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Vici syndrome is a recessively inherited multisystem disorder
characterized by callosal agenesis, cataracts, cardiomyopathy,
combined immunodeficiency and hypopigmentation.
To investigate the molecular basis of Vici syndrome, we carried
out exome and Sanger sequence analysis in a cohort of ?8
affected individuals. We identified recessive mutations in
EPG5 (previously KIAA1632), indicating a causative role in
Vici syndrome. EPG5 is the human homolog of the metazoan-
specific autophagy gene epg-5, encoding a key autophagy
regulator (ectopic P-granules autophagy protein 5) implicated
in the formation of autolysosomes. Further studies showed
a severe block in autophagosomal clearance in muscle and
fibroblasts from individuals with mutant EPG5, resulting in
the accumulation of autophagic cargo in autophagosomes.
These findings position Vici syndrome as a paradigm of human
multisystem disorders associated with defective autophagy and
suggest a fundamental role of the autophagy pathway in the
immune system and the anatomical and functional formation of
organs such as the brain and heart.
Vici syndrome (MIM 242840)1 is a rare multisystem disorder char-
acterized by callosal agenesis, cataracts, cardiomyopathy, combined
immunodeficiency and hypopigmentation. Its occurrence in consan-
guineous families and siblings suggests recessive inheritance1–6.
We identified 18 individuals with Vici syndrome of European
(n = 11), Arab (n = 3), Turkish (n = 2), Japanese (n = 1) and British-
Asian origin (n = 1), 9 of whom were previously reported1,4–9. Clinical
findings from these individuals are summarized in Supplementary
Table 1 and depicted in Supplementary Figure 1.
Muscle biopsies available from eight affected individuals showed
consistent myopathic features5,6, comprising fiber-type dispropor-
tion with type 1 atrophy, increase in internal nuclei and abnormal
glycogen accumulation (data not shown). On electron microscopy
images (Fig. 1), there was redundancy of basal lamina, with material
between layers, suggesting exocytosis of debris. There were
numerous vacuole-like areas and dense bodies, possibly of lysosomal
origin. Myofibrils were lacking in many fibers. Mitochondria were of
variable size and showed abnormal distribution and morphology.
We sequenced the exomes of four individuals with Vici syndrome
from three families, one multiplex consanguineous family and two
non-consanguineous families with one affected child each, and iden-
tified only one gene, EPG5 (NM_020964.2; previously KIAA1632)
on chromosome 18q12.3 in which we found mutations in all affected
individuals. Analysis of all EPG5 coding exons in a total of 15 families
Recessive mutations in EPG5 cause Vici syndrome,
a multisystem disorder with defective autophagy
Thomas Cullup1,28, Ay Lin Kho2,3,28, Carlo Dionisi-Vici4,5, Birgit Brandmeier2,3, Frances Smith1, Zoe Urry6,
Michael A Simpson6, Shu Yau1, Enrico Bertini5, Verity McClelland7, Mohammed Al-Owain8,9, Stefan Koelker10,
Christian Koerner10, Georg F Hoffmann10, Frits A Wijburg11, Amber E ten Hoedt11, R Curtis Rogers12,
David Manchester13, Rie Miyata14, Masaharu Hayashi15, Elizabeth Said16,17, Doriette Soler18, Peter M Kroisel19,
Christian Windpassinger19, Francis M Filloux20, Salwa Al-Kaabi21, Jozef Hertecant21, Miguel Del Campo22,
Stefan Buk23, Istvan Bodi23, Hans-Hilmar Goebel24, Caroline A Sewry25, Stephen Abbs1, Shehla Mohammed26,
Dragana Josifova26, Mathias Gautel2,3,29 & Heinz Jungbluth7,27,29
1DNA Laboratory, Guy’s and St Thomas’ Serco Pathology, Guy’s Hospital, London, UK. 2Randall Division of Cell and Molecular Biophysics, King’s College London,
London, UK. 3Cardiovascular Division, King’s College London British Heart Foundation Centre of Research Excellence, London, UK. 4Division of Metabolism, Bambino
Gesù Children’s Hospital, Istituto di Ricovero e Cure a Carattere Scientifico, Rome, Italy. 5Laboratory of Molecular Medicine, Bambino Gesù Children’s Hospital, Istituto
di Ricovero e Cure a Carattere Scientifico, Rome, Italy. 6Division of Genetics and Molecular Medicine, King’s College London School of Medicine, Guy’s Hospital, London,
UK. 7Department of Paediatric Neurology, Evelina Children’s Hospital, Guy’s and St Thomas’ National Health Service (NHS) Foundation Trust, London, UK. 8Department
of Medical Genetics, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia. 9Faculty of Medicine, Alfaisal University, Riyadh, Saudi Arabia.
