Solving a 50 year mystery of a missing OPA1 mutation: more insights from the first family diagnosed with autosomal dominant optic atrophy.

Abstract

Up to the 1950s, there was an ongoing debate about the diversity of hereditary optic neuropathies, in particular as to whether all inherited optic atrophies can be ascribed to Leber's hereditary optic neuropathy (LHON) or represent different disease entities. In 1954 W. Jaeger published a detailed clinical and genealogical investigation of a large family with explicit autosomal dominant segregation of optic atrophy thus proving the existence of a discrete disease different from LHON, which is nowadays known as autosomal dominant optic atrophy (ADOA). Since the year 2000 ADOA is associated with genomic mutations in the OPA1 gene, which codes for a protein that is imported into mitochondria where it is required for mitochondrial fusion. Interestingly enough, the underlying mutation in this family has not been identified since then.
We have reinvestigated this family with the aim to identify the mutation and to further clarify the underlying pathomechanism. Patients showed a classical non-syndromic ADOA. The long term deterioration in vision in the two teenagers examined 50 years later is of particular note 5/20 to 6/120. Multiplex ligation probe amplification revealed a duplication of the OPA1 exons 7-9 which was confirmed by long distance PCR and cDNA analysis, resulting in an in-frame duplication of 102 amino acids. Segregation was verified in 53 available members of the updated pedigree and a penetrance of 88% was calculated. Fibroblast cultures from skin biopsies were established to assess the mitochondrial network integrity and to qualitatively and quantitatively study the consequences of the mutation on transcript and protein level. Fibroblast cultures demonstrated a fragmented mitochondrial network. Processing of the OPA1 protein was altered. There was no correlation of the OPA1 transcript levels and the OPA1 protein levels in the fibroblasts. Intriguingly an overall decrease of mitochondrial proteins was observed in patients' fibroblasts, while the OPA1 transcript levels were elevated.
The thorough study of this family provides a detailed clinical picture accompanied by a molecular investigation of patients' fibroblasts. Our data show a classic OPA1-associated non-syndromic ADOA segregating in this family. Cell biological findings suggest that OPA1 is regulated by post-translational mechanisms and we would like to hypothesize that loss of OPA1 function might lead to impaired mitochondrial quality control. With the clinical, genetic and cell biological characterisation of a family described already more than 50 years ago, we span more than half a century of research in optic neuropathies.

Full text

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Open Access
RESEARCH ARTICLE
Solving a 50 year mystery of a missing OPA1
mutation: more insights from the first family
diagnosed with autosomal dominant optic atrophy
BioMed Central
© 2010 Fuhrmann et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Com-
mons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduc-
tion in any medium, provided the original work is properly cited.
Research article
Nico Fuhrmann1, Simone Schimpf1, York Kamenisch2, Beate Leo-Kottler3, Christiane Alexander4, Georg Auburger5,
Eberhart Zrenner3, Bernd Wissinger1 and Marcel V Alavi*1
Abstract
Background: Up to the 1950s, there was an ongoing debate about the diversity of hereditary optic neuropathies, in
particular as to whether all inherited optic atrophies can be ascribed to Leber's hereditary optic neuropathy (LHON) or
represent different disease entities. In 1954 W. Jaeger published a detailed clinical and genealogical investigation of a
large family with explicit autosomal dominant segregation of optic atrophy thus proving the existence of a discrete
disease different from LHON, which is nowadays known as autosomal dominant optic atrophy (ADOA). Since the year
2000 ADOA is associated with genomic mutations in the OPA1 gene, which codes for a protein that is imported into
mitochondria where it is required for mitochondrial fusion. Interestingly enough, the underlying mutation in this family
has not been identified since then.
Results: We have reinvestigated this family with the aim to identify the mutation and to further clarify the underlying
pathomechanism. Patients showed a classical non-syndromic ADOA. The long term deterioration in vision in the two
teenagers examined 50 years later is of particular note 5/20 to 6/120. Multiplex ligation probe amplification revealed a
duplication of the OPA1 exons 7-9 which was confirmed by long distance PCR and cDNA analysis, resulting in an in-
frame duplication of 102 amino acids. Segregation was verified in 53 available members of the updated pedigree and a
penetrance of 88% was calculated. Fibroblast cultures from skin biopsies were established to assess the mitochondrial
network integrity and to qualitatively and quantitatively study the consequences of the mutation on transcript and
protein level. Fibroblast cultures demonstrated a fragmented mitochondrial network. Processing of the OPA1 protein
was altered. There was no correlation of the OPA1 transcript levels and the OPA1 protein levels in the fibroblasts.
Intriguingly an overall decrease of mitochondrial proteins was observed in patients' fibroblasts, while the OPA1
transcript levels were elevated.
Conclusions: The thorough study of this family provides a detailed clinical picture accompanied by a molecular
investigation of patients' fibroblasts. Our data show a classic OPA1-associated non-syndromic ADOA segregating in this
family. Cell biological findings suggest that OPA1 is regulated by post-translational mechanisms and we would like to
hypothesize that loss of OPA1 function might lead to impaired mitochondrial quality control. With the clinical, genetic
and cell biological characterisation of a family described already more than 50 years ago, we span more than half a
century of research in optic neuropathies.
