Institute, Houston Methodist Hospital, Houston, Texas; MITOVASC Institute (MF, PR, DM), CNRS 6015, INSERM U1083, University of Angers, France; Singapore National Eye Center (DM), Singapore, Singapore; Singapore Eye Research Institute (DM), Singapore, Singapore; Duke-NUS Medical School (DM), Singapore, Singapore; Copenhagen University Hospital Denmark (DM), Copenhagen, Denmark; Departments of Ophthalmology (AGL), Neurology, and Neurosurgery, Weill Cornell Medicine, New York, New York; Department of Ophthalmology (AGL), University of Texas Medical Branch, Galveston, Texas; University of Texas MD Anderson Cancer Center (AGL), Houston, Texas; Texas A and M College of Medicine (AGL), Bryan, Texas; and Department of Ophthalmology (AGL), The University of Iowa Hospitals and Clinics, Iowa City, Iowa. The authors report no conflicts of interest D. Milea and A. G. Lee contributed equally to this work. Publication Originality Statement. We confirm this publication is original. All named authors meet the International Committee of Medical Journal Editors (ICMJE) criteria for authorship for this article, take responsibility for the integrity of the work as a whole, and have given their approval for this version to be published. Address correspondence to Andrew G. Lee, MD, Department of Ophthalmology, Blanton Eye Institute, Houston Methodist Hospital, 6560 Fannin St. Ste 450 Houston, TX 77030; E-mail: aglee@ houstonmethodist.org Zehden et al: J Neuro-Ophthalmol 2022; 42: 149-153 progressive visual loss. At initial presentation, the patient held books close to her face and reported difficulty seeing pictures. She was diagnosed with astigmatism with superimposed myopia. At age 9, she presented to our neuroophthalmology clinic for further evaluation. She had no medical or surgical history, had normal nutrition, and was not taking any medications. There was no family history of low vision. On examination, her best-corrected visual acuity was 20/100 in both eyes (OU). The pupils were isocoric with no evidence of a relative afferent pupillary defect. External examination and slit-lamp examinations were normal. Motility was full. Dilated fundus examination showed bilateral nonglaucomatous cupping of the optic discs, with temporal pallor of the rim, and shallow saucerization of the cup (Figs. 1A and B). Automated visual fields showed a central scotoma in both eyes. Optical computerized tomography (OCT) of the retinal nerve fiber layer showed advanced thinning, with an average thickness of 71 mm in the right eye and 70 mm in the left eye, with papillomacular bundle dropout (Fig. 1C). MRI of the brain was normal. Testing for OPA1 revealed a novel heterozygous variant, the duplication of an A nucleotide c.1974dup (RefSeq: NM_015560.2) leading to frameshift with premature truncation p.(Gln659Thrfs*4), with no copy number abnormalities detected, raising the question of possible diagnosis of DOA. A full 3-generation pedigree was collected, disclosing no consanguinity and no known family history of optic atrophy, deafness, myopathy or neuropathy, intellectual disability, or learning disabilities. On further molecular testing, the patient’s father was confirmed as a healthy carrier of the same mutation; his neuro-ophthalmic examination was within normal limits. DISCUSSION Establishing firm pathogenicity of a novel mutation in the OPA1 gene in a patient with unexplained optic atrophy requires a systematic approach and may not be straightforward. In most cases, the molecular diagnosis is simple, especially if the mutation has been previously reported and if there is a family history of known or suspected autosomal dominant inherited optic neuropathy. Indeed, DOA, also 149 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Perspective FIG. 1. A, B. Optic disc photographs of the right eye (A) and left eye (B) at the patient’s most recent review showing nonglaucomatous cupping of the optic disc, with temporal pallor of the rim, and shallow saucerization of the cup. (C) Optical coherence tomography showing thinning of the retinal nerve fiber layer, primarily in the papillomacular fibers of the optic disc in both eyes. The average retinal nerve fiber layer thickness is 71 mm in the right eye and 70 mm in the left eye. known as optic atrophy type 1 or Kjer-type optic atrophy, is the most common hereditary optic neuropathy, inherited in an autosomal dominant manner, with a prevalence of 1:50,000 in most populations (1). Penetrance varies from one family to another and between