Weisschuh et al. BMC Medical Genetics(2020) 21:236
https://doi.org/10.1186/s12881-020-01166-z

CASE REPORTOpen AccessFirst submicroscopic inversion of the OPA1
gene identified in dominant optic atrophy– a case report
Nicole Weisschuh1*† , Pascale Mazzola2†, Tilman Heinrich2,3, Tobias Haack2,3, Bernd Wissinger1, Felix Tonagel4 andCarina Kelbsch4

Abstract
Background: Dominant optic atrophy (DOA) is an inherited optic neuropathy that mainly affects visual acuity,
central visual fields and color vision due to a progressive loss of retinal ganglion cells and their axons that form theoptic nerve. Approximately 45–90% of affected individuals with DOA harbor pathogenic variants in the OPA1 gene.
The mutation spectrum of OPA1 comprises nonsense, canonical and non-canonical splice site, frameshift andmissense as well as copy number variants, but intragenic inversions have not been reported so far.
Case presentation: We report a 33-year-old male with characteristic clinical features of DOA. Whole-genome
sequencing identified a structural variant of 2.4 kb comprising an inversion of 937 bp at the OPA1 locus. Fine
mapping of the breakpoints to single nucleotide level revealed that the structural variation was an inversion flanked
by two deletions. As this rearrangement inverts the entire first exon of OPA1, it was classified as likely pathogenic.
Conclusions: We report the first DOA case harboring an inversion in the OPA1 gene. Our study demonstrates that
copy-neutral genomic rearrangements have to be considered as a possible cause of disease in DOA cases.
Keywords: Case report, Inversion, Complex rearrangement, Dominant optic atrophy, OPA1, Non-homologous end
joining (NHEJ)

Background
Dominant optic atrophy (DOA, MIM#165500) and Leberʼshereditary optic neuropathy (LHON, MIM#535000) are thetwo most common entities of inherited optic neuropathies
seen in clinical practice [1]. DOA was first described in the
1950s and is genetically and clinically distinct from LHON
[2, 3]. Clinically, affected individuals with DOA present
with temporally accented pallor of the optic nerve head at
fundus examination, and bilateral central or caeco-central
scotoma at visual field examination. A laterally symmetricaltemporal reduced thickness of the retinal nerve fiber layer* Correspondence: nicole.weisschuh@uni-tuebingen.de†Nicole Weisschuh and Pascale Mazzola contributed equally to this work.
1
Institute for Ophthalmic Research, Centre for Ophthalmology, University of
Tübingen, Tübingen, Germany
Full list of author information is available at the end of the article(RNFL) is seen upon optical coherence tomography
imaging (OCT). Other than in affected individuals diagnosed with LHON, who often show red-green dyschromatopsia, the color vision defect in DOA reflects a generalized
dyschromatopsia or is specific to the tritan axis [4]. Visualacuity can range from 20/20 to light perception, with 40%
of affected individuals having a visual acuity over 20/60 [5].
Depending on the population studied, 45–90% of DOA
cases harbor pathogenic variants in OPA1 [6, 7], which
was the first gene to be described as an underlying causeof DOA [8, 9]. OPA1 encodes a dynamin-related GTPase
which is imported into mitochondria and plays an important role in mitochondrial dynamics and structural maintenance of the cristae junctions [10, 11]. As of August
2020, the Human Gene Mutation Database (HGMD) lists© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to thedata made available in this article, unless otherwise stated in a credit line to the data.

Weisschuh et al. BMC Medical Genetics(2020) 21:236

404 disease-causing variants in OPA1. Most variants are
private and only few constitute founder alleles [7, 12].
Overall, the majority of disease-causing variants are
loss-of-function alleles, indicating that haploinsufficiency is the predominant disease mechanism underlying OPA1-linked DOA [12, 13]. The mutation
spectrum comprises nonsense, canonical splice site,
frameshift and missense variants. In addition, copy
number variants (CNVs) and deep-intronic variants
inducing aberrant splicing have been reported [14–17],
necessitating a comprehensive genetic diagnostic testing including the analysis of read depth and intronic
sequences. So far, submicroscopic inversions in the
OPA1 gene have not yet been reported as a cause of
DOA. Since inversions are copy-neutral, they escapedetection by conventional diagnostic technologies suchas microarrays and read depth methods. We here
present the first affected individual with DOA in whom
whole genome sequencing based on short read technology led to the identification of a complex structural
rearrangement including the first coding exon of OPA1.
Our findings demonstrate that copy-neutral genomic
rearrangements have to be considered as a possible
cause of disease in DOA.