Background
During the first half of the last century, there was a con-
troversial debate about the clinical and etiological unity
of hereditary optic neuropathies. Some ophthalmologists
favored the idea that the majority of cases are part of the
manifestation spectrum of the Optic Atrophy described
by Theodor Leber [1], that we now know as Leber's
hereditary optic neuropathy (LHON) [2-4]. Others
argued that there are different disease entities and sug-
gested discriminating different forms of optic atrophy [5-
7]. In 1954, Wolfgang Jaeger reported his genealogical
* Correspondence: marcel.alavi@googlemail.com
1 Molecular Genetics Laboratory, Institute for Ophthalmic Research, Centre for
Ophthalmology, University of Tuebingen, Germany
Full list of author information is available at the end of the article

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and clinical findings in an extended German family span-
ning five generations. In this pedigree he could clearly
demonstrate that optic atrophy is inherited as a dominant
trait including male-to-male transmission. In addition, he
pointed out the presence of blue-yellow color vision dis-
turbances in the affected subjects in this family that con-
trasts to the red-green defect typically present in families
with LHON [8]. These features, together with a thorough
review of prior clinical reports, enabled him to establish
autosomal dominant optic atrophy (ADOA) as a distinct
disease entity.
Nowadays, ADOA is a well established disease entity
and considered the most frequent hereditary optic atro-
phy besides LHON. ADOA is clinically characterized by a
juvenile onset with a progressive, bilateral reduction of
visual acuity, a cecocentral scotoma, temporal pallor of
the optic disc and tritanopia as the most typical type of
color vision defect [9,10]. There is considerable intra- and
interfamilial variability in progression of the disease as
well as in the severity of the visual impairments, ranging
from very mildly affected subjects to legally blind patients
[11-13]. Moreover, asymptomatic carriers have been
reported in many pedigrees demonstrating reduced pen-
etrance [14-16]. Histopathologic investigations have
shown a loss of retinal ganglion cells (RGCs) and thinning
of the nerve fiber layer [17,18] which later could be con-
firmed in an ADOA mouse model [19]. A major locus for
ADOA was mapped to chromosome 3q28-3q29 by link-
age analysis [20] and subsequently the disease-causing
gene OPA1 was identified by our group and an indepen-
dent group [21,22]. Besides OPA1, two further gene loci
for ADOA, OPA4 [23] and OPA5 [24] have been mapped
but the underlying genes remain unknown so far. In addi-
tion, a small number of families have been reported with
OPA3 mutations that cause ADOA associated with early
onset cataract [25]. Given the large number of reports of
families with OPA1 mutations, the other loci seem to play
only a minor role. There are more than 200 OPA1 muta-
tions listed in the eOPA1 database http://lbbma.univ-
angers.fr/eOPA1[26]. Most of these mutations have been
identified by conventional, qualitative genetic screening
techniques that are insensitive for genomic alterations
(i.e. gross deletions or duplications). Only one family with
a complete OPA1 gene deletion has been reported in the
literature [27] and just recently we have been able to dem-
onstrate that genomic rearrangements may constitute a
considerable proportion of causal OPA1 gene mutations
[28].
The OPA1 protein is a nuclear encoded, dynamin-
related GTPase which is imported into mitochondria and
anchored to the inner membrane of the organelle [29].
Together with mitofusin 1 and 2 it plays a major role in
mitochondrial fusion and therefore is important for the
maintenance of the mitochondrial network morphology
and dynamics [30,31]. Furthermore, OPA1 has been
linked to mitochondrial cristae stability and its remodel-
ing in apoptosis [32-34]. How specific OPA1 mutants
affect these principal functions has only been sparsely
investigated and why mutations in OPA1 cause a mostly
selective degeneration of RGCs is unknown. On the other
hand there are case reports of syndromic forms of ADOA
that include extra-ocular
(sensorineural deafness, ataxia, axonal sensory motor-
polyneuropathy, chronic progressive external ophthal-
moplegia, mitochondrial myopathy) which have been
associated with multiple mitochondrial DNA (mtDNA)
deletions [35,36]. However, mtDNA deletions can not
only be found in muscles from patients with syndromic
ADOA, but seem to be present also in muscle biopsies
from patients with non-syndromic ADOA [37].
Here we report the identification and re-evaluation of
the large family described by W. Jaeger in 1954. Using
copy-number sensitive techniques, we have been able to
finally identify the disease causing mutation in this fam-
ily. We provide an update of the pedigree, a clinical follow
up of a branch of this family as well as a qualitative and
quantitative investigation of mutated OPA1 transcripts
and OPA1 protein in fibroblast cell lines established from
two patients of this family.
neurological features
Results
The original ADOA family described in 1954 was re-con-
tacted with the help of Prof. W. Jaeger (now deceased)
and most of the blood samples and medical records were
collected in a field study in the mid-90s. The pedigree
was updated through several personal interviews with
family members, the latest in 2008 (Figure 1). The recon-
structed pedigree now comprises a total of 216 family
members of whom 57 are affected or were reported to
have been affected. In comparison with the pedigree
reported in 1954 we noted 19 additional family members
affected by ADOA. DNA samples of 22 affected and 31
unaffected family members were collected for this study
and fibroblast cultures for functional investigations were
established from skin biopsies of two affected subjects
(Figure 1, V-24 and V-26).