pathogenic variants, ranging from 43% to 100% (5). Clinical severity also varies between and within families despite having the same muta150 tion, which implies that genetic background, epigenetic, and environmental factors may modulate the expression of the disease (2). About 70% of patients with DOA have pathogenic mutations in OPA1. (2) The locus for OPA1 maps to 3q28-q29 (1). OPA1-linked DOA is due to mutations in a mitochondrial dynamin-related GTPase that plays a role in maintenance of mitochondrial structure and DNA Zehden et al: J Neuro-Ophthalmol 2022; 42: 149-153 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Perspective (1). The key sign of OPA1-linked DOA is symmetric optic atrophy most commonly optic disc pallor temporally, which represents loss of the nerve fibers of the papillomacular bundle and sparing of the peripheral retina (1). OCT has been shown to be highly sensitive in diagnosis of DOA; patients with DOA have markedly reduced perifoveal retinal ganglion cell‒inner plexiform layer thickness compared with mutation-free relatives (6). The spectrum of severity in DOA varies, with visual acuity ranging from 20/20 to light perception; approximately 40% of patients have visual acuity better than 20/60 (2). Optic atrophy is the defining feature of OPA1-linked DOA, but multisystem manifestations, known as DOA-plus, are reported in up to 20% of cases (2,5,7). Although there is no established treatment for DOA, transcriptonomic techniques will likely improve the diagnosis of DOA and help better understand pathological processes that contribute to RGC loss (8). This improved understanding will then be used to develop targeted therapies focused on improving RGC survival (8). The reported pathogenic OPA1 variants include 28% missense variants, 24% induce aberrant splicing, 22% frameshift variants, 15% nonsense variants, and 7% structural variants; most pathogenic variants result in premature termination of translation, leading to null alleles (2). Haploinsufficiency is believed to be the predominant mechanism underlying OPA1-linked DOA (2). Our case is the first report of the pathogenic variant c.1974dup due to the duplication of a A nucleotide at position 1974 of the coding sequence (RefSeq: NM_015560.2), whose protein consequence is p.(Gln659Thrfs*4), a frameshift with premature truncation. As with most other OPA1 cases, this pathogenic variant results in loss of function of one allele. The fact that the patient’s father was positive for the same variant yet did not display symptoms of DOA provides a further reminder that genetic background, epigenetic, and environmental factors modify disease expression. However, establishing the pathogenicity of a mutation is challenging and our case was no exception. The classical criteria for interpreting newly described variants are mainly based on the molecular nature of the variants and on their segregation with the phenotype within families. Here, the nature of the variant is strongly in favor of the pathogenicity because it impairs the synthesis of a complete protein, which leads to haploinsufficiency (lack of expression of one of the 2 alleles), the main pathogenic mechanism of this disease. The presented case was challenging for several reasons: (1) There was no family history of visual loss, (2) DOA is not commonly described in Asian populations (9), and (3) family segregation was not contributory, despite the fact that the father was a carrier of the mutation. However, the intrafamilial variability of the clinical expression is important in this disease, explaining why not all the mutation carriers are clinically affected. Recently, various tools for predicting the pathogenicity of variants have emerged, based on the same molecular criteria Zehden et al: J Neuro-Ophthalmol 2022; 42: 149-153 mentioned above, to already described cases and on the large number of already sequenced genomes worldwide (Table 1). Nomenclature (Consistent and Unambiguous Description) The genetic diagnosis critically depends on accurate, standardized description and sharing of the variants detected. The version 2.0 nomenclature of the Human Genome Variation Society (HGVS) is the gold standard (http://varnomen.hgvs.org): The numbering of the nucleotides reflects that of the cDNA, with “+1” corresponding to the “A” of the ATG translation initiation codon in the reference sequence, according to which the initiation codon is codon 1 (10). OPA1 variants are described according to the OPA1 transcript variant 8 (RefSeq: NM_130837.2), representing the longest transcript. However, it is common to find in the literature the transcript variant 1 (RefSeq: NM_015560.2), the original transcript identified based on an alternate splice pattern characterized by Delettre et al (11), in which the 2 exons 4b and 5b are missing. However, it maintains the same reading frame. Databases (Knowledge Repositories) One of the major criteria for attributing pathogenicity to a variant is its frequency within populations, a pathogenic variant being generally very rarely represented. The so-called locus-specific databases (LSDBs) have proved to be the most complete for reporting pathogenic variations (12) because they benefit from the participation of a curator who is a referent specialist for the gene or disease considered. These databases are often based on isolated initiatives, using various interfaces hosted on different servers, rendering their interoperability and intuitive use rather difficult. The HGVS maintains the LSDB list up to date (https://www. hgvs.org/locus-specific-mutation-databases). For example, there is one database maintained up to date for OPA1 (http://www.LOVD.nl/OPA1) (3,13). On the contrary, variants that are frequently identified in general populations are rarely pathogenic. For example, the Genome Aggregation Database is the aggregation of the highquality exome (protein-coding region) or whole-genome DNA sequence data for about 140,000 individuals (14). Interspecies Conservation (In Silico Prediction) The comparative study of the homologous proteins between different species, as far as possible in evolution, is a powerful means of predicting the pathogenic or nonpathogenic character of a mutation, including if it is not listed in the databases or that its frequency does not allow to decide. Indeed, this prediction capacity is based on the fact that the more a sequence is conserved during evolution, the more its change is likely to be deleterious, while a nonconserved sequence more easily tolerates nonpathogenic variations. 151 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Perspective TABLE 1. Summary of ACMG recommendations suggested resources and results for the new mutation reported in this article Nomenclature HGVS variant nomenclature v2.0(10) (http://varnomen.hgvs.org) Literature mining NCBI PubMed search tool (22) (https://pubmed.gov) Disease-oriented database: OPA1 LSDB (3) (http://www.lovd.nl/OPA1) Population database: gnomAD (14) (https://gnomad.broadinstitute.org/gene/ ENSG00000198836?dataset=gnomad_r3) Missense variants: 0SIFT (18) (https://sift.bii.a-star.edu.sg) PolyPhen-2 (19) (http://genetics.bwh.harvard.edu/pph2/) MutationTaster2 (20) (http://www.mutationtaster.org) VarSome (21) (https://varsome.com) ClinGen (24) (https://search.clinicalgenome.org/kb/ genes/HGNC:8140) Databases Computational (in silico) predictive programs Integrative methods (most advanced method) High-quality clinical relevance of variants by expert consortia NM_015560.2:c.1974dup (transcript variant 1, “historical”) NM_130837.2:c.2139dup (transcript variant 8, “most complete”) Absent from the literature (at the time of its discovery) Absent in population nor disease Useless/Not applicable: obvious pathogenic prediction predicted (duplication of one nucleotide leading to a premature truncation of the protein) Verdict: Pathogenic In progress (nothing available to date) gnomAD, Genome Aggregation Database. Historically, it was based on both protein sequence databases (such as Swiss-Prot (15)) and the software for local search and alignment (such as BLAST (16)) and multiple alignment (such as Clustal (17)), which are certainly the most famous in bioinformatics. Currently, specialized tools greatly enhance the results, particularly the differences in size of the aligned proteins. It is generally recommended that multiple different methods are used to compare results, to allow stronger confidence in predictions when replicated in different models. The state-of-the-art models generally use SIFT (18), PolyPhen-2 (19), and MutationTaster2 (20). Transgenic animal models, incorporating newly described mutations, are additional methods to improve the understanding of their pathogenicity. Aggregators (Integrative Methods) The above bioinformatics resources now tend to be aggregated by algorithms taking into account all the available knowledge and the in silico predictions, integrating them in a weighted manner using artificial intelligence methods. In particular, VarSome (21) is widely used by molecular biologists to interpret the pathogenicity of the variants they identify in patients. In our case, VarSome unambiguously classified the c.1974dup variant as pathogenic. It should be noted that 152 despite the power of these algorithms, the molecular biologist is often confronted with variants classified as VUS (variant of uncertain pathogenicity) for which it is difficult to provide a definitive significance. In this case, the use of the LSDB of the gene or a review of the recent literature (22) can provide significantly updated results that help refine an ambiguous status. Finally, additional tests, exploring the functionality of the mutated protein, can help to establish the pathogenicity of a variant. In the case of OPA1, impaired activity of the mitochondrial respiratory chain or a fragmented mitochondrial network in the fibroblasts of patients is in favor of the pathogenicity. The Near Future of the Variants Interpretation The 2015 report from the American College of Medical Genetics and Genomics (ACMG) provides updated recommendations for the reporting and interpretation of sequence variants for Mendelian disorders in a clinical context (23). These recommendations now reach a level of expertise that addresses them to specialists in each gene, and they far exceed the very fundamental and voluntarily simple description we have made above, to achieve a very high reliability of the status of each variant compatible with molecular diagnosis. For this, the Optic Nerve Atrophy Variant Curation Expert Panel within the Clinical Genome Resource (ClinGen) consortium is currently defining the specific and extremely detailed criteria Zehden et al: J Neuro-Ophthalmol 2022; 42: 149-153 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. Perspective to standardize the status of variants (https://www. clinicalgenome.org/affiliation/50090/) (24). In conclusion, establishing firm pathogenicity of genetic variants in the OPA1 gene is challenging, due the current growing number of identified variants, pathogenic or not. Novel powerful predictive algorithms, in conjunction with comparisons with previously accumulated data are increasingly allowing to determine pathogenicity of a newly described variant in ADOA 11. 12. 13. 14. STATEMENT OF AUTHORSHIP Category 1: a. Conception and design: J. Zehden, S. Raviskanthan, P. W. Mortensen, D. Milea, and A. G. Lee; b. Acquisition of data: J. Zehden, S. Raviskanthan, P. W. Mortensen, D. Milea, and A. G. Lee; c. Analysis and interpretation of data: J. Zehden, S. Raviskanthan, P. W. Mortensen, M. Ferré, P. Reynier, D. Milea, and A. G. Lee. Category 2: a. Drafting the manuscript: J. Zehden, S. Raviskanthan, P. W. Mortensen, D. Milea, and A. G. Lee; b. Revising it for intellectual content: J. Zehden, S. Raviskanthan, P. W. Mortensen, M. Ferré, P. Reynier, D. Milea, and A. G. Lee. Category 3: a. Final approval of the completed manuscript: J. Zehden, S. Raviskanthan, P. W. Mortensen, M. Ferré, P. Reynier, D. Milea, and A. G. Lee. 15. REFERENCES 1. Delettre C, Lenaers G, Pelloquin L, Belenguer P, Hamel CP. OPA1 (kjer type) dominant optic atrophy: a novel mitochondrial disease. Mol Genet Metab. 2002;75:97–107. 2. Lenaers G, Neutzner A, Le Dantec Y, Jüschke C, Xiao T, Decembrini S, Swirski S, Kieninger S, Agca C, Kim US, Reynier P, Yu-Wai-Man P, Neidhardt J, Wissinger B. Dominant optic atrophy: culprit mitochondria in the optic nerve. Prog Retin Eye Res. 2020:100935. doi: 10.1016/ j.preteyeres.2020.100935. (epub ahead of print). 3. Le Roux B, Lenaers G, Zanlonghi X, Amati-Bonneau P, Chabrun F, Foulonneau T, Caignard A, Leruez S, Gohier P, Procaccio V, Milea D, den Dunnen JT, Reynier P, Ferré M. OPA1: 516 unique variants and 831 patients registered in an updated centralized variome database. Orphanet J Rare Dis. 2019;14:214. 4. van de Warrenburg BP, Schouten MI, de Bot ST, Vermeer S, Meijer R, Pennings M, Gilissen C, Willemsen MA, Scheffer H, Kamsteeg EJ. Clinical exome sequencing for cerebellar ataxia and spastic paraplegia uncovers novel gene-disease associations and unanticipated rare disorders. Eur J Hum Genet. 2016;24:1460–1466. 5. Lenaers G, Hamel C, Delettre C, Amati-Bonneau P, Procaccio V, Bonneau D, Reynier P, Milea D. Dominant optic atrophy. Orphanet J Rare Dis. 2012;7:46. 6. Rönnbäck C, Milea D, Larsen M. Imaging of the macula indicates early completion of structural deficit in autosomaldominant optic atrophy. Ophthalmology. 2013;120:2672– 2677. 