Human Molecular Genetics, 2008, Vol. 17, No. 23
doi:10.1093/hmg/ddn261
Advance Access published on August 23, 2008
3663–3674
Mitochondrial processes are impaired in
hereditary inclusion body myopathy
Iris Eisenberg1,{, Noa Novershtern2, Zohar Itzhaki3, Michal Becker-Cohen1,
Menachem Sadeh4, Peter H.G.M. Willems5,6, Nir Friedman2,
Werner J.H. Koopman5,6 and Stella Mitrani-Rosenbaum1,
1
Goldyne Savad Institute for Gene Therapy, Hadassah Hebrew University Medical Center, Jerusalem, Israel,
The Selim and Rachel Benin School of Computer Science and Engineering and 3Department of Molecular Genetics
and Biotechnology, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel, 4Department of
Neurology, Wolfson Hospital, Holon, Israel and 5Department of Biochemistry and 6Microscopical Imaging Centre,
Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen,
The Netherlands
2
Received July 1, 2008; Revised August 13, 2008; Accepted August 21, 2008
Hereditary inclusion body myopathy (HIBM) is an adult onset, slowly progressive distal and proximal myopathy.
Although the causing gene, GNE, encodes for a key enzyme in the biosynthesis of sialic acid, its primary function in HIBM remains unknown. To elucidate the pathological mechanisms leading from the mutated GNE to the
HIBM phenotype, we attempted to identify and characterize early occurring downstream events by analyzing
the genomic expression patterns of muscle specimens from 10 HIBM patients carrying the M712T Persian
Jewish founder mutation and presenting mild histological changes, compared with 10 healthy matched control
individuals, using GeneChip expression microarrays. When analyzing the expression profile data sets by the
intersection of three statistic methods (Student’s t-test, TNoM and Info score), we found that the HIBM-specific
transcriptome consists of 374 differentially expressed genes. The specificity of the HIBM transcriptome was
assessed by the minimal transcript overlap found between HIBM and the transcriptome of nine additional
muscle disorders including adult onset limb girdle myopathies, inflammatory myopathies and early onset
conditions. A strikingly high proportion (18.6%) of the overall differentially expressed mRNAs of known function were found to encode for proteins implicated in various mitochondrial processes, revealing mitochondria
pathways dysregulation. Mitochondrial morphological analysis by video-rate confocal microscopy showed
a high degree of mitochondrial branching in cells of HIBM patients. The subtle involvement of mitochondrial
processes identified in HIBM reveals an unexpected facet of HIBM pathophysiology which could at least
partially explain the slow evolution of this disorder and give new insights in the disease mechanism.
INTRODUCTION
Hereditary inclusion body myopathy (HIBM) is a neuromuscular disorder characterized by adult onset, slowly progressive
distal and proximal muscle weakness and a typical muscle
pathology including cytoplasmatic rimmed vacuoles and
cytoplasmatic or nuclear filamentous inclusions composed
of tubular filaments. This disease is common in the Jewish
Persian community, with a prevalence of 1:1500 (1,2), and
in other Jewish Middle Eastern population clusters, but with
an unusual clinical feature: the sparing of the quadriceps
muscle, even in advanced stages of the disease. The same
disease, termed distal myopathy with rimmed vacuoles
(DMRV), has been described in a cluster of Japanese patients
(3,4). The responsible gene, GNE, presents a single homozygous missense mutation (M712T) in all Persian and other
To whom correspondence should be addressed at: Goldyne Savad Institute of Gene Therapy, Hadassah Hebrew University Medical Center, Hadassah
Hospital, Mount Scopus, Jerusalem 91240, Israel. Tel: þ972 25819134; Fax: þ972 25819134; Email: [email protected]
†
Present address: Howard Hughes Medical Institute, Program in Genomics, Division of Genetics, Children’s Hospital, Harvard Medical School,
Boston, MA, USA.
# The Author 2008. Published by Oxford University Press. All rights reserved.
For Permissions, please email: [email protected]
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Human Molecular Genetics, 2008, Vol. 17, No. 23
Middle Eastern Jewish and non-Jewish HIBM patients (5),
confirming the founder effect hypothesis of this disorder in
Middle Eastern Jews. More than 50 different mutations in
this same gene have now been identified worldwide in quadriceps sparing HIBM non-Jewish patients (5), including Japanese DMRV patients (6,7), establishing HIBM and DMRV
as the same entity.