Long-term follow-up of patients revealed a typical non-
syndromic ADOA
Two affected brothers who were already described in the
original report underwent a full ophthalmological re-
examination more than 50 years after their initial clinical
description (Figure 1A, V-24 and V-26). Patient A (V-24),
now aged 69 years and patient B (V-26), now 66 years old,
were first examined at the age of 15 and the age of 11,
respectively. W. Jaeger reported that both complained of
reduced vision in school and already experienced modest
loss of visual acuity at this age. Since then visual acuity

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Figure 1 Pedigree of the investigated ADOA family and segregation analysis of the OPA1 exon 7-9 duplication. (A) Updated pedigree of the
family. The gray area indicates the original pedigree as described by W. Jaeger 1954. Only persons of whom DNA samples were available are indexed.
(B) Segregation analysis applying a PCR assay specific for the OPA1 exon 7-9 duplication. A fragment covering exon 19 sequences was co-amplified
as internal control. Subjects are numbered according to the pedigree. Ntc - non template control.
1
2
3
4
1
2
4
5
7
8
9
18
1920212223
4
5
6
12
15
16
17
26
40
41
43
45
50
51
52
54
55
56
57
58
60
61
63
66
16
21
24
26
44
45
46
47
48
49
60
59
?
?
A
16 21 24 26 44 45 46 47 48 49 59 60
4
5
6
12 15 16 17 26 40 41 43 45
50 51 52 54 55 56 57 58 60 61 63 66
12
4
5
78918 19 20 21 22 23
12
3
4
ntc
V-
VI-
VI-
VII-
VIII-
Intron 9-6
Exon 19
B

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dropped in both patients from 5/25 and 5/20, respec-
tively, to each 6/120 in both eyes. A slow course of disease
progression was also confirmed by self-reports. Neither
of them remembered periods of fast loss of visual acuity.
Yet both patients have now developed cecocentral sco-
toma and an optic atrophy progressed to a complete pal-
lor of the optic nerve head, especially of the temporal part
of the disc (Figure 2). In comparison with the results of
former color vision tests described in Jaeger's original
paper, both patients reported further deterioration of
their color vision. Using Ishihara's test charts they both
could formerly identify at least 5 of the test plates. At the
recent examination both patients did not see even the ref-
erence plate of the test. Using Stilling-Hertel test charts,
patient A could previously identify 11, patient B 13 of the
plates. At the recent examination patient A could only
identify 2 and patient B 7 plates. Over the period of 54
years, the patients' ability to discriminate between colors
has severely diminished.
In addition patient A suffered from glaucoma; the
intraocular pressure (IOP) was 21/25 mmHG (R/L) with-
out medication, while IOP was still in the normal range in
patient B (R/L 18/19 mmHG). Patient B received a stent
in 2006. Otherwise the medical history was unremarkable
and neither of the patients had diabetes mellitus or hear-
ing impairments, typical symptoms in syndromic forms
of optic atrophy. In conclusion both patients show a clear
and classical non-syndromic ADOA.
Mutation analysis showed a duplication of exons 7 to 9 in
the OPA1 gene
An initial microsatellite marker analysis revealed signifi-
cant linkage with the OPA1 locus (C. Alexander and G.
Auburger, unpublished results). However, subsequent
screening of the OPA1 gene by means of DNA sequenc-
ing of the coding exons and flanking intron sequences
from PCR-amplified genomic DNA did not reveal any
putative pathogenic mutation. We therefore applied Mul-
tiplex Ligation Probe Amplification (MLPA) to analyse
for copy number variations and genomic rearrangements.
Indeed, MLPA showed a significant signal increase for
the OPA1 exon 8 and 9 probe amplicons in the index
patient in three independent experiments (Figure 3A).
Using oligonucleotides placed both - forward and reverse
- in exon 8 for a long distance PCR we were able to
amplify a specific PCR product of approximately 8 kb in
samples of affected individuals only (Figure 3B). Primer
walking revealed the presence of a recombined sequence
that fuses the 5' part of intron 9 (breakpoint at
np194840568, human genome assembly march 2006)
with the 3' part of intron 6 (np194832822) of the OPA1
gene (Figure 3C). From this we conclude that exons 7-9
plus the flanking intron sequences are duplicated in this
family. This mutation was also found in two other ADOA
families [28]. In silico analysis revealed Type I transposon
elements adjacent to both breakpoints (not shown). We
developed a PCR assay that is specific for this duplication
and performed a segregation analysis in all family mem-
bers of whom a DNA sample was available (Figure 1B).
We found that all affected family members carry the
duplication. In addition, three asymptomatic family
members (Figure 1B; V-47, VI-15, VII-5) were mutation
carriers. Taking into account that 22 of the 25 mutation
carriers are affected we calculate a penetrance of 88% in
this family.
The mitochondrial network is fragmented in patients'
fibroblasts
Since OPA1 is necessary for mitochondrial fusion [30,31],
we morphometrically assessed the mitochondrial net-
work in fibroblasts of the two patients after mitotracker
staining and confocal imaging. In comparison with wild
type control cells we found a significantly decreased pro-
portion of cells with a tubular mitochondrial network
when cultured in standard glucose medium (Figure 4A
&4B). Replacement of glucose by galactose in the growth
medium to avoid glycolysis reinforced the decrease in
cells with tubular mitochondrial network (not shown).