Case presentation
The 33-year-old male subject of German descent presented
with a history of bilateral slowly progressive vision loss. His
younger brother was reported to have “bad vision” but was
not available for phenotypic and genetic analysis. Parents
and grandparents were apparently unaffected. Cerebral
imaging (magnetic resonance imaging with contrast) had
not revealed any underlying cause, particularly no spaceoccupying lesions compromising the visual pathway.
Clinical investigation

At examination, best-corrected visual acuity was 0.5(right eye) and 0.4 (left eye). According to previous
eye examinations, visual acuity was documented toPage 2 of 6

have been 0.8 in both eyes 10 years ago with acontinuous slowly progressive decline.
Anterior segment slit-lamp examination was unremarkable. Intraocular eye pressure was within normal
limits, and no relative afferent pupillary defect was
found. Fundus ophthalmoscopy unveiled bilateral symmetric, temporally accented optic disc pallor, which is atypical and characteristic finding of DOA (Fig. 1). Correspondingly, the temporal optic atrophy could be confirmed by bilaterally reduced RNFL in OCT (Fig. 2).
Visual field examination of the central 30° of the visual
field was relatively unremarkable with only slight paracentral relative defects R > L (Additional file 1). Color
testing with Panel-D15 was significant for a dyschromatopsia to the tritan axis L > R (Additional file 2).
In summary, the affected individual presented withcharacteristic clinical findings consistent with a diagnosis
of DOA.
Genetic analysis

Genomic DNA was extracted from peripheral blood using
standard protocols. Whole genome sequencing (2 × 150 bp
paired-end reads) was performed on an Illumina platform(NovaSeq6000). The average coverage on target was 54.8x(99.76% > 20x). Bioinformatic processing of raw read data,
annotation and variant calling was performed as described
previously [18]. For details refer to the megSAP pipeline(https://github.com/imgag/megSAP) developed at theInstitute of Medical Genetics and Applied Genomics,
University Hospital Tübingen, Germany. Bioinformatic analysis and genomic coordinates given in this manuscript are
based on the GRCh37 genome (hg19). The Manta Structural
Variant Caller [19] identified a ~ 1 kB spanning inversion
that changes the orientation of the first coding exon of
OPA1. Manual inspection of split reads using the Integrative
Genomics Viewer (IGV, version 2.3, see Fig. 3) revealed acomplex rearrangement. Subsequent breakpoint PCR andSanger sequencing was performed in order to resolve thevariant configuration to single nucleotide resolution.

Fig. 1 Eye fundi. Temporal pallor of discs is seen in the right eye (a) and the left eye (b) of the affected proband

Weisschuh et al. BMC Medical Genetics(2020) 21:236

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Fig. 2 Optical coherence tomography (OCT) of the retinal nerve fiber layers (RNFLs). The right eye (a) and left eye (b) OCT of the affected
proband show temporal thinning of RNFLs

Figure 4a gives a schematic overview of the structuralvariant, and Fig. 4b shows the Sanger sequencing of the
breakpoint junctions. The nomenclature for the variant
according to the guidelines established by the Human
Genome Variation Society (HGVS) [20] was established asfollows: NC_000003.11:g.193,310,511_193,312,932delins193,
310,605_193,311,825[193,310,605_193,311,541inv].
It was not possible to obtain an RNA sample from theproband, excluding transcript analysis. However, since
the inversion comprises the first coding exon of the
OPA1 gene including the translational start site, it most
likely constitutes a loss-of-function allele.