GNE encodes the enzyme UDP-N-acetylglucosamine
2-epimerase/N-acetylmannosamine kinase (UDP-GlcNAc
2-epimerase/ManNAc kinase; GNE), which is the key
enzyme in the biosynthetic pathway of sialic acid (8). Sialic
acid, the most abundant terminal monosaccharide on glycoproteins and glycolipids of eukaryotic cells, is involved in
multiple biological pathways, playing an important role in
many cellular functions, such as adhesion processes, cell
migration, inflammation, wound healing and also in metastasis
(9,10). GNE catalyzes the first two steps of the sialic acid
biosynthesis, the formation of N-acetylmannosamine from
UDP-N-acetylglucosamine and the consecutive phosphorylation of the sugar at C6. As a bifunctional enzyme, it consists
of two functional domains, an epimerase domain and a kinase
domain (11). Most Middle Eastern patients are homozygous
for a single mutation, whereas most of the others are found
as compound heterozygotes, carrying mutations either both
at the epimerase domain, both at the kinase domain, or one
in each domain of the enzyme. The broad distribution of
mutations along the gene tends to exclude the impairment of
a solitary aspect of the enzyme’s function as the underlying
cause for HIBM. It suggests instead a more non-specific disruption that results in a general loss of function. Although
the role of GNE has been thoroughly recognized as a key
enzyme in the biosynthetic pathway of sialic acid, the
process by which the mutations in the enzyme lead to
muscle disease is not understood. In spite of a slight reduction
in the enzymatic activity of most mutated GNE proteins, and
some reports of slightly reduced sialylation in histological
sections of some patients, there is no clear effect of the
mutations upon the overall production of sialic acid in
HIBM patients and therefore the data do not support a priori
an explanation for the pathology of the disease through the
conventional sialic acid pathway (12 –17). Although a
mutated GNE transgenic mouse model generated on a
GNE 2/2 cell basis (using a D176V GNE missense mutation
occurring in the epimerase domain of the enzyme, a prevalent
mutation in Japan) showed a strong hyposialylation of most
organs in vivo (18), the primary function of GNE in HIBM,
impaired by the different mutations, remains to be elucidated.
In recent years, microarray studies and genome-wide
expression profiling of normal and diseased skeletal muscle
in human and in mouse models have generated a detailed
insight into the molecular processes accompanying different
conditions (19 – 24) such as nemaline myopathy (NM) or
Duchenne muscular dystrophy (DMD), facilitating our understanding of the underlying disease mechanism. In an effort to
elucidate the pathological mechanisms of the mutated GNE
gene leading to the HIBM phenotype, we have attempted to
identify and characterize the early occurring downstream
events of this pathway by analyzing the genome scale
expression patterns in the disease target tissue. We have compared gene expression patterns of muscle specimens from
HIBM patients carrying the M712T mutation, presenting
with mild histological changes, against healthy matched
control individuals, using HG-U133A GeneChip expression
microarrays. Clusters of genes functionally related to the
mutated GNE, and possibly involved in the phenotype of
HIBM, were defined and analyzed. In particular functional
assessment of mitochondrial involvement was performed by
refined mitochondrial morphology analysis.
RESULTS
A distinct expression signature for HIBM-affected muscle
To define gene expression patterns and pathways specifically
dysregulated in HIBM and involved early in the disease
process, the global mRNA expression profile from 10 HIBM
patients carrying the founder Persian Jewish mutation in GNE
along with 10 muscle samples from matched healthy individuals
(Table 1; Supplementary Material, Fig. S1) was analyzed using
Affymetrix HG-U133A GeneChip arrays. Following array
signal quantitation and normalization, a set of 7530 active transcripts which represent 34% of the 22 000 different genes
present on the array were identified as expressed in muscle
tissue, and showed variance among subjects. The differential
expression pattern of HIBM was generated from these data by
statistical analysis. A signature of 374 transcripts, constituting
5% of the 7530 valid active genes represented on the microarray, were defined as significantly differentially expressed in
HIBM compared with control muscles with a minimum geometric fold change of 1.2 (P , 0.05) (Supplementary Material,
Table S1). Of these, 194 transcripts were found as overexpressed and the remaining 180 were underexpressed in HIBM
versus unaffected muscle tissue. The highest fold change of
more than 2.7 was observed for transferrin receptor (TFRC)
(P ¼ 0.00034) and with the mitochondrial proton carrier uncoupling protein 3 (UCP3) transcript with a downregulation of
2.3-fold (P ¼ 0.0021). Interestingly, the fold change of the
remaining 372 disease signature transcripts was only of a
small magnitude, with most of the transcripts being dysregulated in the range of 1.5 to 21.6-fold change; only six genes
had a fold higher than 2, and 34 genes higher than 1.5-fold.
Table 2 details the 35 genes with the highest fold difference in
expression between healthy and HIBM muscle tissue.
Notably, a strikingly high number of 56 transcripts,
representing 18.6% of the overall differentially expressed
mRNAs of known function, were found to encode for proteins
implicated in various mitochondrial processes (Table 3).
Differentially expressed transcripts in HIBM
To detect a finer structure of transcriptional variation within
each group, hierarchical clustering (Fig. 1) performed on the
374 mRNAs differentially expressed genes selected by the
intersection analysis, identified separate clusters for HIBM
muscles and normal controls and demonstrated clear segregation of muscle biopsies taken from HIBM patients from
the control unaffected samples. The two normal outliers
classified with the affected individuals were both from relatively adult individuals (48 and 56 years old) but no direct
explanation could be given for their outlier clustering, together
Human Molecular Genetics, 2008, Vol. 17, No. 23
3665
Quantitative real-time PCR confirmed the differential
expression identified by the microarray analysis
Table 1. Clinical information
Sample
id
Pathology
stage
Tissue type
Clinical
status
Age
Gender
Anesthesia
B150
B143
B146
B153
B118
B155
B149
B144
B81
B77
2
3
2
1
1
2
2
2
2
3
Affected
Affected
Affected
Affected
Affected
Affected
Affected
Affected
Affected
Affected
26
55
45
29
29
59
40
46
25
26
M
F
M
M
M
F
M
F
F
M
Local
Local
Local
Local
Local
Local
Local
Local
Local
Local
B100
B101
B162
B127
B119
B158
B163
B165
B129
B170
1
1
1
1
1
1
1
1
1
1
Deltoid
Deltoid
Deltoid
Deltoid
Biceps
Quadriceps
Deltoid
Deltoid
Biceps
Tibialis
anterior
Quadriceps
Quadriceps
Gluteus
Deltoid
Deltoid
Paraspinal
Triceps
Deltoid
Quadriceps
Triceps
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
Normal
18
52
56
26
19
73
31
74
21
48
M
F
M
M
M
F
F
F
M
F
Local
Local
General
Local
Local
General
General
General
Local
General
1, no pathological changes; 2, mild changes; 3, moderate– severe changes.
with the oldest affected individuals. On the normal branch of
the cluster, samples distribution did not correlate with either
age or muscle type and no obvious correlation between the
disease state and muscle type was observed within the group
of affected individuals (Fig.1, top panel). Conversely, the
disease expression distribution as displayed in the cluster
does correlate with the severity of muscle pathology, from
specimens with moderate changes on the left side of the
cluster (B77 and B143), then mild changes and finally with
very mild or no pathological changes (Fig. 1). This expression
pattern suggests that disease state has a stronger impact on
gene expression clustering compared with other clinical and
demographic factors examined.
Notably, when applying an unsupervised Principal Component Analysis (PCA) approach to the data set (data not
shown), the 20 samples did not cluster into affected versus
control groups, but rather most of the HIBM-affected
samples clustered together, whereas the healthy controls
seemed to cluster by age, thus suggesting a common process
in HIBM muscles, independent of the variable degrees of
pathology. In order to address the possibility that differential
expression could be associated with the age of the donor, as
particularly in the normal control group age was not entirely
uniform, we estimated the effect of age on gene expression.
Among the 374 differentially expressed genes, Pearson correlation test pointed to correlation between age and expression
of 12 genes (LYPLA1, MRPS33, BNIP3, CL40, JMID3,
MGC4504, UROS, SDBCAG84, CDON, GBAS, TMX2,
MRPL13). However, covariance analysis of the expression
of these 12 genes for age in all examined muscle specimens
remained significantly different between the affected
and the control groups (P ¼ 0.004; P ¼ 0.001; P 0.001;
P ¼ 0.005; P ¼ 0.007; P ¼ 0.002; P ¼ 0.001; P 0.001;
P ¼ 0.034; P ¼ 0.046; P ¼ 0.023; P ¼ 0.005, respectively).
To validate the differential expression identified by microarray
analysis, using an independent method, we performed quantitative real-time PCR analysis for a subset of eight genes with
the highest fold change (LDHB, S100A4, UCP3, BNIP3,
COX7A2, ATP5E, TFRC, TXNIP). Four of the samples
previously analyzed on the arrays (two HIBM patients and
two control muscle samples) were assayed in this experiment.