These results demonstrate that mutant cells are clearly
impaired in their ability to fuse mitochondria. We tested
also the integrity of the mtDNA in these cell lines by the
amplification of a 12.5 kb fragment, but did not notice
any differences from controls (Figure 4C).
Qualitative and quantitative cDNA analyses revealed an in-
frame duplication, a reduced expression level of mutant
transcripts and an overall increase of OPA1 transcripts in
fibroblasts
To study the consequences of this mutation we analysed
cDNA from patients' fibroblast cell lines. Thereby we
found minor fragments that may represent aberrantly
spliced mutant transcripts. Subcloning and sequencing of
the major fragment of increased length revealed that this
cDNA species had the additional exons correctly inte-
grated as a duplication of 306 bp (c.678-984dup306). This
duplication results in an in-frame duplication of 102
amino acids (p.L227_K328dup102). To test if there is an
imbalance between the transcripts of the mutated and the
WT allele in our patients we quantified allelic transcript
levels by pyrosequencing as described previously [38].
Analyses of the OPA1 exon 21 polymorphism (c.2109C >
T) revealed a mean percentage ratio for the two alleles of
48.7% to 51.3% (S.D. = 1.2). This shows that both alleles
are transcribed similar. We also quantified different
OPA1 exon-exon junctions in relation to GAPDH by real
time PCR to verify this result. If both alleles are tran-
scribed similarly one would expect a ratio of exon 9-7 (x)
to exon 6-7 (y) to exon 7-8 (z) of one-to-two-to-three (x :

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Figure 2 Clinical picture of two patients that had been examined already by W. Jäger. (A) Funduscopy of patient B (V-26) showed pallor of the
optic disc with temporal prominence. (B) Visual field of patient A (V-24) showed a clear central scotoma. (C) Patient B showed a decentered central
scotoma (excentric retinal locus of fixation, note conjugate displacement of the blind spot).
A A
B
C
RARALA LA

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y : z = 1 : 2 : 3; see Figure 5A &5B, line 1 for an overview).
However, we found a ratio x : y : z of 0.60 (S.D. = 0.05) : 2 :
2.85 (S.D. = 0.25) in patient A and a ratio x : y : z of 0.62
(S.D. = 0.07) : 2 : 2.24 (S.D. = 0.18) for patient B, respec-
tively (Figure 5B, line 2 and 3). Controls displayed a ratio
y : z of 2 : 1.98 (S.D. = 0.14) thus proving the reliability of
this method (Figure 5B, lane 4). This inconsistency in the
results of both assays can be interpreted in a way that a
fraction of the transcripts derived from the mutant allele
may be spliced correctly (i.e. eliminating the 3 duplicated
exons and presenting as wild type) or spliced aberrantly,
or both. Of note, the overall steady-state levels of OPA1
transcripts in patients' fibroblasts was more than two-
fold increased in comparison to the control fibroblasts
(Figure 5C).
Qualitative and quantitative protein analysis reveals
reduced levels of OPA1 and altered processing of the
different OPA1 isoforms
For a qualitative and quantitative analysis of OPA1 pro-
tein we used total cell lysates from fibroblast cell lines of
patients V-24 and V-26 and four control fibroblast cell
lines derived from healthy individuals. The duplicated
exons 7-9 of the mutant OPA1 allele result in an in-frame
duplication of 102 amino acid residues within the GTPase
domain of the OPA1 protein (p.L227_K328dup102), that
is expected to increase its molecular weight by 11.8 kDa.
We consistently found a reduction of all OPA1 protein
isoforms in both patients' cell lines in relation to actin
(Figure 6A). The different OPA1 isoforms were assigned
according to their size as described before [39]. Still we
are aware that the assignment is rather hypothetical and
Figure 3 Identification and characterization of the exon 7-9 duplication in OPA1 by MLPA. (A) Typical read out of the MLPA experiment of index
patient (VIII-4) depicting a significant increase for the exon 8 and 9 probes. (B) Long distance PCR amplification with forward and reverse primer lo-
cated both in exon 8 yielded a fragment of approximately 8 kb in affected family members only (line 1: patient A, line 2: patient B, line 3: not affected
control, line 4: no template control). (C) Sequencing of the amplicon shown in (B) reveals the genomic breakpoint and demonstrates that exon 7 is
duplicated as well. The numbers above the arrows give the genomic position (human genome assembly, build 36.3), black capital letters indicate re-
tained sequences of intron 9-10 and intron 6-7, respectively, while small red letters show the deleted sequence portion. The underlined 3 nucleotides
can be found as reminiscence of the deletion event.
B
...TGAGTGGTGATAAcaa
...tacaaaaaattagctg
Intron 6-7
gttgggtatac... gca
GGCGTGGTGGCAGG...