Discussion and conclusions
This is the first report of a submicroscopic inversion involving the OPA1 gene. Inversions are mainly mediated by threemechanisms: While non-allelic homologous recombination(NAHR) and the fork stalling and template switching(FoSTeS) mechanism require homologous sequences at the
breakpoints, non-homologous end joining (NHEJ) is a repair
mechanism for double-strand breaks [21–24]. In the latter,the inverted sequence directly ligates to the breakpoint, independent of sequence homology. Using theonline tools Blast 2 Sequences (http://polyp.biochem.
uci.edu/blast/wblast2.html) and Repeat Masker (http://
www.repeatmasker.org), we analyzed the sequences
flanking the breakpoints for similarities to each otherand to repeat elements that can mediate genomic rearrangements (data not shown). The lack of sequencehomology at the breakpoints and the sequencing results of breakpoint junctions indicate that the structural variant described in this study most likely
involved multiple double-strand breaks and the rejoining of DNA fragments by NHEJ. Of note, we followedWeisschuh et al. BMC Medical Genetics(2020) 21:236

Page 4 of 6

Fig. 3 Integrative Genomics Viewer (IGV) screenshot of the genomic region containing the structural variant identified in the affected proband.
From top to bottom the read coverage track, the alignment track (reads colored by insert size and pair orientation and grouped by pairorientation) and the gene track are shown. Colored reads represent discordant reads with unexpected insert size and/or pair orientation whenaligned to the reference genome indicating the structural variant. Blue read pairs/lines represent read pairs with right-right pair orientation, tealreads represent read pairs with left-left pair orientation and red read pairs/lines represent read pairs with aberrant insert size. Split reads with softclipped bases span the breakpoints of the structural variant

Fig. 4 Fine mapping of the structural variant by sequencing of breakpoint junctions. a Schematic representation of the structural variant. The
color of the arrows indicates normal sequence (grey), inverted sequence (yellow), inserted sequence (blue) and deleted sequence (pink). b Sanger
sequencing of breakpoint amplicons. The vertical dashed lines highlight the 5´ and 3′ breakpoints. The background colors of theelectropherogram correspond to the scheme shown in (A). The deleted sequences are indicated by pink triangles. Note that the inserted
sequence is actually “leftover original sequence” between the deletion and inversion events

Weisschuh et al. BMC Medical Genetics(2020) 21:236

the guidelines of the HGVS when assigning the nomenclature to the variant. However, from a mechanistic point of view, the inserted sequence is basically“leftover original sequence” between the deletion andinversion events.
Screening for the structural variant in a cohort of 350
optic atrophy cases that had been previously tested negative
for single nucleotide variants and CNVs in OPA1 was
performed using patient-specific breakpoint PCRs (primer
pairs OPA1-Inv-BP1-f: 5′-tgctcagcactaggcatctg-3′ / OPA1Inv-BP1-r: 5′-ttctggacgcctctcaatct-3′, OPA1-Inv-BP2-f: 5′ggaacgggaagggctaaa-3′ / OPA1-Inv-BP2-r: 5′-tcgaatcaccgt
ctctgaca-3′, and OPA1-Inv-BP3-f: 5′-tgtcttctttttcttccatttccac-3′ / OPA1-Inv-BP3-r: 5′-atgcttatgctctcatctgttaggg-3′,
respectively). All patient-specific breakpoint PCRs were
performed as a duplex PCR reaction with primers that bindto the reference sequence and amplify exon 1 of OPA1
(OPA1-Ex1-F: 5′-actgagtacgggtgcctgtc-3′ / OPA1-Ex1-R:
5’gccagattagagcctgcactt-3′). PCR products were resolved on
a 2% agarose gel. No additional cases could be identified
among the 350 unsolved cases. The variant was also absent
in our internal cohort of 1400 genomes, and in the public
databases gnomAD, ClinVar and DECIPHER. The lack ofadditional cases indicates that the variant is private, and isin line with the proposed molecular mechanism, sincerecurrent genomic rearrangements are most often mediatedby NAHR, and not by NHEJ [25]. DNA samples were notavailable for familial co-segregation analysis. Hence, we
could not establish whether the rearrangement was a denovo event in our proband.
Compared to single nucleotide variants and small insertions and deletions, the mapping of structural variants(SVs) is much more challenging as they cover a large portion of a read or are even larger than the read length [26].
As a consequence, the importance of SVs in Mendeliandiseases is not well defined. In a recent comprehensive
study, an average of 27,622 SVs was identified per humangenome, including 156 inversions per genome, many ofwhich intersected with genomic regions associated withgenetic disease syndromes [27]. Whole genome sequencing detects SVs more reliably than exon-based
approaches since most of the functionally relevant junctions will have both ends contained within introns orintergenic regions. Given that sequencing costs are constantly decreasing, whole genome sequencing is commissioned into routine clinical care pathways more and more
frequently, offering the potential to detect SVs. Thirdgeneration sequencing technologies will probably further
improve the diagnostic sensitivity as long read lengths are
more likely to contain the whole SV (i.e. both breakpoints), providing less potential for error and facilitating
mapping. Future studies will show whether the feasibility
to detect copy-number neutral rearrangements can boost
the diagnostic rate in Mendelian disorders like DOA.