10Division of Inherited Metabolic Diseases, University Children’s Hospital, Heidelberg, Germany. 11Department of Pediatrics, Academic Medical Centre, University of
Amsterdam, Amsterdam, The Netherlands. 12Greenwood Genetic Center, Greenville, South Carolina, USA. 13Department of Pediatrics, Clinical Genetics and Metabolism,
University of Colorado School of Medicine, Children’s Hospital Colorado, Aurora, Colorado, USA. 14Department of Pediatrics, Tokyo Kita Shakai Hoken Hospital, Tokyo,
Japan. 15Department of Brain Development and Neural Regeneration, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan. 16Section of Medical Genetics,
Mater dei Hospital, Msida, Malta. 17Department of Anatomy & Cell Biology, University of Malta, Msida, Malta. 18Department of Paediatrics, Mater dei Hospital, Msida,
Malta. 19Institute of Human Genetics, Medical University of Graz, Graz, Austria. 20Division of Pediatric Neurology, University of Utah School of Medicine, Salt Lake
City, Utah, USA. 21Department of Pediatrics, Tawam Hospital, Al-Ain, UAE. 22Genetics Department, Hospital Vall d’Hebron, Barcelona, Spain. 23Department of Clinical
Neuropathology, Academic Neuroscience Centre, King’s College Hospital, London, UK. 24Department of Neuropathology, Johannes Gutenberg University Medical Centre,
Mainz, Germany. 25Dubowitz Neuromuscular Centre, Institute of Child Health, University College London, London, UK. 26Department of Clinical Genetics, Guy’s Hospital,
London, UK. 27Clinical Neuroscience Division, Institute of Psychiatry, King’s College London, London, UK. 28These authors contributed equally to this work.
29These authors jointly directed this work. Correspondence should be addressed to H.J. (email@example.com) or M.G. (firstname.lastname@example.org).
Received 20 April; accepted 15 November; published online 9 December 2012; doi:10.1038/ng.2497
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with Vici syndrome with 18 affected individuals showed homozygo-
sity or compound heterozygosity for truncating mutations (including
mutations affecting the invariant splice donor and acceptor recogni-
tion sites) in 10 individuals and compound heterozygosity for truncat-
ing and missense mutations in 2 families. Subject 7.1 was homozygous
for a mutation affecting the penultimate base of exon 2, predicted to
cause aberrant splicing (Table 1). Parental studies suggested recessive
inheritance with no carrier manifestations.
Subjects 14.1 and 15.1 did not have any pathogenic sequence vari-
ants; quantitative fluorescent PCR (QF-PCR) did not identify any
copy-number variations in subject 14.1.
Although the absence of EPG5 mutations
in these individuals indicates the possibility
of locus heterogeneity (supported by the
presence of heterozygous SNPs in EPG5 in
subject 14.1, who comes from a consanguin-
eous partnership), we cannot exclude the
possibility of splice-site or promoter variants
or mutations affecting other regulatory ele-
ments. Sequencing of VMP1 (NM_030938.3)
and EI24 (NM_004879.3), the human
homologs of C. elegans epg-3 and epg-4,
respectively, identified by Tian et al.10, did
not reveal any mutations.
EPG5 consists of 44 exons, encodes a
protein of 2,579 amino acids11 and is pre-
dominantly expressed in the central nervous
system (CNS), skeletal and cardiac muscle,
thymus, immune cells, lung and kidneys.
EPG5 is the human homolog of epg-5 in
Caenorhabditis elegans, which encodes a
protein with a key role in the autophagy
pathway of multicellular organisms10. EPG5
has previously been found to be mutated
in human cancer tissue12, a finding shared
with other genes implicated in the autophagy
Autophagy is an evolutionarily highly con-
served lysosomal degradation pathway with
fundamental roles in cellular homeostasis,
including nutrient provision during fasting
or increased metabolic demands, removal of
defective proteins or organelles, and defense
against infection14–19. A role in normal
embryonic development has been suggested20
and is supported by the recent observation of
a link between autophagy and early differen-
tiation events in human embryonic stem cells21. Autophagy has also
emerged as a key regulator of cardiac and skeletal muscle homeostasis
and functional remodelling22–24. The process of autophagy involves
several tightly regulated steps: (i) formation of an isolation membrane
(the phagophore), (ii) fusion of the phagophore to form a double-
membranated autophagosome and (iii) fusion of the autophagosome
with a lysosome to form the autolysosome, where both captured mem-
brane and contents are degraded. Defects in the autophagy pathway
can be broadly divided into those causing impaired autophagosome
formation and those causing decreased autolysosome clearance.