Intron 9-10
T G A G T G G TG A T A A G C A G G C G T G G TG G C A G G
194840568 194832822
patient A
A
12
3
4
8 kbp
1,4
1,6
1
1,2
atio
Ra
0,6
0,8
0,2
0,4
0
OPA1 Exon 01A
OPA1 Exon 01B
OPA1 Exon 02A
OPA1 Exon 02B
OPA1 Exon 05A
OPA1 Exon 05B
OPA1 Exon 06
Exon 08
OPA1
Exon 09
OPA1
Exon 10
OPA1
Exon 13
OPA1
Exon 14
OPA1
Exon 17
OPA1
Exon 18
OPA1
Exon 20
OPA1
Exon 21
OPA1
Exon 22
OPA1
Exon 23
OPA1
Exon 24
OPA1
Exon 26
OPA1
Exon 27
OPA1
Intron 28
OPA1
Exon 29
OPA1
RDS Exon 01
RDS Exon 02A
RDS Exon 02B
Exon 03
RDS
VMD2 Exon 01
Exon 02
VMD2
Exon 03
VMD2
Exon 04
VMD2
Exon 05
VMD2
Exon 06
VMD2
Exon 07
VMD2
Exon 08
VMD2
Exon 09
VMD2
Exon 10
VMD2
Exon 11
VMD2
Control
Control
Control
Control
C

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additional experiments would be necessary to define pre-
cisely the single bands. However, according to its size the
P/M band could represent either OPA1 precursor or
mutant protein or both. Densitometric analysis of the dif-
ferent isoforms revealed that the S3 and S4 isoforms were
reduced to approx. 50% of their levels in controls while
L1, L2 and S5 were reduced not that strong (Figure 6B).
The overall OPA1 protein level (excluding the precursor/
mutant form) was reduced to approx. 66%. The observed
changes in the relative abundance of the different OPA1
isoforms implicate altered proteolytic processing of
OPA1 in mitochondria (e.g. reduced processing when L1
and S3 constitutes splice variant 7 and L2, S4 and S5 con-
stitute splice variants 1 and 5 as suggested before [39,40]).
Since we found a reduction of all OPA1 isoforms in
relation to actin, we assessed several other mitochondrial
proteins (Tom40, CoxIX, ATPase β, Hsp60) in total cell
lysates by westernblot analyses and subsequent densito-
metric measurements to clarify whether other mitochon-
drial proteins are reduced in their abundance as well. In
comparison to four control fibroblast cell lines we found a
reduction of all tested mitochondrial proteins of 20 to
55% consistent in both patients' fibroblast cell lines (Fig-
ure 6C &6Ds). This finding shows that together with
OPA1 other mitochondrial proteins seem to be reduced
as well.
Discussion
Here we report the identification of a large intragenic
duplication in the OPA1 gene in a historic family with
ADOA that was first described in 1954 and that was deci-
sive for the definition of ADOA as a separate disease
entity [8]. The updated pedigree now comprises a total of
216 family members of whom 57 are affected or were
reported to suffer from ADOA. The family thus consti-
tutes probably one of the largest reported in the ADOA
literature and may enable further investigations of sec-
ondary factors that modulate disease expression and pen-
etrance. Based on our genotyping results we calculated a
penetrance of 88% in this family. This is in line with a pre-
vious thorough study of several Australian families with
ADOA that reports also a penetrance of 88% and 82.5%
for the fully ascertained sibships, respectively [14,15].
Notably, all unaffected mutation carriers in the family
presented herein are females and we previously reported
two more unaffected female mutation carriers in other
families, one with the same exon 7-9 duplication muta-
tion and one with a complete deletion of the OPA1 gene
[28]. All of these unaffected mutation carriers have
affected siblings or children which share by definition the
same mtDNA haplogroup. This shows that the mtDNA
background does not play a major role in governing pene-
trance in ADOA and we would like to speculate instead
about an x-chromosomal inherited QTL or environmen-
tal factors. In summary, the studied family presents a
well-defined clinical picture for non-syndromic ADOA.
There is no evidence of associated extra-ocular symp-
toms, either in the thorough clinical investigations by W.
Jaeger or in our recent re-examinations.
Figure 4 Assessment of the mitochondrial network morphology. (A) Representative micrographs of cells with different mitochondrial network
morphology. The different categories are indicated. Arrow heads accentuate tubular structures in the mitochondrial network classified as "intermedi-
ate". Scale bar, 20 μm. (B) Cells have been classified by two independent qualified persons in a blinded experiment with a third person taking the
micrographs (n > 100 for each group). The number of cells that exhibit tubular mitochondrial network morphology is reduced in patients' fibroblasts
compared to a control. (C) Amplification of a 12.5 kb mtDNA fragment did not reveal any specific mtDNA deletions for patients or controls. Ntc - non
template control.
cells [%]
20
40
60
80
100
control
patient A
patient B
0
fragmented
intermediate
tubular
BA
tubular
intermediate
fragmented
12.5
2
0.5
12
3
4
C
control
patient A
patient B
ntc

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In contrast to other inherited blinding diseases, in
ADOA the affected gene is expressed ubiquitously
[21,41]. With limitations, this offers the possibility to
study the consequences of the disease causing mutation
on cellular level in any tissue or cell line derived from
patients. For the study presented here, we have estab-
lished two fibroblast cell lines from skin biopsies of the
two affected brothers at the age of 69 and 66, respectively
and compared them with four different control fibroblast
cell lines with the same passage number. Two of the latter
were obtained from young children's circumcision speci-
mens whereas the other two derived from skin biopsies of
a middle-aged donor and a donor in his late sixties, which
served as the primary control. All donors were healthy
individuals. Unfortunately we were not able to obtain skin
biopsies of the unaffected brothers. However, to our
knowledge, donor age has only a minor effect on the rep-
licative lifespan of fibroblasts, in contrast to passage
numbers of the cells and/or health status of the donors
[42,43], which was both carefully controlled in this study.
Donor age shows also only minor effects on mitochon-
drial enzyme activity [44]. In agreement with this, we
didn't find any specific mtDNA deletions in patients' cell
lines or control cells that would refer to a premature
aging effect of the cells.
Nevertheless, we found a more fragmented and less
tubular morphology of the mitochondrial network in the
patients' cell lines. Knockout mutants of the OPA1-
homologue mgm1/msp1 in yeast show also fragmenta-
tion of mitochondria and a reduction of mtDNA content
[45,46]. A quite similar mitochondrial phenotype could
also be observed by down-regulation of OPA1 expression
by RNA interference in HeLa cells, which caused frag-
mentation of the mitochondrial network, loss of mito-
chondrial membrane potential and disorganization of the
mitochondrial cristae [33,47]. Spinazzi and colleagues
also reported fragmentation of the mitochondrial net-
work in fibroblasts as well as myotubes from patients that
harbor a deletion in the GTPase domain of OPA1
(c1410_144314del38) [48].
We found reduced OPA1 protein levels in fibroblasts
obtained from patients with non-syndromic ADOA that
harbor a 3-exon-duplication in the GTPase domain of
OPA1 when compared to controls. This is in line with
previous reports on other OPA1 mutations in patients
with ADOA, including the deletion in the GTPase
domain of OPA1 mentioned above, or Opa1 mouse mod-
els, which all report reduced OPA1 protein levels, regard-
less of the underlying OPA1 mutation [48-52]. Based on
this and the description of different families with non-
syndromic ADOA that segregate heterozygous deletions
of the complete OPA1 gene [27,28], haploinsufficiency is
believed to be a major pathomechanism in OPA1 associ-
ated non-syndromic ADOA.
On the other hand our data show that there might be an
accumulation of OPA1 transcripts in the patients' cells,
which would be in accordance with the observed aug-
mentation of the precursor/mutant protein (P/M band),
but in contrast to the reduction of the other OPA1 iso-
forms in these cells. Previously, we have reported reduced
Opa1 protein levels in various tissues obtained from
Opa1 mutant mice. In these mice, the Opa1 transcript
levels were not altered compared to littermate controls
[49]. Taken together, there seems to be no correlation of
the transcript levels and the protein levels for OPA1,
which suggests that OPA1 is regulated on protein levels.
Figure 5 Quantification of the different OPA1 transcripts derived
from the mutant and the WT allele. (A) A clarifying scheme about
the strategy for the relative quantification with boxes indicating the
different amplicons of exon/exon junctions and their relative ratios if
one assumes that both transcripts are equally abundant. (B) One
would expect a ratio of exon 7-8 to exon 6-7 to exon 9-7 of 3 to 2 to 1
when transcripts of both alleles are equally abundant (expected); pa-
tients clearly presented with reduced levels of exon 7-8 and 9-7 ampl-
icons indicating an imbalance in favor of the transcripts derived from
the WT allele (patient A & patient B); controls show no difference in the
quantification of the exon 7-8 and 6-7 amplicons, proving that the
method works very accurate. (C) The readout of a typical quantification
of the exon 6-7 junction in relation to GAPDH shows a significant up
regulation of OPA1 in patients' fibroblasts.
exon6-7
exon 6-7
exon 7-8
exon 9-7
exon7-8
exon9-7
A
B
expected patient Apatient Bcontrol
1
2
3
n.d.
Ex9Ex6
Ex7
Ex8Ex10
Ex9Ex6
Ex7
Ex8Ex10 Ex9
Ex7
Ex8
WT
c.678-984dup306
6-77-8
6-7 7-8 7-89-7
relative copies
patient Apatient Bcontrolcontrol
1
5
10
C
relative copies

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This is supported by a study on OPA1 in conjunction
with heart failure that also found no correlation of OPA1
transcript and OPA1 protein levels [53]. In conclusion,
this suggests that this 102 amino acid duplication in the
GTPase domain of OPA1 leads to altered OPA1 function
rather than haploinsufficiency. Taking into account that
loss of one complete OPA1 allele leads to a comparable
clinical picture (i.e. non-syndromic ADOA) [27,28], one
might speculate that OPA1 function is reduced by this
particular mutation. Then why and how are OPA1 protein
levels reduced in ADOA? The ubiquitine-proteasome
pathway is not involved in the degradation of OPA1 as
could be shown in Opa1 mutant mice [49].
Recently, it has been proposed that mitochondrial fis-
sion and selective fusion serves as mitochondrial quality
control. Experiments with photo labeled mitochondria
revealed that selective fusion separates functional from
dysfunctional mitochondria [54-56]. Dysfunctional mito-
chondria display a reduced membrane potential ΔΨ [56],
which results in increased proteolytic cleavage of OPA1
[57-62]. As a consequence dysfunctional mitochondria
are not capable to fuse anymore and are degraded by
autophagy [54,56]. According to this, a change in OPA1
function which reduces mitochondrial capability to fuse
should also result in increased mitochondrial degradation
by autophagy. Indeed, increase in autophagy has been
observed in the optic nerve of Opa1 mutant mice [63].
Figure 6 Reduced OPA1 protein amount and altered processing in fibroblasts from patients with ADOA. (A) Quantitative western blot against
OPA1 (upper panel) and actin (lower panel) of the indicated probes. Arrows indicate the corresponding bands; P/M: precursor/mutant protein, L1 to
S5 bands according to Duvezin-Caubet et al. 2007 [39]. (B) Densitometric evaluation of the different OPA1 isoforms from the western blot shown in
A as mean ± S.D. from patients vs. control. OPA1 protein levels are reduced in the patients' cell lines and the unequal reduction of the different corre-
sponding OPA1 isoforms suggest altered proteolytic cleavage of OPA1. (C) Quantitative western blot against indicated mitochondrial proteins in total
cell lysates of the indicated probes. (D) Densitometric quantification of the western blot shown in C as mean ± S.D. from patients vs. controls. The
analysis shows that all tested mitochondrial proteins are less abundant in patients' cell lines compared to controls.
average [%]
Opa1
Tom40
CoxIV
ATPase
Hsp60
20
40
60
80
100
0
A
B
Actin
Opa1
Tom40
CoxIV
ATPase
Hsp60
patient A
patient B
control 3
control 1
control 4
control 2
L1 L2
S3
S4 S5P/M
100
50
average [%]
C
D
L1
L2
S3
S4
S5
P/M
110 kDa
85 kDa
50 kDa
α- ctinA
α-Opa1
patient A
patient B
control 1
patients
control
patients
controls

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This might be an explanation for the discrepancy of
OPA1 transcript and OPA1 protein levels. In addition,
this would be in agreement with the observed fragmented
mitochondrial network, the reduced proteolytic process-
ing and the reduction of OPA1 and other mitochondrial
proteins in the cell lines from patients with ADOA found
in this study. Altered mitochondrial quality control would
also explain mitochondrial dysfunction and/or the accu-
mulation of mtDNA deletions as described for patients
with ADOA [37].
Still it needs to be shown, if loss of OPA1 function
impairs mitochondrial function, which leads to more dys-
functional mitochondria and subsequent removal of
these dysfunctional mitochondria, or if loss of OPA1
function impairs mitochondrial quality control directly,
which then leads to increased clearance of apparently
functional mitochondria, too.
Methods
Patients and Subjects
We included 53 family members in this study who have
been recruited in a field study in the mid-nineties. An
update of the pedigree was obtained from recent personal
interviews with various family members. Two of the
patients underwent a full ophthalmological examination
at the University Ophthalmic Hospital in Tuebingen in
2008. Informed consent was obtained for blood samples
and skin biopsies. The study was performed in accor-
dance to the tenets of the Declaration of Helsinki and was
approved by the local ethics committee.
Cell culture and staining of the mitochondrial network
Fibroblast cell lines were established from skin biopsies
from patients and compared to 4 different healthy control
cell lines with the same passage number. All cell lines
were free of mycoplasma contaminations. Cells were
propagated in minimal essential medium supplemented
with 10% fetal calf serum (FCS) and antibiotics under
standard conditions. For assessment of the mitochondrial
network structure, cells were cultured for 24 hours in glu-
cose-free DMEM supplemented with either 10% FCS and
0.45% (w/v) glucose or 10% FCS, 5 mM galactose and 5
mM pyruvate prior to staining with 10 nM Mitotracker
for 45 min at 37°C in the corresponding medium. The
morphology of the mitochondrial network was qualita-
tively assessed by two independent people who did not
know either the condition or the origin of the pictures
which had been taken by a third person with a fluores-
cence microscope (AXIO Imager Z1 with ApopTome,
Zeiss, Germany).
Isolation of DNA and RNA
Genomic DNA was extracted from venous blood samples
applying standard salting-out procedure, or from culti-
vated fibroblasts applying High Pure PCR Template Prep-
aration Kit (Roche, Mannheim, Germany). Total RNA was
isolated from whole blood drawn in PAXgene tubes using
the PAXgene blood RNA kit (Qiagen, Hilden, Germany).
RNA from fibroblasts was obtained using the RNeasy Kit
(Qiagen) and subsequently treated with DNAse (New
England Biolabs, Frankfurt, Germany). RNA was reverse
transcribed using either the Long Range 2 Step RT-PCR
Kit (Qiagen) or the Super Script III First-Strand Synthesis
System for RT-PCR (Invitrogen, Carlsbad, USA) with
oligo-dT-primers or random hexamers.
Multiplex ligation-dependent probe amplification (MLPA)
MLPA reactions were performed using the P97 and P229
kit from MRC-Holland (Amsterdam, The Netherlands)
following the manufacturers' instructions and applying an
exactly defined amount of sample DNA (100 ng). Reac-
tions were performed in triplicates and compared with
parallel processed controls. The amplified MLPA prod-
ucts were separated on a 3100 Capillary Sequencer
(Applied Biosystems, Darmstadt, Germany) and analysed
using the Coffalyser spreadsheet (control probe analysis,
MRC-Holland).
Breakpoint identification and segregation analysis
The exon 7-9 duplication was confirmed by long distance
PCR, carried out with the TaKaRa LA Taq™ kit (TAKARA
BIO INC., Shiga, Japan) applying both forward and
reverse oligonucleotides placed in Exon 8, Exon 8L: 5'-
GAT GTT CTC TCT GAT TAT GAT GCC-3' and Exon
8R: 5'-CAG ATG ATC TTG CGT ATT ATA ACT GG-3'.
The duplication breakpoint was identified by direct
sequencing of the ExoSAP (USB, Staufen, Germany) puri-
fied long distance PCR product applying a primer walk-
ing strategy. Sequencing was done employing BigDye
Terminator Chemistry 1.1 (Applied Biosystems) and
products separated on an ABI 3100 DNA Sequencer. Seg-
regation analysis was done applying a duplication specific
PCR assay with oligonucleotides: Dupl-Ex7-9-L: 5'-TTC
TTG ACA GGT TTG ATA TGG AGA-3' and Dupl-Ex7-
9-R: 5'-AAT TAA TGC ATG TCA GTG TCA CCT-3'. In
this case PCR was performed using standard Taq poly-
merase and a short extension time of not more than 30
sec. Numbering of exons and introns was based on OPA1
transcript variant 1 (NM_015560).
Real-Time PCR
Real Time PCR was performed using cDNA generated
from total fibroblast RNA with the following oligonucle-
otides: RT-Ex6-7-f: 5'-AAG AAC TTC TGC ACA CTC
AGT TGA A-3' & RT-Ex6-7-r: 5'-TTC TAA TCG TTC
CAA GAT TCT CTG ATA C-3'; RT-Ex7-8-f: 5'-GGC
ATT CAT CAT AGA AAG CTT AAG AA-3' & RT-Ex7-
8-r: 5'-AAG AAC TTC AGA ATA CAT GTC AAT CAA
AG-3'; RT-Ex9-7-f: 5'-GGA GAT GAT GAC ACG TTC

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TCC A-3' & RT-Ex9-7-r: 5'-TTC CAA GAT TCT CTG
ATA CTT CAA CTT AA-3'. Assays were carried out with
the Power SYBR Green PCR Master Mix on a real time
7500 PCR instrument (Applied Biosystems). Relative copy
numbers were calculated applying the ΔΔCt method with
GAPDH as reference. The pyrosequencing assay has been
described in details before [38].
Amplification of mtDNA
A 12.5 kb mtDNA fragment covering most of the coding
region was amplified using TaKaRa LA Taq reagents
(TAKARA BIO INC., Shiga, Japan) under recommended
cycling PCR conditions and applying the following
primer pair: MIT-LR1F: 5'-ACA ACC CTT CGC TGA
CGC CAT A-3'; MIT-LR2R: 5'-GGT GGT ACC CAA
ATC TGC TTC C-3'.
Quantitative westernblot analysis
For quantitative Westernblot analysis 15 μg total protein
solubilized in RIPA was separated on NuPAGE 4-12%
Bis-Tris Gels (Invitrogen). Blots were processed for
immunodecoration with antibodies against OPA1 (BD-
Transduction, LA, CA, USA), Tom40 (a kind gift of D.
Rapaport, Tuebingen), CoxIV (Abcam, Cambridge, MA,
USA), ATPase β (BD-Transduction), Hsp60 (Stressgen,
Victoria, Canada) or actin (Chemicon, Temecula, CA,
USA) applying the ECL chemiluminescence system
(Pierce, Rockford, IL, USA). According bands were quan-
tified using ImageJ http://rsb.info.nih.gov/ij/. All protein
levels were normalized to actin levels as loading control.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
NF performed all genetic analyses including real time experiments; he assisted
in the study design, interpreted the data and wrote the paper. SS contributed
to the cDNA analysis. YK extracted patients' and control fibroblasts propagated
them and provided antibodies. BLK performed all ophthalmological examina-
tions. CA and GA did the field study and provided patients' DNA samples. EZ
critically read through the paper. BW critically read through the paper. MVA
designed the study, performed all western blot analyses as well as the mito-
chondrial network analyses, he interpreted the data and wrote the paper. All
authors read and approved the final manuscript.
Acknowledgements
In memoriam Prof.Wolfgang Jäger. We are indebted to the members of this
family for their friendly cooperation and support of this study. We furthermore
thank Tao Wei for help with the morphological assessment of the mitochon-
drial network in stained fibroblasts. We appreciate helpful discussion and com-
ments from Jörg Tatzelt and Konstanze Winklhofer. This work was supported in
part by the Friedrich-Spicker-Stiftung, Essen, Germany and the Fritz-Thyssen-
Stiftung Köln, Germany. We thank Judy Howell for carefully reading through
the manuscript.
Author Details
1Molecular Genetics Laboratory, Institute for Ophthalmic Research, Centre for
Ophthalmology, University of Tuebingen, Germany, 2Department of
Dermatology, Eberhard Karls University Tuebingen, Germany, 3Centre for
Ophthalmology, Institute for Ophthalmic Research, University of Tuebingen,
Germany, 4Department of Neurodegeneration, Max-Delbrück-Center for
Molecular Medicine, Berlin, Germany and 5Section Molecular Neurogenetics,
Department of Neurology, Goethe University, Frankfurt am Main, Germany
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This article is available from: http://www.molecularneurodegeneration.com/content/5/1/25 © 2010 Fuhrmann et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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doi: 10.1186/1750-1326-5-25
Cite this article as: Fuhrmann et al., Solving a 50 year mystery of a missing
OPA1 mutation: more insights from the first family diagnosed with auto-
somal dominant optic atrophy Molecular Neurodegeneration 2010, 5:25