Calpain-5 mutations cause autoimmune uveitis, retinal neovascularization, and photoreceptor degeneration.
ABSTRACT Autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV) is an autoimmune condition of the eye that sequentially mimics uveitis, retinitis pigmentosa, and proliferative diabetic retinopathy as it progresses to complete blindness. We identified two different missense mutations in the CAPN5 gene in three ADNIV kindreds. CAPN5 encodes calpain-5, a calcium-activated cysteine protease that is expressed in retinal photoreceptor cells. Both mutations cause mislocalization from the cell membrane to the cytosol, and structural modeling reveals that both mutations lie within a calcium-sensitive domain near the active site. CAPN5 is only the second member of the large calpain gene family to cause a human Mendelian disorder, and this is the first report of a specific molecular cause for autoimmune eye disease. Further investigation of these mutations is likely to provide insight into the pathophysiologic mechanisms of common diseases ranging from autoimmune disorders to diabetic retinopathy.
- SourceAvailable from: Vinit B Mahajan[Show abstract] [Hide abstract]
ABSTRACT: The purpose of this project was to identify short hairpin RNA (shRNA) sequences that can suppress expression of human CAPN5 in which gain-of-function mutants cause autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV). We created HEK293T cells that stably express an ADNIV disease allele, CAPN5-p.R243L. Transfection protocols were optimized for neuroblastoma SHSY5Y cells. The gene silencing effect of four different shRNA plasmids that target CAPN5 was tested. RNA and protein expression was determined using quantitative RT-PCR and immunoblot analysis.BMC Research Notes 09/2014; 7(1):642.
- [Show abstract] [Hide abstract]
ABSTRACT: Exome sequencing indicated that the gene encoding the calpain-5 protease, CAPN5, is the likely cause of retinal degeneration and autoimmune uveitis in human patients with autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV, OMIM #193235). To explore the mechanism of ADNIV, a human CAPN5 disease allele was expressed in mouse retinas with a lentiviral vector created to express either the wild-type human (h) CAPN5 or the ADNIV mutant hCAPN5-R243 L allele under a rhodopsin promoter with tandem green fluorescent protein (GFP) expression. Vectors were injected into the subretinal space of perinatal mice. Mouse phenotypes were analyzed using electroretinography, histology, and inflammatory gene expression profiling. Mouse calpain-5 showed high homology to its human ortholog with over 98% sequence identity that includes the ADNIV mutant residue. Calpain-5 protein was expressed in the inner and outer segments of the photoreceptors and in the outer plexiform layer. Expression of the hCAPN5-R243 L allele caused loss of the electroretinogram b-wave, photoreceptor degeneration, and induction of immune cell infiltration and inflammatory genes in the retina, recapitulating major features of the ADNIV phenotype. Intraocular neovascularization and fibrosis was not observed during the study period. Our study shows that expression of the hCAPN5-R243 L disease allele elicits an ADNIV-like disease in mice. It further suggests that ADNIV is due to CAPN5 gain-of-function rather than haploinsufficiency, and retinal expression may be sufficient to generate an autoimmune response. Genetic models of ADNIV in the mouse can be used to explore protease mechanisms in retinal degeneration and inflammation as well as therapeutic testing in pre-clinical models.Human Molecular Genetics 12/2013; · 6.68 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: To highlight major advancements in ocular genetics from the year 2013.Asia-Pacific journal of ophthalmology (Philadelphia, Pa.). 05/2014; 3(3):181-193.
Calpain-5 Mutations Cause Autoimmune Uveitis, Retinal
Neovascularization, and Photoreceptor Degeneration
Vinit B. Mahajan1, Jessica M. Skeie1, Alexander G. Bassuk2,3, John H. Fingert1, Terry A. Braun1,4,
Heather T. Daggett1, James C. Folk1, Val C. Sheffield1,2,5, Edwin M. Stone1,5*
1Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, Iowa, United States of America, 2Department of Pediatrics, University of Iowa, Iowa
City, Iowa, United States of America, 3Department of Neurology, University of Iowa, Iowa City, Iowa, United States of America, 4Department of Biomedical Engineering,
University of Iowa, Iowa City, Iowa, United States of America, 5Howard Hughes Medical Institute, Iowa City, Iowa, United States of America
Autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV) is an autoimmune condition of the eye that
sequentially mimics uveitis, retinitis pigmentosa, and proliferative diabetic retinopathy as it progresses to complete
blindness. We identified two different missense mutations in the CAPN5 gene in three ADNIV kindreds. CAPN5 encodes
calpain-5, a calcium-activated cysteine protease that is expressed in retinal photoreceptor cells. Both mutations cause
mislocalization from the cell membrane to the cytosol, and structural modeling reveals that both mutations lie within a
calcium-sensitive domain near the active site. CAPN5 is only the second member of the large calpain gene family to cause a
human Mendelian disorder, and this is the first report of a specific molecular cause for autoimmune eye disease. Further
investigation of these mutations is likely to provide insight into the pathophysiologic mechanisms of common diseases
ranging from autoimmune disorders to diabetic retinopathy.
Citation: Mahajan VB, Skeie JM, Bassuk AG, Fingert JH, Braun TA, et al. (2012) Calpain-5 Mutations Cause Autoimmune Uveitis, Retinal Neovascularization, and
Photoreceptor Degeneration. PLoS Genet 8(10): e1003001. doi:10.1371/journal.pgen.1003001
Editor: Gregory S. Barsh, Stanford University School of Medicine, United States of America
Received June 8, 2012; Accepted August 14, 2012; Published October 4, 2012
Copyright: ? 2012 Mahajan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Howard Hughes Medical Institute (EMS, VCS); NIH Grants K08EY020530 (VBM), R01EY016822 (EMS), and R01NS064159
(AGB); the Roy J. Carver Charitable Trust; the Foundation Fighting Blindness; and Research to Prevent Blindness. The funders had no role in study design, data
collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Autosomal dominant neovascular inflammatory vitreoretino-
pathy (ADNIV) is a heritable autoimmune condition. It is
characterized by various stages that mimic several much more
common eye diseases, including: uveitis, retinitis pigmentosa,
proliferative diabetic retinopathy and proliferative vitreoretino-
pathy [1,2]. Together, these diseases account for a significant
fraction of visual morbidity and human blindness [3,4]. Identifi-
cation of a gene that generates the varied pathological features of
these common conditions could have a significant impact on the
understanding and treatment of blindness . Although there are
numerous causative genes for retinitis pigmentosa, only a handful
of genes have previously been associated with intraocular
inflammation, neovascularization and fibrotic disease .
Because of its similarity to other common eye diseases, ADNIV
patients are often misdiagnosed, unless the familial nature of their
disease is recognized. Bennett and co-workers described the
original ADNIV family, ADNIV-1 in this study , in which the
characteristic clinical findings were transmitted in an autosomal
dominant fashion through eight-generations. The disease onset in
this family varies between 10 and 30 years of age and the disease
course can be divided into five stages, each lasting approximately
ten years (Figure 1, Figure S1) . In the first stage, ADNIV is
clinically indistinguishable from an autoimmune, non-infectious
uveitis . Although the retina appears normal, an abnormality is
detectable with electroretinography very early in the course of the
disease. In the second stage, retinitis pigmentosa-like photorecep-
tor degeneration is apparent. In the third stage, retinal neovas-
cularization develops, which is very similar to the pathologic
angiogenesis of proliferative diabetic retinopathy . In the fourth
stage, intraocular fibrosis leads to retinal detachment, similar to
that seen in proliferative vitreoretinopathy . In the fifth stage,
continued inflammation, intraocular hemorrhage, neovascular
glaucoma, fibrosis and retinal detachment eventually lead to
phthisis and complete blindness. There are no systemic features in
this condition. This combination of overlapping clinical conditions
is unusual and suggests that the disease-causing mutations may act
through multiple pathways.
Stone and co-workers previously mapped the genetic locus for
ADNIV to chromosome 11q13 . In this study, we identified
two new ADNIV families, and these additional subjects provided
an opportunity to refine the genetic interval and identify the
causative gene. Two different mutations were identified among
three ADNIV families in the gene encoding calpain-5, an
intracellular calcium-activated cysteine protease with an unknown
The new ADNIV families displayed a phenotype very similar to
the original ADNIV-1 pedigree. Specifically, affected members
showed all of the previously reported clinical signs of the disease
PLOS Genetics | www.plosgenetics.org1 October 2012 | Volume 8 | Issue 10 | e1003001
(Figure 1, Figure S1) including: non-infectious uveitis (Figure 1A,
1B), early loss of the b-wave on electroretinography (Figure 1C),
pigmentary retinal degeneration (Figure 1D, 1E), cystoid macular
edema, (Figure 1F), retinal and iris neovascularization, vitreous
hemorrhage (Figure 1H), epiretinal membrane formation, prolif-
erative vitreoretinopathy (Figure 1G), retinal detachment, cata-
ract, neovascular glaucoma and ultimately phthisis and complete
blindness (Figure 1I) . Each pedigree was consistent with
autosomal dominant inheritance with complete penetrance
(Figure 2). There were sixty-one affected subjects in ADNIV-1,
seven in ADNIV-2, and thirty-one in ADNIV-3 (Figure 2). Forty-
two of these 99 affected individuals (42%) were male. The clinical
severity of the disease was indistinguishable between affected males
and females. With the exception of psoriasis in one individual,
there were no other systemic, autoimmune or inflammatory
conditions present in any of the affected family members.
Prior linkage analysis of the ADNIV-1 family mapped the
disease-causing mutation to a 22-megabase (chr11: 91,760,018–
69,339,635) interval on chromosome 11q13 (Figure 3A) .
Genotyping of the ADNIV-2 and ADNIV-3 families with short
tandem repeat polymorphisms was consistent with linkage to the
same locus. Haplotype analysis was suggestive of an ancestral
relationship between ADNIV-1 and ADNIV-2. In addition, two
affected individuals in the ADNIV-3 family were found to be
recombinant within the disease interval, narrowing it to 6.5
megabases between D11S4139 and D11S1789. High resolution
SNP genotyping of ADNIV-1 and ADNIV-3 further reduced the
interval to the 6 megabases between rs879380 and D11S1789, a
region harboring 86 known genes (Figure 3A).
Whole-exome sequencing was performed using DNA from two
affected family members from the ADNIV-1 pedigree who were
separated by seven meioses. Only one of the resultant sequence
variations met the following four criteria: located within the 6 Mb
ADNIV interval, shared by the two affected members of ADNIV-
1, not previously reported as a polymorphism, and nonsynon-
ymous. This variant is a guanine to thymine nucleotide change
(c.728G.T, p.Arg243Leu) in exon 6 of the CAPN5 gene
(NM_004055) (Figure 3B–3D). A combination of SSCP and
Sanger sequencing of CAPN5 exon 6 verified that this mutation
was present in all the affected members and none of the unaffected
members of ADNIV-1.
The coding sequence of CAPN5 was then sequenced in affected
members of the two other ADNIV pedigrees. All affected
members of ADNIV-2 were found to harbor the same heterozy-
gous variant (c.728G.T, p.Arg243Leu) found in ADNIV-1,
supporting the suspected ancestral relationship between these
two families (Figure 3D). All affected members of the ADNIV-3
family, were found to harbor a heterozygous variant in the
adjacent codon, a thymine to cytosine change (c.731T.C,
p.Leu244Pro) (Figure 3E). Both of these putative disease-causing
variants in exon 6 of CAPN5 were easily detectable by SSCP in
ADNIV patients, but were absent from all unaffected adult
members of the ADNIV families (no asymptomatic minors were
tested) as well as 272 ethnically similar control individuals
(Figure 3F). None of the three variants were listed in the dbSNP
or 1000 Genome databases. In addition, none of the variant alleles
were found in the over 10,700 CAPN5 alleles sequenced in the
NHLBI Exome Sequencing Project (http://evs.gs.washington.
Structural Analysis of Calpain-5 ADNIV Mutations
Calpain-5 is an intracellular calcium-activated cysteine protease
(NP_004046) with evolutionarily conserved domains required for
protease activity. Both ADNIV-causing mutations were found in
exon 6, which encodes a major part of the catalytic domain and
contains two of the three catalytic residues that compose the active
site (Figure 4). Modeling of secondary structure suggests that both
the ADNIV-1/2 (p.Arg243Leu) and ADNIV-3 (p.Leu244Pro)
mutations lie within a nearby alpha helical domain. The first of
these mutations removes a charged residue while the second
disrupts the putative helical structure (Figure 4A).
The amino acid sequence of CAPN5 exon 6 is highly conserved
across vertebrate species (Figure 4B). The catalytic residues show
100% conservation among CAPN5 orthologs. Interestingly, there is
also 100% conservation of the Arg243 residue mutated in
ADNIV-1/2 and 88% conservation of the Leu244 mutated in
ADNIV-3. This small evolutionary divergence at the latter codon
is also quite conservative: methionine for leucine in the frog. In
contrast, the disease-causing mutation at this codon introduces a
new proline bend within a putative alpha helix.
Previous comparisons of calpain-5 to its human paralogs
demonstrated that it has diverged significantly and now belongs
to its own subfamily with calpain-6 . This divergence is also
evident within exon 6 alone, where the calpain-5 catalytic domain
shows relatively low homology to other human calpains
(Figure 4C). Each of the ADNIV mutant residues is conserved
in four or fewer of 12 calpain paralogs, suggesting that the residues
mutated in ADNIV are specifically important to calpain-5
function and may physiologically distinguish it from the other
calpains. The PolyPhen2 sequence analysis program predicted
both ADNIV mutations to have damaging effects on protein
function (0.999 for Arg243Leu and 0.998 for Leu244Pro)
comparable to an active site Cys81Ser mutation (1.0). The SIFT
program predicted the Leu244Pro mutation to be comparably
pathogenic (0.04) to Cys81Ser (0.03) but predicted the Arg243Leu
mutation to be better tolerated (0.1).
To better examine the relationship of ADNIV mutations within
the calpain-5 catalytic domain, homology modeling to calpain-2
(m-calpain) was used to generate a three-dimensional structure for
calpain-5 (Figure 4D) [12,13]. Both mutations were outside the
active site cleft and relatively far removed from the calcium-
binding domains and the binding site of the endogenous inhibitor
calpastatin. Interestingly, both the ADNIV-1/2, and ADNIV-3
mutations fell into a region of low electron density, suggesting the
presence of a flexible loop (Figure 4E). In calpain-1 (m-calpain)
models, the homologous loop undergoes calcium-induced confor-
mational changes that regulate the proximity of catalytic residues
We care for several families with an inherited form of
autoimmune inflammation inside the eye. The patients
also develop bleeding, scar tissue, and eventually blind-
ness. Using advanced gene analysis methods, we discov-
ered the cause of this disease is gene mutations in the
CAPN5 gene. This gene makes a protein, calpain-5, which
belongs to a family of calcium-activated enzymes that slice
other proteins inside cells. Calpain-5 is expressed in the
retina, and the disease mutations alter its location inside
the cell. Future studies to understand how this protein
causes inflammation and bleeding inside the eye will help
us develop treatments for this condition and more
common eye diseases with inflammation and bleeding.
Calpain-5 Mutations Cause ADNIV
PLOS Genetics | www.plosgenetics.org2 October 2012 | Volume 8 | Issue 10 | e1003001
within the active site cleft . This putative loop contains both
ADNIV mutants and is highly conserved among all calpain-5
orthologs (Figure 4B).
Calpain-5 Expression in the Retina
We evaluated the CAPN5 transcript in human retinal tissue
using RNA sequencing. The transcript was observed at a level of
4.63 fragments per kilobase of exon per million, which places it
between the first quartile and the median level of expression for all
transcripts observed in the retina. No significant splice variants
were detected. Two antibodies against calpain-5 were used to
determine whether calpain-5 protein could also be detected in
human retinal tissue sections. Both antibodies showed strong
calpain-5 expression in the photoreceptor cells (Figure 5A, 5B).
There was a punctate pattern of labeling over the nuclei and inner
segments with less expression along the outer segments and outer
plexiform layer. There was no significant expression in the nerve
fiber layer, ganglion cell layer, inner nuclear layer, inner plexiform
layer, or retinal pigment epithelium. The localization to the
photoreceptor cells is consistent with both the early electrophys-
iologic abnormalities and the later photoreceptor degeneration
seen in ADNIV patients.
Effect of Calpain-5 Mutations on Intracellular Location
Intracellular compartmentalization is a key regulatory mecha-
nism for calpains [15,16,17]. For example, mutations that disturb
localized protein interactions with calpain-3 cause limb-girdle
muscular dystrophy type 2A [17,18]. To determine the effect of
the ADNIV-causing mutations on the intracellular compartmen-
talization of calpain-5, HEK293T cells were transfected with
normal and mutant CAPN5 constructs. A western blot with anti-
myc antibody revealed a single protein species of the expected size
for myc-tagged calpain-5 (Figure 5C). Immunocytochemistry of
HEK293T cells showed normal calpain-5 to be localized near the
cell surface (Figure 5E). In contrast, both ADNIV mutants were
found largely within the cytoplasm (Figure 5F and Figure S2). This
suggests that the ADNIV-causing mutations may alter a mem-
brane binding property of the protein.
The calpains are an evolutionarily ancient family of calcium
dependent intracellular proteases that utilize a cysteine residue in
the active site to mediate limited proteolysis. The multifunctional
calpains require careful regulation, since they target multiple
intracellular proteins and pathways [15,16,17,19,20]. Their
activity is regulated by intracellular calcium, lipid and protein
interactions, subcellular localization, autocatalysis and inhibition
by the endogenous peptide calpastatin [18,20,21]. There is no
consensus amino acid sequence or structural motif that is targeted
for cleavage by calpains, and as a result it is often difficult to
identify the physiologic substrates of these enzymes, including
calpain-5 [22,23]. Capn5 is expressed during nematode and mouse
embryogenesis [24,25]. In adults, CAPN5 is highly expressed in the
colon, kidney, liver, trachea, uterus, eye and brain [11,25].
Calpains have been implicated in the pathogenesis of a wide range
of human diseases including cancer, multiple sclerosis, Alzheimer’s
disease, cataract, diabetes and muscular dystrophy [17,26]. Some
polymorphisms in CAPN10 and CAPN5 have been shown to be risk
factors for type II diabetes [27,28]. However, prior to this report,
limb girdle muscular dystrophy (LGMD) type 2A was the only
disease shown to be caused in a monogenic fashion by variations in
a calpain’s protein sequence .
Figure 1. ADNIV phenotype. A–B. Clusters of autoimmune
reactive leukocytes are visible in the vitreous cavity (inset, arrows).
C. Electroretinography shows loss of the b-wave. D. Fundus image
of the normal retina. E. Fundus image of the ADNIV retina shows
pigmentary degeneration (arrow) similar to retinitis pigmentosa. F.
Fluorescein angiography reveals cystoid macular edema at the
fovea (arrow), a consequence of autoimmune intraocular inflam-
mation. G. Preretinal fibrosis leads to tractional retinal detach-
ments. H. Vitreous hemorrhage (arrow) from retinal neovascular-
ization. I. Phthisis bulbi and involution of eye tissues in end-stage
Calpain-5 Mutations Cause ADNIV
PLOS Genetics | www.plosgenetics.org3 October 2012 | Volume 8 | Issue 10 | e1003001
The evidence that the two missense mutations we observed in
CAPN5 are responsible for ADNIV is compelling. The gene lies
within the critical region previously linked to the disease, and all
living subjects in the study who are clinically affected were found
to harbor a CAPN5 mutation in exon 6. Each of these two
mutations alters an amino acid in the catalytic domain that has
been highly conserved throughout evolution. Neither of these
mutations was found among any of the clinically unaffected
members of the three kindreds we studied, or among thousands of
There are interesting similarities and differences between
calpain-associated LGMD and ADNIV. In both disorders, the
affected cells (skeletal muscle fibers and photoreceptor cells)
experience large changes in membrane potential and intracellular
calcium concentration as part of their normal behavior. In both
disorders, the non-mutant calpain molecules display functionally
critical subcellular localization [17,18,20,30]. A subset of LGMD
is associated with leukocyte infiltration into the tissue , and all
cases of ADNIV are marked by severe intraocular inflammation.
The differences in these diseases are also noteworthy. Calpain-
associated LGMD is inherited in a recessive fashion and appears to
result from loss of calpain-3 function . In contrast, ADNIV is
inherited in an autosomal dominant fashion and is caused by
missense mutations near the active site. Although these mutations
could cause disease through haploinsufficiency, it seems more
likely that they result in a gain of function of calpain-5 that causes
harm to the photoreceptor cells. Capn5 knockout mice have no
observable phenotype , and several human neurological
disorders have been associated with excess calpain activity
[17,33], including photoreceptor degeneration .
A gain of function mechanism for ADNIV is also supported by
the unusual inflammation and neovascularization associated with
the disease. There are dozens of monogenic disorders that cause
the apoptotic death of photoreceptor cells without causing severe
intraocular inflammation or neovascularization of the retina. The
latter is much more typical of proliferative diabetic retinopathy
than it is heritable photoreceptor disease . It is easier to imagine
an unregulated or mislocalized calpain promiscuously activating
different signaling pathways, or being released into the extracel-
lular space after photoreceptor death and causing inappropriate
angiogenesis and leukocyte recruitment, than it is to imagine a
50% reduction of such a protein causing these dramatic
Whether caused by a gain or loss of function of CAPN5, it is
likely that the further elucidation of the pathogenic mechanism of
ADNIV will provide important new insight into some of the most
important causes of irreversible human blindness: autoimmune
uveitis, retinitis pigmentosa, proliferative vitreoretinopathy and
diabetic retinopathy. The latter condition alone is responsible for
as much as 17% of blindness in some regions of the world . The
fact that an amino acid change in a single protein can lead to such
a phenotype raises the possibility that a common, therapeutically
accessible pathway may be shared among these conditions that
could be targeted with drugs, antibodies or gene transfer
approaches. It is possible that variations in the structure or
expression of CAPN5 cause or modify some of these common
disorders and this hypothesis will be important to test in future
studies. However, given the extreme phenotypic heterogeneity of
these disorders, it will be important to study a large number of
subjects in such an experiment, to subdivide the patient cohorts
into clinically well-characterized groups, and to screen an equal
number of ethnically matched controls for each of these groups.
Materials and Methods
The study was approved by the University of Iowa’s Institu-
tional Review Board and adheres to the tenets set forth in the
Declaration of Helsinki. Informed consent was obtained from all
study participants. Informed consent was obtained from all study
Figure 2. ADNIV pedigrees. A–C. Three families with clinical features of ADNIV exhibit a dominant pattern of inheritance. Black symbols represent
clinically affected subjects. Open symbols represent unaffected subjects. Deceased individuals are marked by a slash.
Calpain-5 Mutations Cause ADNIV
PLOS Genetics | www.plosgenetics.org4 October 2012 | Volume 8 | Issue 10 | e1003001
Figure 3. The CAPN5 gene harbors mutations in exon 6 of ADNIV subjects. A. The ADNIV locus was previously mapped to chromosome
11q13 (green bar). STRP and SNP array mapping narrowed the interval (red bar). B. CAPN5 gene structure is composed of 13 exons. C. Chromatogram
of normal CAPN5 DNA sequence in exon 6. D–E. Chromatograms of ADNIV subjects shows mutations in CAPN5 exon 6. F. SSCP distinguishes between
normal sequence and ADNIV mutations.
Calpain-5 Mutations Cause ADNIV
PLOS Genetics | www.plosgenetics.org5October 2012 | Volume 8 | Issue 10 | e1003001
Figure 4. Protein structure modeling of calpain-5 and ADNIV mutants. A. Both ADNIV mutations (red arrows) are located in exon 6, which
encodes a portion of the catalytic domain and two of three catalytic residues (blue arrows). Primary protein sequence analysis shows the ADNIV-1/2
and ADNIV-3 mutations to be 8–9 amino acids upstream of the catalytic histidine residue. Secondary structure modeling shows that the two
mutations are within a putative alpha helical domain. One mutated codon results in the loss of a basic residue, while the other introduces a proline
into the putative alpha helix. B. Alignment of calpain-5 orthologs shows very high evolutionary conservation of the mutated residues (red arrows).
Amino acid mismatches are color-highlighted. C. Twelve human calpain paralogs show significant differences in exon 6 (Black, 100% similarity; Dark
grey, 80–100% similarity; Light grey, 60–80% similarity; White, less than 60% similarity). D. Three-dimensional modeling of the catalytic domain shows
Calpain-5 Mutations Cause ADNIV
PLOS Genetics | www.plosgenetics.org6October 2012 | Volume 8 | Issue 10 | e1003001
Phenotypic ascertainment of the pedigrees included complete
ocular examination as previously described . Following
genotyping of all adult members of the three ADNIV pedigrees
with short tandem repeat polymorphisms within the original
disease interval, three members of ADNIV-1 and three members
of ADNIV-3 were also genotyped with an Affymetrix GeneChip
Human Mapping 50K Array. Exome sequencing was performed
using NimbleGen’s SeqCap EZ Human Exome v2.0 capture and
paired-end (26100) sequencing on an Illumina HiSeq 2000
instrument at Otogenetics (Norcross, GA). The putative disease-
causing mutations in CAPN5 were evaluated in the ADNIV
kindreds and controls using Sanger sequencing and single-strand
conformational polymorphism analysis (SSCP) . Sequence
analysis of human retinal cDNA was performed using the Illumina
HiSeq 2000 instrument.
Primary and secondary structure protein alignments and trees
were created with Geneious Pro 5.4.6 (http://www.geneiouspro.
com). Yasara Structure (version 11.3.2) was used to generate a
homology model of human calpain-5 using calpain-2 (m-calpain)
structures (pdb id: 3BOW, 1DF0, 1U5I, 1KFU) as templates
[12,13]. Sequence alignment with the templates was first used to
build a backbone model for aligned residues followed by loop
modeling and side chain optimization using a combination of
steepest descent and simulated annealing minimization. The top
ranking of the 20 models generated was used as the homology
model of calpain-5. The above steps were automated using
Yasara’s hm_build macro (http://www.yasara.org). Another
homology model was generated using the Phyre server (version
0.2) (http://www.sbg.bio.ic.ac.uk/,phyre), which showed good
agreement with the Yasara model for the domain folds and the
the location of the active site cleft (red outline). E. Magnified view of the active site cleft shows the catalytic triad (dashed line – blue text) and location
of the two mutations (red arrows and text). Both mutations are located within a peptide loop that is homologous to a flexible loop of calpain-1 that
undergoes a calcium-induced conformational change in association with regulation of the active site cleft (see text).
Figure 5. Calpain-5 expression in human retinal photoreceptor cells and cultured cells. A. No significant signal was detected in control
retinal sections probed with secondary antibody or primary antibody blocked with recombinant calpain-5. DAPI highlights the cell nuclei (blue) B.
Calpain-5 was detected in photoreceptor cells (green). A punctate expression pattern was most prominent overlying the photoreceptor nuclei in the
outer nuclear layer (ONL) and inner and outer segments (IS, OS). C. Anti-myc antibody western blot detects a single species (black arrow) of the
appropriate size in HEK293T cells transfected with a vector bearing normal, myc-tagged CAPN5. D. No significant anti-myc signal was detected in the
nuclei (white arrowhead) or cytoplasm (white arrow) of control cells that were treated with transfection reagent alone, vector alone, or secondary
antibody alone. E. Anti-myc antibody shows that calpain-5 (green) is expressed in a ruffled pattern (white arrow) that obscures the underlying nuclei
(white arrowhead) suggesting a location near the cell surface in these very thin cultured cells. F. Transfection with a mutant CAPN5 (Arg243Leu)
exhibits a more uniform anti-myc signal that does not obscure the nuclei (white arrowhead) and is thus more compatible with localization to the
cytoplasm (white arrow).
Calpain-5 Mutations Cause ADNIV
PLOS Genetics | www.plosgenetics.org7 October 2012 | Volume 8 | Issue 10 | e1003001
active site region. The putative alpha-helix region in the Yasara
model also formed a helix in the best Phyre model although it was
positioned further away from the calpastatin binding site region
than the Yasara model. PolyPhen2 (http://genetics.bwh.harvard.
edu/pph2/) and SIFT (http://sift.jcvi.org/) sequence analysis
software were used to predict the functional effect of mutations.
RNA sequence analysis was performed by extracting RNA from
the retina of a human eye donor using an RNeasy kit from Qiagen
(Valencia, CA) according to the manufacturer’s instructions,
preparing the sequencing library using the Illumina (San Diego,
CA) RNA TruSeq sample preparation kit, and sequencing the
latter on the Illumina HiSeq 2000 instrument at the Hudson
Alpha Institute in Huntsville Alabama. The resulting sequence
data were mapped using TopHat  and analyzed using
Cufflinks . The retinal expression of CAPN5 was compared
to the expression of all other genes expressed in the retina.
Donor eyes were received from Iowa Lions Eye Bank (Iowa
City, IA). Tissue was fixed in 4% paraformaldehyde solution
diluted in 10 mM phosphate buffered saline (PBS), pH 7.4, and
7 mm sections underwent immunohistochemistry (IHC) using a
polyclonal anti-calpain 5 primary antibody (Santa Cruz Biotech-
nology, Inc., Santa Cruz, CA), AlexaFluor 488 donkey anti-rabbit
secondary antibody (Invitrogen, Carlsbad, CA) and 49,6-diami-
dino-2-phenylindole (DAPI; Invitrogen). Images were captured
using an Olympus BX41 microscope equipped with fluorescent
filters and the SPOT Advanced software package.
HEK293T cells (ATCC, Manassas, VA) were transfected with
normal and mutant CAPN5 pCMV6-Entry vector plasmids using
Turbofectin 8.0 (Origene) transfection reagent according to the
manufacturer’s instructions. Cells were incubated for 48 hours
post transfection. For immunocytochemistry, cells were blocked
using 5% bovine serum albumin (Amresco, Solon, OH) diluted in
PBS with 0.1% Triton X-100. The polyclonal primary antibody,
anti-Myc-tag, was diluted in PBS at 1:500 and applied to the cells.
Alexa Fluor 488 donkey anti-rabbit secondary, at concentration
10 mg/mL, and 0.0001 mg/mL of the counterstain, 49, 6-
diamidino-2-phenylindole (DAPI) (both from Molecular Probes,
Eugene, OR), were applied to the cells, Images were captured
using a Zeiss LSM 710 equipped with Zen2009 software (Zeiss,
New York, NY).
vitreoretinal conditions. A. In Stage I disease, there are mild cells
(white dots) in the vitreous (orange) and a reduced b-wave on
electroretinography (ERG). B. In Stage II disease, the anterior
chamber (blue) shows mild inflammatory cells and there is early
development of cataract. The posterior segment shows moderate
cells, retinal pigmentary changes, and some edema in the macula
or optic nerve head. There is selective loss of the b-wave in the
scotopic bright flash ERG. C. In Stage III disease, the anterior
segment shows moderate cells, progressive cataract, and iris
synechiae. Progressive inflammation in the posterior segment
shows development of vitreous bands and epiretinal membranes,
and more posterior retinal pigmentary changes. There is reduction
of the a-wave on ERG. D. In Stage IV disease, inflammation in
the anterior segment causes neovascular and angle closure
glaucoma. Neovascularization develops in the retina with vitreous
hemorrhage and progressive retinal detachment that may include
features of anterior or posterior proliferative vitreoretinopathy.
The ERG becomes non-recordable. E, In Stage V disease, the eye
becomes phthisical. (Blue bar, anterior chamber features; orange
bar: posterior chamber features; black bar, ERG features)
Illustrations by Alton Szeto, MFA.
Disease stages of ADNIV phenocopy common
Transfection with the ADNIV-3 mutant CAPN5 (Leu244Pro)
exhibits a more uniform anti-myc signal that does not obscure the
nuclei (arrowhead) and is thus more compatible with localization
to the cytoplasm (arrow).
Calpain-5 mutant expression in cultured cells.
Peter Sonkin MD and Jeremiah Brown MD assisted with patient
examinations, Benjamin Roos provided assistance with DNA array
analysis, Lokesh Gakhar helped with protein structure modeling, and
Alton Szeto MFA prepared Figure S1.
Conceived and designed the experiments: VBM JMS JHF TAB VCS
EMS. Performed the experiments: VBM JMS JHF HTD VCS EMS.
Analyzed the data: VBM JMS AGB JHF TAB HTD JCF VCS EMS.
Contributed reagents/materials/analysis tools: VBM AGB TAB VCS
EMS. Wrote the paper: VBM JMS VCS EMS. Medical examination of
patients and phenotype ascertainment: VBM JCF EMS.
1. Bennett SR, Folk JC, Kimura AE, Russell SR, Stone EM, et al. (1990)
Autosomal dominant neovascular inflammatory vitreoretinopathy. Ophthalmol-
ogy 97: 1125–1135; discussion 1135–1126.
2. Mahajan VB, Folk J.C., Fingert J.H, Skeie J.M., Kinnick T.R., Scheetz T.C.,
Bassuk A.G., B J.R.M., Sheffield V.C., and Stone E.M. (2011) Genetic Analysis
and Phenotypic Staging of Autosomal Dominant Neovascular Inflammatory
Vitreoretinopathy. ARVO May 01: 62/A175.
3. Pascolini D, Mariotti SP (2012) Global estimates of visual impairment: 2010.
Br J Ophthalmol 96: 614–618.
4. Resnikoff S, Pascolini D, Etya’ale D, Kocur I, Pararajasegaram R, et al. (2004)
Global data on visual impairment in the year 2002. Bull World Health Organ
5. Sheffield VC, Stone EM (2011) Genomics and the eye. N Engl J Med 364:
6. Berger W, Kloeckener-Gruissem B, Neidhardt J (2010) The molecular basis of
human retinal and vitreoretinal diseases. Prog Retin Eye Res 29: 335–375.
7. Caspi RR (2010) A look at autoimmunity and inflammation in the eye. J Clin
Invest 120: 3073–3083.
8. Frank RN (2004) Diabetic retinopathy. N Engl J Med 350: 48–58.
9. Pastor JC, de la Rua ER, Martin F (2002) Proliferative vitreoretinopathy: risk
factors and pathobiology. Prog Retin Eye Res 21: 127–144.
10. Stone EM, Kimura AE, Folk JC, Bennett SR, Nichols BE, et al. (1992) Genetic
linkage of autosomal dominant neovascular inflammatory vitreoretinopathy to
chromosome 11q13. Hum Mol Genet 1: 685–689.
11. Dear N, Matena K, Vingron M, Boehm T (1997) A new subfamily of vertebrate
calpains lacking a calmodulin-like domain: implications for calpain regulation
and evolution. Genomics 45: 175–184.
12. Hanna RA, Campbell RL, Davies PL (2008) Calcium-bound structure of calpain
and its mechanism of inhibition by calpastatin. Nature 456: 409–412.
13. Moldoveanu T, Gehring K, Green DR (2008) Concerted multi-pronged attack
by calpastatin to occlude the catalytic cleft of heterodimeric calpains. Nature
14. Moldoveanu T, Hosfield CM, Lim D, Elce JS, Jia Z, et al. (2002) A Ca(2+)
switch aligns the active site of calpain. Cell 108: 649–660.
15. Leloup L, Shao H, Bae YH, Deasy B, Stolz D, et al. (2010) m-Calpain activation
is regulated by its membrane localization and by its binding to phosphatidy-
linositol 4,5-bisphosphate. J Biol Chem 285: 33549–33566.
16. Michetti M, Salamino F, Tedesco I, Averna M, Minafra R, et al. (1996)
Autolysis of human erythrocyte calpain produces two active enzyme forms with
different cell localization. FEBS Lett 392: 11–15.
17. Zatz M, Starling A (2005) Calpains and disease. N Engl J Med 352: 2413–
Calpain-5 Mutations Cause ADNIV
PLOS Genetics | www.plosgenetics.org8 October 2012 | Volume 8 | Issue 10 | e1003001
18. Ermolova N, Kudryashova E, DiFranco M, Vergara J, Kramerova I, et al.
(2011) Pathogenity of some limb girdle muscular dystrophy mutations can result
from reduced anchorage to myofibrils and altered stability of calpain 3. Hum
Mol Genet 20: 3331–3345.
19. Croall DE, DeMartino GN (1991) Calcium-activated neutral protease (calpain)
system: structure, function, and regulation. Physiol Rev 71: 813–847.
20. Goll DE, Thompson VF, Li H, Wei W, Cong J (2003) The calpain system.
Physiol Rev 83: 731–801.
21. Kawasaki H, Kawashima S (1996) Regulation of the calpain-calpastatin system
by membranes (review). Mol Membr Biol 13: 217–224.
22. DuVerle DA, Ono Y, Sorimachi H, Mamitsuka H (2011) Calpain cleavage
prediction using multiple kernel learning. PLoS ONE 6: e19035. doi:10.1371/
23. Waghray A, Wang DS, McKinsey D, Hayes RL, Wang KK (2004) Molecular
cloning and characterization of rat and human calpain-5. Biochem Biophys Res
Commun 324: 46–51.
24. Barnes TM, Hodgkin J (1996) The tra-3 sex determination gene of
Caenorhabditis elegans encodes a member of the calpain regulatory protease
family. EMBO J 15: 4477–4484.
25. Dear TN, Boehm T (1999) Diverse mRNA expression patterns of the mouse calpain
genes Capn5, Capn6 and Capn11 during development. Mech Dev 89: 201–209.
26. Huang Y, Wang KK (2001) The calpain family and human disease. Trends Mol
Med 7: 355–362.
27. Horikawa Y, Oda N, Cox NJ, Li X, Orho-Melander M, et al. (2000) Genetic
variation in the gene encoding calpain-10 is associated with type 2 diabetes
mellitus. Nat Genet 26: 163–175.
28. Saez ME, Martinez-Larrad MT, Ramirez-Lorca R, Gonzalez-Sanchez JL,
Zabena C, et al. (2007) Calpain-5 gene variants are associated with diastolic
blood pressure and cholesterol levels. BMC Med Genet 8: 1.
29. Richard I, Broux O, Allamand V, Fougerousse F, Chiannilkulchai N, et al.
(1995) Mutations in the proteolytic enzyme calpain 3 cause limb-girdle muscular
dystrophy type 2A. Cell 81: 27–40.
30. Marcilhac A, Raynaud F, Clerc I, Benyamin Y (2006) Detection and localization
of calpain 3-like protease in a neuronal cell line: possible regulation of apoptotic
cell death through degradation of nuclear IkappaBalpha. Int J Biochem Cell Biol
31. Krahn M, Lopez de Munain A, Streichenberger N, Bernard R, Pecheux C, et al.
(2006) CAPN3 mutations in patients with idiopathic eosinophilic myositis. Ann
Neurol 59: 905–911.
32. Franz T, Winckler L, Boehm T, Dear TN (2004) Capn5 is expressed in a subset
of T cells and is dispensable for development. Mol Cell Biol 24: 1649–1654.
33. Vanderklish PW, Bahr BA (2000) The pathogenic activation of calpain: a marker
and mediator of cellular toxicity and disease states. Int J Exp Pathol 81: 323–
34. Azuma M, Shearer TR (2008) The role of calcium-activated protease calpain in
experimental retinal pathology. Surv Ophthalmol 53: 150–163.
35. Tucker BA, Scheetz TE, Mullins RF, DeLuca AP, Hoffmann JM, et al. (2011)
Exome sequencing and analysis of induced pluripotent stem cells identify the
cilia-related gene male germ cell-associated kinase (MAK) as a cause of retinitis
pigmentosa. Proc Natl Acad Sci U S A 108: E569–576.
36. Trapnell C, Pachter L, Salzberg SL (2009) TopHat: discovering splice junctions
with RNA-Seq. Bioinformatics 25: 1105–1111.
37. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, et al. (2010)
Transcript assembly and quantification by RNA-Seq reveals unannotated
transcripts and isoform switching during cell differentiation. Nat Biotechnol 28:
Calpain-5 Mutations Cause ADNIV
PLOS Genetics | www.plosgenetics.org9October 2012 | Volume 8 | Issue 10 | e1003001