Figure 1 Ultrastructural abnormalities in Vici
syndrome. (a–f) Muscle biopsy from subject 4.1
subjected to electron microscopy (transverse
sections). In many fibers, there is material
between the layers of basal lamina (a, arrows;
scale bar, 500 nm) or overt exocytic vacuoles
(b, arrow; scale bar, 2 µm). Some fibers show a
single centralized nucleus (c; scale bar, 2 µm).
Mitochondria are of variable size and distribution.
In some fibers, they form a loop around the
nucleus in the periphery of the fiber, resembling
a necklace as in c; in others, they form clusters
(d; scale bar, 2 µm). The appearance of cristae
is often abnormal (e; scale bar, 2 µm;
f; scale bar, 500 nm). N, nucleus; M, mitochondrion.
table 1 Genetic findings in individuals with Vici syndrome
Family Subject Consanguinity
Variant 1Variant 2
NucleotideAmino acid NucleotideAmino acid
p.Phe1604Glyfs*20 c.4952+1G>A p.Phe1604Glyfs*20
c.6005_6006dupAG p.Leu2003Serfs*30 c.6112T>C
Nucleotide numbering for EPG5 (KIAA1632) is based on GenBank RefSeq NM_020964.2, with the adenosine of the
annotated translation start codon defined as nucleotide position +1. The genotype in the original probands reported
by Vici et al.1 (family 1) is an implied genotype based on heterozygous EPG5 variants identified in the parents.
On the basis of sequencing of cDNA derived from fibroblast cultures from affected individuals, the homozygous
intronic variants identified in subject 4.1 were predicted to result in a frameshift and the introduction of a premature
stop codon (p.Phe1604Glyfs*20). Further qPCR and protein blot studies on tissue from this subject indicated the
presence of an unstable or quickly degraded polypeptide (data not shown). The heterozygous missense variants
identified in subject 6.1 in trans were absent from in-house exome data and from 372 control chromosomes.
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Membrane dynamics during autophagy are highly conserved
from yeast to mammals, but, along the evolutionary trajectory, they
involve an increasingly complex molecular machinery. In yeast,
a set of more than 30 ATG (autophagy-related) genes encode proteins
involved in the autophagic cascade18; this conserved group is modu-
lated in higher organisms by mammalian-specific factors, mainly
with a role in autophagosome-lysosome fusion. Epg-5 is one of four
higher eukaryote–specific autophagy genes in C. elegans (in addition
to epg-2, epg-3 and epg-4), which regulate specific autophagy steps
in multicellular organisms and are ubiquitously expressed in early
development10. Epg-3, Epg-4 and Epg-5 have an important role in
starvation-induced autophagy, as suggested by the reduced survival
of animals with mutations in these genes in the absence of food.
Epg-5 seems to be specifically involved in a late step of autophagy,
the autophagic degradation of protein aggregates, as evidenced by
defects in phagolysosome formation in the absence of Epg-5 (ref. 25),
accumulation of non-degradative autolysosomes following knock-
down of mammalian EPG5 in HeLa cells and accelerated autophagic
degradation following epg-5 overexpression10.
To further investigate the autophagy pathway in Vici syndrome,
we performed additional immunofluorescence studies on skeletal
muscle tissue from two affected individuals (subjects 2.1 and 3.1).
We observed marked fiber atrophy and fiber size heterogeneity
with upregulation of the sarcomere-associated autophagy proteins
SQSTM1 (p62) and NBR1 (refs. 26–28) (Fig. 2), with numerous posi-
tive puncta (autophagosomes) in many fibers compared to normal
controls that were also positive for LC3 but not for sarcomeric
proteins. The same fibers also showed strong upregulation of MURF2,
a SQSTM1-linked muscle ubiquitin E3 ligase29. Immunofluorescence
microscopy and analysis of electron microscopy images detected no
obvious myofibrillar abnormalities, in contrast to the secondary
autophagy defects due to mutations in BAG3 (ref. 30) or the kinase
domain of titin26,31. This suggests that EPG5 may not be involved in
the autophagic degradation of myofibrillar components.
We hypothesized that the increases in SQSTM1 and NBR1 in
atrophic muscle fibers reflect a block in the autophagy pathway,
in keeping with the accumulation of autolysosomes observed after
epg-5 knockdown in multicellular organisms10 and the observation of
severe muscle atrophy and cardiac failure following autophagy inhi-
bition22,32,33. To further investigate whether the increased SQSTM1
and NBR1 levels are reflective of increased upstream induction or
downstream blockade of the autophagy pathway, we exposed fibro-
blasts derived from affected individuals and healthy controls for
12 h to the autophagy inducer rapamycin (an inhibitor of the mTORC1
complex) and the autophagy inhibitor bafilomycin (an inhibitor of
the autolysosomal H+ ATPase required for acidification and, hence,
degradation of lysosomal contents34). Untreated cells from affected
individuals showed higher amounts of SQSTM1, NBR1 and LC3, par-
ticularly of processed, lipidated LC3-II. Induction of autophagy by
rapamycin or blockade of autophagosomal clearance by bafilomycin
led to strong accumulation of NBR1 and SQSTM1 in control cells after
12 h, whereas the combination of upstream activation by rapamycin
and blockade of clearance by bafilomycin led to further accumulation
of these proteins as well as of LC3-II after 12 h (Fig. 3), as expected.
In contrast, the higher amounts of NBR1 and SQSTM1 were not fur-
ther increased in cells from individuals with Vici syndrome after treat-
ment with rapamycin or combination treatment. This suggests that the
induction of early steps in autophagy, including the processing of LC3-
I to LC3-II, is not impaired in Vici syndrome, whereas the clearance
of autophagosomal cargo is severely impaired. These observations
are supported by immunofluorescence microscopy in fibroblasts from
individuals with Vici syndrome: we observed a large number of base-
line NBR1- and SQSTM1-positive puncta (Supplementary Fig. 2)
as well as LC3- and SQSTM1-positive puncta (Supplementary
Fig. 3) compared to control cells, indicative of the accumulation of
autophagosomes in the EPG5-deficient cells. In contrast, we found
that the fusion of LC3-positive puncta with lysosomes, as indicated
by the colocalization of LC3 with lysosome-associated membrane
proteins (LAMP1), was reduced in fibroblasts from individuals
with Vici syndrome (Fig. 4). Similarly, there was much less fusion
of NBR1-positive puncta with LAMP1-positive lysosomal vesicles
(Supplementary Fig. 4). Lastly, baseline amounts of protein poly-
ubiquitinated at lysine 63 (a measure of ubiquitination products
Figure 2 Accumulation of NBR1-positive
puncta in the skeletal muscle of an individual
with Vici syndrome. Transverse sections
from muscle biopsies from subject 3.1
(top) and a normal control (bottom) were
stained with monoclonal antibody against
NBR1 or polyclonal antibody to MURF2 and
counterstained with antibody to the M-band
titin, titin M8. Accumulation of NBR1 in puncta
(autophagosomes, arrowheads) and of MURF2,
as well as fiber inhomogeneity with marked
fiber atrophy, characterizes Vici syndrome
muscle. Numerous fibers of very small
cross-sectional area (arrows) with high
content of MURF2 and NBR1 puncta are
frequently seen. Scale bar, 10 µm.
Figure 3 Autophagy is blocked at a late stage in Vici syndrome.
Accumulation of autophagy adaptors NBR1 and SQSTM1 and the
phagophore membrane component LC3 is induced in fibroblasts
from two controls and a subject with Vici syndrome (subject 4.1)
after 12 h of treatment with rapamycin or dual treatment with rapamycin
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destined for autolysosomal degradation) were higher in cells from
individuals with Vici syndrome (Supplementary Fig. 5). Taken
together, these results indicate a severe deficit in autophagosomal
clearance associated with mutations in EPG5, which results in the
accumulation of autophagic cargo in NBR1- and SQSTM1-positive
autophagosomes and impaired fusion with lysosomes.
To determine whether these changes also involve upstream regu-
latory mechanisms of autophagy, we analyzed the AKT-mTOR pro-
tein kinase pathway, a major pathway controlling the expression of
autophagy proteins, by inhibiting the transcriptional activity of FOXO
transcription factors35. We observed markedly lower phosphoryla-
tion of the two activating sites in AKT (Ser308 and Ser473) and, as a
result, lower phosphorylation of the AKT substrate GSK-3β on the
inhibitory Ser21 site (Supplementary Fig. 6). Whereas total FOXO3
protein amounts were about 5-fold higher, the ratio of phosphorylated
to total FOXO was decreased to about 0.6 (Supplementary Fig. 7).
These observations suggest a profound derailment of autophagy in
Vici syndrome on multiple regulatory levels, including transcriptional
regulation via the AKT pathway.
These findings are all in agreement with histopathological fea-
tures consistent with defective autophagy, such as the prominence
of autophagic vacuoles, storage of abnormal material and secondary
mitochondrial abnormalities, the latter of which likely reflects the
evolutionarily conserved role of the autophagy pathway in maintain-
ing mitochondrial quality and function36. On the clinical level, skeletal
and cardiac muscle involvement also features prominently in other
conditions with primary disturbance of autophagy, such as X-linked
recessive Danon disease (MIM 300257), caused by abnormal autolyso-
some formation due to mutations in the lysosomal LAMP-2 gene37.
Disrupted autophagy is also an important secondary pathogenetic
mechanism in other neuromuscular disorders26,38–40. In the heart,
autophagy has an important role in the constant renewal of post-
mitotic cardiomyocytes41 and functions to meet nutrient requirements
at times of increased metabolic demand42,43. Of note, microscopic
cardiac changes observed post mortem in one individual with Vici
syndrome were much more pronounced in the left compared to
the right ventricle9, suggesting a relationship between the inten-
sity of physiological workload and the severity of histopathological
Additional defects in CNS development, immune regulation
and skin pigmentation in individuals with Vici syndrome impli-
cate autophagy in a wider range of cellular processes. In the brain,
autophagy has been extensively investigated as a key pathogenic
mechanism in various neurodegenerative disorders (for reviews,
see refs. 17,44). The observation of callosal agenesis and disturbed
neuromigration in individuals with Vici syndrome identifies aberrant
autophagy also as a cause of disturbed embryonic CNS development,
a notion so far only supported by the observation of increased
neuronal proliferation and severe neural tube defects in mice with
null mutations in Ambra1, a positive autophagy regulator with
predominant expression in neural tissues45. In the immune system,
autophagy is generally involved in the delivery of microorganisms to
lysosomes (xenophagy) and, more specifically, in trafficking events
that activate immunity46. As indicated by observations in the T cell–
specific Atg5-knockout mouse, autophagy is directly important for
T-cell survival and proliferation47, and our findings also indicate a
role for autophagy in B-cell homeostasis. The finding of generalized
hypopigmentation in Vici syndrome supports a functional relation-
ship between melanogenesis and the autophagic machinery, as has
been suggested recently48.
In conclusion, our findings identify Vici syndrome as a paradigm
for human multisystem disorders associated with defective autophagy.
The wide range of associated clinical manifestations suggests that
EPG5 is a pivotal protein within the autophagic machinery in different
tissues. Other genes within the same pathway are plausible candidates
in Vici syndrome cases not linked to the EPG5 locus.
URLs. Novoalign, http://novocraft.com/; Primer3, http://frodo.wi.mit.
edu/primer3/; Merlin, http://www.sph.umich.edu/csg/abecasis/Merlin/
index.html; Alamut, http://www.interactive-biosoftware.com/; SIFT,
http://sift.jcvi.org/; Align GVGD, http://agvgd.iarc.fr/agvgd_input.
php; PolyPhen-2, http://genetics.bwh.harvard.edu/pph2/; NHLBI
Exome Variant Server, http://evs.gs.washington.edu/EVS/; IDT, http://
Methods and any associated references are available in the online
version of the paper.
Note: Supplementary information is available in the online version of the paper.
We are grateful to the individuals with Vici syndrome and their families for their
participation in this study. We would like to thank our colleagues at the Genomics
Facility of the Comprehensive Biomedical Research Centre of Guy’s and
St Thomas’ NHS Foundation Trust for their support. We would also like to
thank the physicians D. Creel, R.O. Hoffman and L. Al-Gazali for their input
and productive discussions. H.J. was supported by a grant from the Guy’s and
St Thomas’ Charitable Foundation (grant 070404). M.G. and A.L.K. were
supported by the Leducq Foundation Transatlantic Network Proteotoxicity
(11 CVD 04) and the Medical Research Council of Great Britain (MR/J010456/1).
M.G. holds the British Heart Foundation Chair of Molecular Cardiology.
H.J. would like to dedicate this work to the memory of Rahul Ghosh, his first
patient with Vici syndrome.
T.C. designed the experiments, performed whole-exome capture, Sanger
sequencing, cDNA sequencing and quantitative PCR (qPCR) analysis, analyzed
data and wrote the manuscript. A.L.K. and B.B. performed immunostaining,
confocal microscopy, cell culture studies and protein blotting. Z.U. performed
Figure 4 Fusion of LC3-positive puncta with lysosomes in Vici syndrome.
In control fibroblasts subjected to 6 h of bafilomycin treatment, lysosomal
structures were detected by staining with monoclonal antibody to
LAMP1. Numerous LC3-positive autophagosomes are found engulfed
by the LAMP1-positive vesicular structures (arrowheads). In contrast,
fibroblasts from individuals with Vici syndrome consistently show smaller
LC3-positive puncta that only sporadically colocalize with LAMP1, with
many isolated LC3-positive puncta (arrows). Note that LC3 signal in Vici
syndrome cells occurs mostly at the rim of LAMP1-positive structures, not
centrally. Scale bar, 5 µm.
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qPCR analysis. F.S., M.A.S., S.Y. and S.A. prepared and performed whole-exome
capture and analyzed the exome sequencing data. C.D.-V., E.B., V.M., M.A.-O.,
S.K., C.K., G.F.H., F.A.W., A.E.t.H., R.C.R., D.M., R.M., M.H., E.S., D.S., P.M.K.,
C.W., F.M.F., S.A.-K., J.H. and M.D.C. provided clinical data. S.B., I.B., H.-H.G.
and C.A.S. provided and analyzed neuropathological data. S.M. and D.J. provided
clinical data and oversaw genetic aspects of the research. M.G. analyzed data
obtained from immunostaining, confocal microscopy, cell culture studies
and protein blotting and wrote the manuscript. H.J. provided clinical and
neuropathological data, analyzed exome and Sanger sequencing data,
oversaw all aspects of the research and wrote the manuscript.
COMPETInG FInAnCIAL InTERESTS
The authors declare no competing financial interests.
Published online at http://www.nature.com/doifinder/10.1038/ng.2497.
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Subjects. A total of 18 individuals with Vici syndrome were included in this
study. Affected individuals were included in the study if four of the five major
diagnostic criteria (callosal agenesis, cataracts, cardiomyopathy, hypopigmen-
tation and immunodeficiency) were fulfilled. Clinical features of the individuals
included in this study are summarized in Supplementary Table 1 and depicted
in Supplementary Figure 1. Research ethics committee approval (reference
09/H0807/089, Identification of Genes Underlying Neurodevelopmental
Disorders, and reference 06/Q0406/33, Setting Up of a Rare Diseases Biological
Samples Biobank for Research to Facilitate Pharmacological, Gene and Cell
Therapy Trials in Neuromuscular Disorders) was obtained. Informed consent
was obtained from all individuals and the legal guardians of minors.
Exome capture and sequencing. Library construction. Libraries were prepared
according to Agilent’s SureSelect Human All Exon Capture v.1.0.1 protocol
(Illumina Paired-End Sequencing, Agilent Technologies), with the following
modifications: 3 µg of genomic DNA from subjects 2, 4, 5.1 and 5.2 were
sheared using adaptive focused acoustics (Covaris S2), using the following
conditions: 20% duty cycle, 5 intensity, 200 cycles per burst and frequency
sweeping mode for 90 s at 4 °C, to give an average fragment size of 200 bp.
Fragmented DNA was then subjected to end repair, phosphorylation, A-base
addition and adaptor ligation followed by six cycles of PCR amplification.
Solution capture. Solution capture was carried out according to Agilent’s
v.1.0.1 protocol with the following modifications: 500 ng of prepped library
were mixed with 7.5 µg of human Cot-1 DNA and dried down to a pellet
using a Savant SpeedVac 120 (Thermo Fisher Scientific). The pellet was resus-
pended in 3.4 µl of water to give a final library concentration of 147 ng/µl.
After hybridization and selection, libraries were subjected to ten cycles of
Sequencing. Target-enriched sequencing libraries were quantified by Qubit
(Invitrogen). Denatured libraries were loaded onto an Illumina Genome
Analyzer IIx flow cell at a concentration of 10 pM.
Sequencing of flow cells was carried out as described in the manufacturer’s
instructions, using one lane of 76-bp paired-end sequencing per sample.
Read conversion and alignment. Read conversion, alignment and variant
calling were conducted in parallel using both the NextGENe (SoftGenetics)
and Linux-based tools on the King’s College London (KCL) cluster as follows.
Sequencing metrics are described in Supplementary Table 2.
NextGENe read conversion and alignment. Reads were obtained in qseq
format from the Genome Analyzer IIx instrument. NextGENe software was
used to quality filter and convert the reads into fasta format. The following
filtering settings were used: median phred score of 15, maximum number of
uncalled bases of 10, called base number greater than or equal to 25, and trim
or reject reads where 10 or more bases have a phred score of 15 or less. Reads
not fulfilling these quality criteria were omitted from alignment.
Quality-filtered fasta-converted reads were aligned to the whole human
genome reference sequence (GRCh37) using the preindexed reference in
KCL Linux alignment. Sequence reads were aligned to the reference genome
(hg18) with Novoalign (see URLs). Duplicate reads, resulting from PCR
clonality or optical duplicates, and reads mapping to multiple locations were
excluded from downstream analysis. Single-nucleotide substitutions and small
insertions or deletions were identified and quality filtered within the SAMtools
software package49 and in-house software tools. Variants were annotated
with respect to genes and transcripts with the SNPClassifier tool50. Filtering
of variants for novelty was performed by comparison to dbSNP131 and
1000 Genomes Project pilot SNP calls (March 2010).
Gene identification strategy and results. Analogous gene identification strat-
egies were employed for the output from both the NextGENe and KCL Linux
pipelines. Removal of previously identified variants (NextGENe: dbSNP; KCL
pipeline: dbSNP, 1000 Genomes Project and in-house exomes) and synony-
mous changes resulted in the identification of 9 and 11 genes, respectively, with
homozygous sequence variants in both individuals (subjects 5.1 and 5.2) from
the consanguineous kindred (Supplementary Table 3). Of these genes, five
were shared, and ten were identified by a single analysis pipeline. Differences
between the two data sets can be attributed to alternative variant calling set-
tings and algorithms and to the subtraction of in-house exome data in the KCL
pipeline. The sequencing data for candidate genes identified in both analyses
were then interrogated in the remaining two exomes (from subjects 2.1 and
4.1). For both data sets, a single gene, EPG5 (KIAA1632), was identified in
which all affected individuals had two truncating mutations in keeping with
expected inheritance patterns (Table 1).
The homozygous sequence variants in subjects 5.1, 5.2 and 4.1 were present
at read depths of 48, 52 and 113, respectively. The heterozygous variants in
subject 2.1, c.2413–2A>G and c.6724delA, were present in reference to variant
read depth ratios of 35:22 and 68:50, respectively.
Sanger sequencing. All EPG5 mutations identified by exome sequencing
were confirmed by bidirectional Sanger sequencing. Sequencing primers
were designed to EPG5 (NM_020964.2), EI24 (NM_004879.3) and VMP1
(NM_030938.3) using Primer3 (see URLs) to cover all coding exons and
intron-exon boundaries. PCR and sequencing reactions were performed as
described previously5. Affected individuals without truncating mutations in
EPG5 were subjected to sequencing of the human homologs of the Epg genes
identified previously10, EI24 and VMP1. All sequencing primers used in this
study are listed in Supplementary Table 4.
Inheritance studies and mutation frequency in the general population.
ABI traces for mutant alleles identified in families 1–13 are shown in
Supplementary Figure 8. Parental samples were available in 11 of the 13 families
in which sequence variants were detected. In all cases, sequence variants
exhibited segregation in accordance with autosomal recessive inheritance.
Missense variants identified in subjects 6.1 and 12.1 segregated on the opposite
allele to the truncating mutations identified in these families (Supplementary
Fig. 8). Additionally, unaffected siblings were available for segregation ana-
lysis in families 5 and 9 and were shown to have genotypes consistent with
the expected inheritance pattern. Using Merlin (see URLs), we calculated an
LOD score of 8.304 across all families (assuming a recessive model with full
penetrance and no phenocopies, a marker allele frequency of 0.000444444 and
a disease frequency of 0.00001).
Sequence variant in silico analysis and control chromosome studies. The
missense variants, p.Leu457Pro and p.Gln784Pro, identified in subject 6.1
were subjected to in silico analysis using the Alamut software suite (see URLs).
The affected amino acids showed moderate and low levels of conservation,
respectively, with Grantham distances of 98 and 76. The SIFT and Align GVGD
prediction tools indicated that neither change was likely to be disease caus-
ing, but PolyPhen-2 predicted that both changes were probably damaging.
Neither variant was predicted to cause aberrant splicing. Both variants were
absent from 374 in-house control chromosomes and from more than 9,000
chromosomes on the Exome Variant Server (see URLs). It is therefore unclear
whether either or both of these variants are responsible for the presumed
loss of function of this allele found in trans with the mutation encoding the
p.Cys1945* alteration identified in this subject. Undetected mutations, such as
large deletions, duplications, inversions, deep intronic splice-site mutations or
promoter mutations in cis with these missense variants cannot be eliminated
as the true causative change on this allele.
The p.Cys2038Arg missense variant identified in subject 12.1 was subjected
to in silico analysis in the same manner. Cys2038 is highly conserved (from
Drosophila melanogaster). The cysteine-to-arginine Grantham distance is 180.
PolyPhen-2 and SIFT predicted that the change affected protein function,
although, conversely, Align GVGD predicted that the change was unlikely to be
pathogenic. This amino-acid change is absent from the Exome Variant Server,
although an amino-acid change at the same location (p.Cys2038Gly) has been
observed with a frequency of 1 in 9,990 alleles. Other mutations on this allele
that are undetectable by sequencing of coding regions cannot be eliminated,
but the high degree of conservation and the disruption of a cysteine residue,
residues that are often involved in protein domain structure, indicate that this
variant may be the second disease-causing alteration in this subject.
Sequence variants affecting canonical splice donor and acceptor sites
were observed in four families (families 2–4 and 10) and were predicted to
abolish splicing, according to the prediction tools in Alamut, namely
© 2012 Nature America, Inc. All rights reserved. Download full-text
SpliceSiteFinder-like, MaxEntScan, NNSPLICE and Human Splicing Finder.