7. Leruez S, Milea D, Defoort-Dhellemmes S, Colin E, Crochet M, Procaccio V, Ferré M, Lamblin J, Drouin V, Vincent-Delorme C, Lenaers G, Hamel C, Blanchet C, Juul G, Larsen M, Verny C, Reynier P, Amati-Bonneau P, Bonneau D. Sensorineural hearing loss in OPA1-linked disorders. Brain. 2013;136:e236. 8. Gilhooley MJ, Owen N, Moosajee M, Yu Wai Man P. From transcriptomics to treatment in inherited optic neuropathies. Genes (Basel). 2021;12:147. 9. Loo JL, Singhal S, Rukmini AV, Tow S, Amati-Bonneau P, Procaccio V, Bonneau D, Gooley JJ, Reynier P, Ferré M, Milea D. Multiethnic involvement in autosomal-dominant optic atrophy in Singapore. Eye (Lond). 2017;31:475–480. 10. den Dunnen JT, Dalgleish R, Maglott DR, Hart RK, Greenblatt MS, McGowan-Jordan J, Roux AF, Smith T, Antonarakis SE, Taschner Zehden et al: J Neuro-Ophthalmol 2022; 42: 149-153 16. 17. 18. 19. 20. 21. 22. 23. 24. PE. HGVS recommendations for the description of sequence variants: 2016 update. Hum Mutat. 2016;37:564–569. Delettre C, Griffoin JM, Kaplan J, Dollfus H, Lorenz B, Faivre L, Lenaers G, Belenguer P, Hamel CP. Mutation spectrum and splicing variants in the OPA1 gene. Hum Genet. 2001;109:584–591. Brookes AJ, Robinson PN. Human genotype-phenotype databases: aims, challenges and opportunities. Nat Rev Genet. 2015;16:702–715. Ferre M, Amati-Bonneau P, Tourmen Y, Malthiery Y, Reynier P. eOPA1: an online database for OPA1 mutations. Hum Mutat. 2005;25:423–428. Karczewski KJ, Francioli LC, Tiao G, Cummings BB, Alföldi J, Wang Q, Collins RL, Laricchia KM, Ganna A, Birnbaum DP, Gauthier LD, Brand H, Solomonson M, Watts NA, Rhodes D, Singer-Berk M, England EM, Seaby EG, Kosmicki JA, Walters RK, Tashman K, Farjoun Y, Banks E, Poterba T, Wang A, Seed C, Whiffin N, Chong JX, Samocha KE, Pierce-Hoffman E, Zappala Z, O’Donnell-Luria AH, Minikel EV, Weisburd B, Lek M, Ware JS, Vittal C, Armean IM, Bergelson L, Cibulskis K, Connolly KM, Covarrubias M, Donnelly S, Ferriera S, Gabriel S, Gentry J, Gupta N, Jeandet T, Kaplan D, Llanwarne C, Munshi R, Novod S, Petrillo N, Roazen D, Ruano-Rubio V, Saltzman A, Schleicher M, Soto J, Tibbetts K, Tolonen C, Wade G, Talkowski ME. Genome Aggregation Database Consortium, Neale BM, Daly MJ, MacArthur DG. The mutational constraint spectrum quantified from variation in 141,456 humans. Nature. 2020;581:434–443. Consortium UniProt. UniProt: the universal protein knowledgebase in 2021. Nucleic Acids Res. 2021;49:D480– D489. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403–410. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J, Thompson JD, Higgins DG. Fast, scalable generation of high-quality protein multiple sequence alignments using clustal omega. Mol Syst Biol. 2011;7:539. Sim NL, Kumar P, Hu J, Henikoff S, Schneider G, Ng PC. SIFT web server: predicting effects of amino acid substitutions on proteins. Nucleic Acids Res. 2012;40:W452–W457. Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, Kondrashov AS, Sunyaev SR. A method and server for predicting damaging missense mutations. Nat Methods. 2010;7:248–249. Schwarz JM, Cooper DN, Schuelke M, Seelow D. MutationTaster2: mutation prediction for the deep-sequencing age. Nat Methods. 2014;11:361–362. Kopanos C, Tsiolkas V, Kouris A, Chapple CE, Albarca Aguilera M, Meyer R, Massouras A. VarSome: the human genomic variant search engine. Bioinformatics. 2019;35:1978–1980. Sayers EW, Barrett T, Benson DA, Bolton E, Bryant SH, Canese K, Chetvernin V, Church DM, Dicuccio M, Federhen S, Feolo M, Geer LY, Helmberg W, Kapustin Y, Landsman D, Lipman DJ, Lu Z, Madden TL, Madej T, Maglott DR, Marchler-Bauer A, Miller V, Mizrachi I, Ostell J, Panchenko A, Pruitt KD, Schuler GD, Sequeira E, Sherry ST, Shumway M, Sirotkin K, Slotta D, Souvorov A, Starchenko G, Tatusova TA, Wagner L, Wang Y, John Wilbur W, Yaschenko E, Ye J. Database resources of the national center for biotechnology information. Nucleic Acids Res. 2010;38:D5–D16. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, Grody WW, Hegde M, Lyon E, Spector E, Voelkerding K, Rehm HL; ACMG Laboratory Quality Assurance Committee. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the american college of medical genetics and genomics and the association for molecular pathology. Genet Med. 2015;17:405–424. Rehm HL, Berg JS, Brooks LD, Bustamante CD, Evans JP, Landrum MJ, Ledbetter DH, Maglott DR, Martin CL, Nussbaum RL, Plon SE, Ramos EM, Sherry ST, Watson MS, ClinGen. ClinGen–the clinical genome resource. N Engl J Med. 2015;372:2235–2242. 153 Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited.