Optic atrophy plus phenotype due to mutations in the OPA1 gene:
Two more Italian families
Michela Ranieria, Roberto Del Boa, Andreina Bordonia, Dario Ronchia, Irene Colomboa, Giulietta Riboldia,
Alessandra Cosia, Maura Servidaa, Francesca Magria, Maurizio Moggioa, Nereo Bresolina,b,c,
Giacomo P. Comia,b, Stefania Cortia,b,⁎
aDino Ferrari Centre, Department of Neurological Sciences, University of Milan, IRCCS Foundation Ca' Granda, Ospedale Maggiore Policlinico, Milan, Italy
bCentre of Excellence in Neurodegenerative Diseases, University of Milan, Milan, Italy
cIRCCS Eugenio Medea, Bosisio Parini, Lecco, Italy
a b s t r a c t a r t i c l ei n f o
Received 22 June 2011
Received in revised form 24 November 2011
Accepted 2 December 2011
Available online 22 December 2011
Autosomal Dominant Optic Atrophy
Optic Atrophy 1 gene
Autosomal Dominant Optic Atrophy (ADOA) is characterized by the selective degeneration of retinal ganglion
cells. The occurrence of mutations in the gene encoding the dynamin-like GTPase protein Optic Atrophy 1
(OPA1) has been observed in about 60–70% of ADOA cases. A subset of missense mutations, mostly within
the GTPase domain, has recently been associated with a syndromic ADOA form called “OPA1 plus” phenotype
presenting, at muscle level, mitochondrial DNA (mtDNA) instability.
In this study we disclosed two OPA1 gene mutations in independent probands from two families affected
by OPA1 plus phenotype: the previously reported c.985-2A>G substitution and a novel microdeletion
The correlation between genotype and phenotype and the effects of these variants at the transcript level and
in the muscle tissue were investigated, confirming the broad complexity in the phenotypic spectrum associ-
ated with these OPA1 mutations.
© 2011 Elsevier B.V. All rights reserved.
AutosomalDominantOptic Atrophy (ADOA) is due in about 60–70%
mapping to chromosome 3q28-29. ADOA is the most common form of
hereditaryoptic neuropathy,witha prevalenceof1/50,000[1,2].Classi-
cal ADOA usually begins before 10 years of age with slowly progressive
ral optic disc atrophy [3,4]. The disease has an incomplete penetrance
and variable phenotypic expression, ranging from mild visual impair-
ment to blindness . Up to 20% of OPA1-mutated patients also devel-
op, during clinical history, additional neuromuscular complications
leading to the so-called “OPA1 plus” phenotype [6,7].
More than200 pathogenic mutationsin theOPA1 genehave beenso
far described . Half of these variants are predicted to result in a trun-
cated protein producing haploinsufficiency and are usually associated
to the classical non-syndromic form of optic neuropathy. Missense mu-
tations within or closeto the GTPase domain,preservingthe expression
of OPA1 transcript are responsible for both the classical and syndromic
The present study further extends the mutational spectrum of
OPA1 with the report of a novel heterozygous deletion within the
GTPase effector domain (GED). We also confirmed a previously pub-
lished mutation in an Italian family suggesting the existence of a mu-
tational hot spot in OPA1 sequence sited in the surroundings of exon
10 splice acceptor site.
2. Material and methods
2.1. Case reports
2.1.1. Family 1
The proband is an adult Italian male with a clinical history charac-
terized by visual impairment since childhood.
He came to our attention at 48 years of age, complaining of general-
ized fatigue and progressive visual loss since childhood. Neurological
examination showed a mild bilateral ptosis and ophthalmoparesis; he
also presented pes cavus on the left side with a decreased/absent achil-
les tendon reflex bilaterally. Visual field revealed a peripheral concen-
tric narrowing. Fundus oculi examination showed mild bilateral
temporal pallor of the optic disc.
Journal of the Neurological Sciences 315 (2012) 146–149
⁎ Corresponding author at: Department of Neurological Sciences, University of
Milan, IRCCS Foundation Ca' Granda Policlinico, Ospedale Maggiore Policlinico, Via
Francesco Sforza 35, 20122 Milan, Italy. Tel.: +39 0255033817; fax: +39 0250320430.
E-mail address: firstname.lastname@example.org (S. Corti).
0022-510X/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
Contents lists available at SciVerse ScienceDirect
Journal of the Neurological Sciences
journal homepage: www.elsevier.com/locate/jns
He underwent ocular computerized tomography, which disclosed
a thinning of the peripapillar bundle nervous layers in both superior
and inferior temporal quadrant bilaterally.
His father, a paternal aunt and her two sons were also affected by
visual loss starting in childhood (Fig. 1A).
Brachial biceps muscle biopsy showed at the histochemical inves-
tigation three cytochrome c oxidase (COX) negative fibers; Succinate
Dehydrogenase (SDH) activity was normal. The sequential applica-
tion of these two reactions to the muscle section revealed abnormal
COX deficient fibers appearing blue.
2.1.2. Family 2
consanguineous parents. A 15-year-old female patient was affected
by a slowly progressive visual loss with an onset at the age of 11.
Her current visual acuity is 7/10 on the right eye and 6/10 on the
She underwent visual evoked potentials (VEPs), which showed a
pattern characterized by increased latencies of cortical responses,
with a moderate signal dispersion. Fundus examination disclosed
mild temporal pallor of the optic disc bilaterally.
Her brother is an 11-year-old boy who developed a progressive vi-
sual loss at the age of 10 years with a current visual acuity of 4/10
Fig. 1. Pedigrees of the described families. Black symbols indicate affected subjects. The
described probands are indicated by arrows.
Fig. 2. (A) PCR analysis of muscle-derived mtDNA showing the presence of multiple deleted mitochondrial genomes in Patient 1. Controls are age-matched muscle biopsies.
(B) Sequence analysis of OPA1 gene in Patient 1 disclosing the microdeletion c.2819-1_2921del at genomic and transcript level. PCR fragments obtained using genomic DNA as
a template were subcloned to discriminate between mutated and wildtype alleles. (C) Sequence analysis of OPA1 gene in Family 2 discloses the point mutation c.985-2A>G in
affected members. This substitution results in the skipping of exon 10 as showed by electrophoresis on agarose gel of RT-PCR fragments and sequence analysis of cDNA clones.
M. Ranieri et al. / Journal of the Neurological Sciences 315 (2012) 146–149
No other pathological signs were found at their neurological
Their mother suffered from poor vision and died at the age of 38
for breast cancer. A 41-year old maternal aunt has presented a deteri-
oration of visual acuity since childhood; current visual acuity is 2/10
bilaterally; a mild sensorineural hearing loss was observed in the
last years (Fig. 1B).
Muscle specimens from probands of Family 2 were not available,
since diagnosis was performed at the genetic level.
3. Molecular analysis
Total DNA was isolated from muscle and peripheral blood accord-
ing to the standard protocols. Southern blot and long-range polymer-
ase chain reaction analysis of muscle-derived mtDNA were performed
The following mitochondrial DNA variants, previously associated
to LHON (Leber Hereditary Optic Neuropathy) were ruled out by
PCR-RFLP and sequence analysis: m.11778G>A, m.13708G>A,
m.3460G>A, m.3994T>C, m.15812G>A, m.15257G>A, m.7444G>A,
m.5244G>A, m.14884T>C, m.14459G>A, m.14596A>T. All OPA1
exons and at least 30 bp of flanking intronic sequences were amplified
by PCR; fragments were purified and directly sequenced using BigDye
Terminator protocol on an automated 3100 ABI Prism Genetic Analyzer
Total RNA was obtained from skeletal muscle (proband, Family1)
or peripheral blood mononuclear cells (probands and healthy sub-
jects, Family 2), according to standard procedures and reverse-
transcribed. RT-PCR fragments were purified and directly sequenced.
PCR products carrying the mutations were subcloned into TOPO-TA
vector (Invitrogen). Nucleotide numbering of OPA1 gene mutations
reflects cDNA numbering with +1 corresponding to the A of the
ATG translation initiation codon of the GenBank reference sequence
NM_015560.2. The initiation codon is Met 1.
Long PCR analysis of muscle-derived mtDNA of Family 1 proband
disclosed several PCR products corresponding to mtDNA deletions.
Southern blot analysis of muscle mtDNA did not show multiple-
deleted genomes (Fig. 2A).
Sequence analysis of OPA1 gene showed two different mutations
in the affected probands.
Proband of Family 1 carried the 4 nucleotide-deletion c.2819-
1_2821del, involving the last nucleotide of intron 27 and the first
three nucleotides of exon 28 (Fig. 2B). This unreported rearrange-
ment results in the activation of an exonic cryptic donor site leading
(r.2819_2824del) and producing a microdeletion within the GED do-
main (p.Lys940_Val942delinsIle). This variant was not detected in
120 ethnic-matched control subjects.
Patients from Family 2 showed the heterozygous nucleotide sub-
stitution c.985-2A>G in intron 9, previously described with incom-
plete penetrance, in a Chinese pedigree . The same mutation
was also confirmed in DNA derived from the affected maternal aunt.
This mutation is associated with the in-frame skipping of exon 10,
as detected by mRNA analysis, resulting in the loss of 27 amino acids
within the GTPase domain (Fig. 2C).
skipping ofexon 28
OPA1 is a dynamin-like GTPase protein anchored to the mito-
chondrial inner membrane which controls the fusion of mitochondri-
mitochondrial network [1,12]. Up to now more than 200 pathogenic
mutations, along OPA1 coding region, have been associated to
physiological remodeling of
human disease. Beside optic atrophy, about 20% of patients bearing
OPA1 mutations also develop additional neuromuscular complica-
tions, mostly including deafness, progressive external ophthalmople-
gia and myopathy, starting from the third decade of life onwards
Our study further extends the mutational spectrum of OPA1 lead-
ing to the discovery of a novel mutation in a proband with a clinical
picture of syndromic optic atrophy. This patient harbors a microdele-
tion located within the GED sequence confirming the importance of
the integrity of this domain, responsible for the interaction between
OPA1 and its partners involved in mitochondrial fusion process. The
GED domain and its flanking regions in fact represent an OPA1 muta-
tional hotspot, since about 28% of mutations are located in this region.
These findings suggest that not only missense mutation but also in
frame-deletion preserving a terminal abnormal transcript, could
lead to the extraneurological features observed in OPA1-plus patients
Skeletal muscle analysis in our patient reveals mild mitochondrial
defects consisting in the presence of COX-negative fibers and the oc-
currence of multiple deletions in mtDNA as detected by PCR analysis.
The occurrence of these histological findings is frequently detected in
OPA1-mutated patients with a 4-to-1 ratio in OPA1-plus patients re-
spect to individuals with pure optic nerve involvement .
The mutation identified in Family 2 has been previously described
in a Chinese pedigree showing an incomplete penetrance . On the
contrary, in our family the c.985-2A>G mutation was associated with
an early-onset and a complete penetrance disease. The disclosure of
the same variant in the affected members of the Italian and Chinese
pedigrees supports its pathogenic nature, since it arose independent-
ly in independent genetic backgrounds. Whereas in Chinese family no
mutated subjects developed any additional extraocular symptom
even in late adulthood, a member of our family (the proband's
aunt) showed an early onset sensorineural hearing impairment.
In our probands, transcript analysis was fundamental to characterize
the effectof the genomic variants on OPA1 mRNA, but this tool is not al-
ways able to predict the resulting phenotype. In fact, in frame-deletions
also in a multisystemic disorder in the absence of optic atrophy .
Recently, multiplex ligation probe amplification (MLPA) assay has
allowed to detect OPA1 rearrangements in a large cohort of Danish
ADOA probands, revealing that heterozygous deletions involving
whole exons represent a remarkable proportion among OPA1 muta-
tions, ranging between 10% and 19% . These defects are usually
missed by standard sequencing methods which are not able to detect
large scale deletions as well as variants located within promoter or
In our opinion a combined strategy involving different techniques
applied to genomic and transcript analysis could offer the most valu-
able option to investigate the OPA1 defects underlining the several
forms of inherited optic neuropathy .
Thefinancialsupport of thefollowingresearchgrantisgratefullyac-
knowledged:Telethon-UILDMProjectGUP09004 “Constructionof ada-
tabase for a nation wide Italian collaborative network of mitochondrial
diseases”, Associazione Amici del Centro Dino Ferrari, University of
Milan, the Telethon project GTB07001, the Eurobiobank project QLTR-
2001-02769 and R.F. 02.187 Criobanca Automatizzata di Materiale
Conflict of interest
The authors report no competing interests.