Page 5 of 6

Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s12881-020-01166-z.
Additional file 1: Figure 1. Results of perimetry.
Additional file 2: Figure 2. Results of panel D15 test.

Abbreviations
CNV: Copy number variant; DOA: Dominant optic atrophy; FoSTeS: Forkstalling and template switching; IGV: Integrative Genomics Viewer;
LHON: Leber’s hereditary optic neuropathy; NAHR: Non-allelic homologous
recombination; NHEJ: Non-homologous end joining; OCT: Optical coherence
tomography imaging; RNFL: Retinal nerve fiber layer; SV: Structural variant
Acknowledgements
We acknowledge support by the Deutsche Forschungsgemeinschaft and theOpen Access Publishing Fund of the University of Tübingen.
Authors’ contributions
CK and FT collected the clinical data. PM and TiH performed the sequencing
data analysis. NW performed the breakpoint analysis and wrote themanuscript. BW and ToH supervised the study and reviewed the data. All
authors read and approved the manuscript.
Funding
This study was in part funded by a grant of the Deutsche
Forschungsgemeinschaft (Wi1189/11–1) as part of a joint research project‘TreatOPON’ within the framework of the ERA-NET E-Rare 3 call. The funding
body had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript. Open Access funding enabled andorganized by Projekt DEAL.
Availability of data and materials
The data that support the findings of this study are available from theInstitute for Medical Genetics and Genomics (IMGAG) in Tübingen but
restrictions apply to the availability of these data, which were used underlicense for the current study, and so are not publicly available. Data are
however available from the authors upon reasonable request and withpermission of the IMGAG.
The datasets analysed during the current study are available at gnomAD
https://gnomad.broadinstitute.org/, ClinVar (https://www.ncbi.nlm.nih.gov/),
and DECIPHER (https://decipher.sanger.ac.uk/). The accession numbers usedin this study and obtained from the National Center for Biotechnology
Information (NCBI) include: NM_015560.2, and the NCBI37/hg19 assembly(https://www.ncbi.nlm.nih.gov/assembly/GCF_000001405.13/).
Ethics approval and consent to participate
DNA samples were recruited in accordance with the principles of the
Declaration of Helsinki and were obtained with written informed consent
accompanying the samples. The study was approved by the institutional
review board of the Ethics Committee of the University Hospital of Tübingen(study number 637/2017BO1, dated October 23, 2017).
Consent for publication
Written informed consent to publish clinical details was obtained from theproband.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Institute for Ophthalmic Research, Centre for Ophthalmology, University of
Tübingen, Tübingen, Germany. 2Institute of Medical Genetics and Applied
Genomics, University of Tübingen, Tübingen, Germany. 3Centre for Rare
Diseases, University of Tübingen, Tübingen, Germany. 4University Eye
Hospital, Centre for Ophthalmology, University of Tübingen, Tübingen,Germany.

Weisschuh et al. BMC Medical Genetics(2020) 21:236

Received: 14 August 2020 Accepted: 10 November 2020

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