The homozygous variant c.1007A>G (p.Gln336Arg) detected in subject 7
affects the penultimate base of exon 2 and was predicted by SpliceSiteFinder-
like and NNSPLICE to abolish the donor splice site; scores for MaxEntScan
and Human Splicing Finder also indicated reduced splicing. Additionally,
this variant was absent from the Exome Variant Server data set. This variant
therefore seems highly likely to be the pathogenic mutation in this subject.
Quantitative fluorescent PCR. QF-PCR was used to interrogate exon copy
number in subject 14.1. PCR was performed using a two-stage reaction with
fluorescently labeled primers complementary to a tag sequence incorpo-
rated into the exon-specific primers in a multiplex reaction (full details are
available upon request)51.
cDNA sequencing. Nested PCR primers flanking exon 28 of EPG5 were
designed using Primer3. RNA was extracted from fibroblast culture from
subject 4.1 using the RNeasy kit (Qiagen NV) and reverse transcribed using
SuperScript III First-Strand Synthesis SuperMix (Life Technologies) with
oligo(dT) primer. The resulting cDNA was subjected to two-stage nested
PCR and sequenced. PCR and sequencing reactions were performed as
Quantitative RT-PCR. qPCR was used to interrogate relative transcript
abundance in subject 4.1. Dual-labeled probes and primers were designed
for EPG5 and ACTB using the IDT RT-PCR assay design tool (see URLs).
qPCR reactions were carried out using TaqMan Gene Expression Master Mix
in a 25-µl reaction on an ABI 7900 instrument (Life Technologies). Analysis
was conducted using the ∆∆CT method (according to the Applied Biosystems
protocol Performing Relative Quantitation of Gene Expression Using
Real-Time Quantitative PCR).
Immunostaining and confocal microscopy. Skeletal muscle sections were
lightly fixed in 4% paraformaldehyde, permeabilized in 0.05% Tween-20 in
PBS and blocked in 10% goat serum in PBS. Sections were immunostained
using antibodies against SQSTM1 (Abcam, ab56416), NBR1 (Abcam, ab55474)
and MURF2 (Abcam, ab4387) and counterstained with an affinity-purified
rabbit polyclonal antibody against M-band titin (T-M8ra)52 using standard
procedures53. Fibroblasts were cultured for the times indicated, washed briefly
in PBS and fixed in methanol for 10 min at −20 °C. Cells were immunostained
using antibodies against SQSTM, NBR1 (Novus Biologicals, NBP1-71703),
LAMP1 (monoclonal H4A3, Abcam, ab25630) and LC3B (Cell Signaling
Technology, 2775S). Fluorescent secondary antibodies were from Jackson
Immunoresearch and Molecular Probes. Specimens were analyzed by confocal
microscopy using a Zeiss LSM-510 Meta microscope with 63× magnification
and 2–4× zoom, and identical gain settings were used for image recording for
all related samples.
Cell culture studies and protein blotting. Fibroblast cultures were main-
tained in DMEM supplemented with 10% FBS, 2 mM glutamine, penicillin
(100 U/ml), streptomycin (100 µg/ml) and 50 µg/ml uridine in 3- or 10-cm
culture dishes in an atmosphere with 5% CO2. Cells were treated with 100 nM
rapamycin and/or 200 nM bafilomycin for 5–12 h before lysis or received
equal concentrations of vehicle (DMSO). Cells were harvested after wash-
ing twice with PBS by direct addition of Laemmli sample buffer, and lysates
were separated by 10–15% SDS-PAGE. Protein loading was normalized to
actin staining and GAPDH immunoreactivity in protein blots. Protein blots
were performed using standard procedures53, and antibodies to LC3B (Cell
Signaling Technology), NBR1 (Abcam) and SQSTM1 (Abcam). All antibodies
to phosphorylated proteins and pan antibodies (AKT, FOXO, GSK-3β, mTOR
and p70S6K) were obtained from Cell Signaling Technology. The catalog
numbers for the antibodies used were as follows: AKT (P-S473 AKT, 4060;
P-T308 AKT, 2965; pan AKT, 4691); FOXO (P-Thr24 FOXO1/Thr32 FOXO3a,
9464; P-S253 FOXO3a, 9466; P-S318/321 FOXO3a, 9465; pan FOXO3a, 9467);
GSK-3β (P-S9 GSK-3β, 9322; pan GSK-3β, 9315); mTOR (P-S2481 mTOR,
2974; P-S2448 mTOR, 2971; pan mTOR, 2983); and p70S6K (P-T389 p70S6K,
9234; pan p70S6K, 2708). Densitometric quantification was carried out as
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defined domains of myomesin, M-protein, and the 250-kD carboxy-terminal region
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