M. Ranieri et al. / Journal of the Neurological Sciences 315 (2012) 146–149
Acknowledgments Download full-text
Gratitude has to be expressed to the patient for participating in
this research. We wish to thank especially the ‘Associazione Amici
del Centro Dino Ferrari’ for their support.
 Olichon A, Guillou E, Delettre C, Landes T, Arnauné-Pelloquin L, Emorine LJ, et al.
Mitochondrial dynamics and disease, OPA1. Biochim Biophys Acta May-Jun
 Cohn AC, Toomes C, Potter C, Towns KV, Hewitt AW, Inglehearn CF, et al. Autoso-
mal dominant optic atrophy: penetrance and expressivity in patients with OPA1
mutations. Am J Ophthalmol Apr 2007;143(4):656–62.
 Kerrison JB. Hereditary optic neuropathies. Ophthalmol Clin North Am. 2001
 Votruba M, Moore AT, Bhattacharya SS. Clinical features, molecular genetics, and
pathophysiology of dominant optic atrophy. J Med Genet. 1998. Oct;35(10):
 Pesch UE, Leo-Kottler B, Mayer S, Jurklies B, Kellner U, Apfelstedt-Sylla E, et al.
OPA1 mutations in patients with autosomal dominant optic atrophy and evidence
for semi-dominant inheritance. Hum Mol Genet Jun 15 2001;10(13):1359–68.
 Amati-Bonneau P, Valentino ML, Reynier P, Gallardo ME, Bornstein B, Boissière A,
et al. OPA1 mutations induce mitochondrial DNA instability and optic atrophy
‘plus’ phenotypes. Brain Feb 2008;131(Pt 2):338–51.
 Hudson G, Amati-Bonneau P, Blakely EL, Stewart JD, He L, Schaefer AM, et al. Mu-
tation of OPA1 causes dominant optic atrophy with external ophthalmoplegia,
ataxia, deafness and multiple mitochondrial DNA deletions: a novel disorder of
mtDNA maintenance. Brain Feb 2008;131(Pt 2):329–37.
 Ferré M, Amati-Bonneau P, Tourmen Y, Malthièry Y, Reynier P. eOPA1: an online
database for OPA1 mutations. Hum Mutat May 2005;25(5):423–8.
 ZevianiM,GelleraC,PannacciM,UzielG,PrelleA,ServideiS,etal.Tissue distribution
and transmission of mitochondrial DNA deletions inmitochondrial myopathies. Ann
 Moraes CT, Atencio DP, Oca-Cossio J, Diaz F. Techniques and pitfalls in the detec-
tion of pathogenic mitochondrial DNA mutations. J Mol Diagn 2003;5:197–208.
 Li Y, Deng T, Tong Y, Peng S, Dong B, He D. Identification of two novel OPA1 mu-
tations in Chinese families with autosomal dominant optic atrophy. Mol Vis
 Frezza C, Cipolat S, Martins de Brito O, Micaroni M, Beznoussenko GV, Rudka T,
et al. OPA1 controls apoptotic cristae remodeling independently from mitochon-
drial fusion. Cell Jul 14 2006;126(1):177–89.
 Amati-Bonneau P, Milea D, Bonneau D, Chevrollier A, Ferré M, Guillet V. OPA1-
associated disorders: phenotypes and pathophysiology. Int J Biochem Cell Biol
 Yu-Wai-Man P, Trenell MI, Hollingsworth KG, Griffiths PG, Chinnery PF. OPA1 mu-
tations impair mitochondrial function in both pure and complicated dominant
optic atrophy. Brain Apr 2011;134(Pt 4):e164.
 Milone M, Younge BR, Wang J, Zhang S, Wong LJ. Mitochondrial disorder with
OPA1 mutation lacking optic atrophy. Mitochondrion Jul 2009;9(4):279–81.
 Almind GJ, Grønskov K, Milea D, Larsen M, Brøndum-Nielsen K, Ek J. Genomic de-
letions in OPA1 in Danish patients with autosomal dominant optic atrophy. BMC
Med Genet Apr 4 2011;12(1):49.
 Yu-Wai-Man P, Griffiths PG, Hudson G, Chinnery PF. Inherited mitochondrial
optic neuropathies. J Med Genet 2009;46:145–58.
M. Ranieri et al. / Journal of the Neurological Sciences 315 (2012) 146–149