As illustrated in Fig. 2, the expression level of six of the
transcripts, as assessed by real-time PCR, correlated with the
microarray results and verified the directional change mode
and the magnitude of the change. COX7A2, ATP5E, LDHB,
BNIP3 and TFRC were overexpressed in the range from
2 to 6-fold in the HIBM muscle biopsies, whereas UCP3
was downregulated 3-fold in muscle biopsies taken from
HIBM patients. In contrast to the microarray results,
S100A4 and TXNIP expression evaluated by real-time PCR
did not clearly distinguish between normal and HIBM
muscle in those samples.
Functional annotation revealed mitochondria
pathways dysregulation
Our analyses identified a signature of 374 differentially
expressed transcripts whose expression levels correlate with
HIBM muscle phenotypes. To gain a better understanding of
these transcripts and to uncover common functions among the
differentially expressed genes, we examined their functional
classifications and analyzed for enrichment in the gene ontology
(GO) (25) categories for cellular processes using the DAVID
bioinformatics resource (26). Although the largest resulting category regrouped 73 genes of unknown function, several functional clusters were identified among the additional genes
representing the disease signature. Most notably, genes related
to mitochondrial processes and structure (P ¼ 6.6E214) and
transcripts encoding for proteins involved in transport activity
(P ¼ 6.8E25) were most significantly enriched among the
genes dysregulated in HIBM patients. Genes related to oxidative
phosphorylation, including ATP5E, ATP5H, ATP6V1G1,
COX4I1, COX6C, COX7A2, COX7B, NDUFA1, NDUFB2,
NDUFB6, NDUFB8 and SDHC (P ¼ 7.5E25) and intracellular transport (P ¼ 7.0E24) as well as transcripts encoding
for signal recognition particle (P ¼ 1.8E24) and cytoplasmic
vesicles (P ¼ 1.9E23) were also enriched in the set of differentially expressed genes.
To identify functional modules of gene expression and
interacting partners that are relevant to the HIBM gene
expression signature, we applied annotation enrichment
analysis through the Ingenuity Pathways Knowledge Base
(IPA) (27). Fisher’s exact test was performed to determine
the likelihood of the 374 genes to participate in a given
function or pathway, relative to the total number of occurrences of these genes in all functional annotation in the IPA.
Principal functions associated with the signature were cell
cycle (P ¼ 2.6E24), molecular transport (P ¼ 4.81E24),
cell morphology (P ¼ 7.6E24) and cellular growth and
proliferation (P ¼ 5.21E24) in which four out of the 51
molecules in the group, ALOX15, GNAQ, HBEGF and
3666
Human Molecular Genetics, 2008, Vol. 17, No. 23
Table 2. Top differentially expressed transcripts in HIBM
Probe ID
Symbol
Gene description
Biological functiona
Fold change
P-valueb
208737_at
213379_at
201193_at
212328_at
201849_at
212224_at
218428_s_at
201273_s_at
206765_at
204041_at
218263_s_at
209656_s_at
213019_at
203811_s_at
217801_at
209732_at
208652_at
218139_s_at
201597_at
200777_s_at
217869_at
216814_at
201005_at
203789_s_at
220556_at
200884_at
203186_s_at
201744_s_at
215000_s_at
213765_at
213564_x_at
208691_at
219827_at
201010_s_at
203068_at
ATP6V1G1
CL640
IDH1
KIAA1102
BNIP3
ALDH1A1
REV1L
SRP9
KCNJ2
MAOB
LOC58486
TM4SF10
RANBP6
DNAJB4
ATP5E
CLECSF2
PPP2CA
C14orf108
COX7A2
BZW1
HSD17B12
ACTR3
CD9
SEMA3C
ATP1B4
CKB
S100A4
LUM
FEZ2
MFAP5
LDHB
TFRC
UCP3
TXNIP
KIAA0469
ATPase, Hþ transporting, lysosomal 13 kDa, V1 subunit G isoform 1
Hypothetical protein CL640
Isocitrate dehydrogenase 1 (NADPþ), soluble
KIAA1102 protein
BCL2/adenovirus E1B 19 kDa interacting protein 3
Aldehyde dehydrogenase 1 family, member A1
REV1-like (yeast)
Signal recognition particle 9 kDa
Potassium inwardly rectifying channel, subfamily J, member 2
Monoamine odxidase B
Transposon-derived Buster1 transposase-like protein
Transmembrane 4 superfamily member 10
RAN-binding protein 6
DnaJ (Hsp40) homolog, subfamily B, member 4
ATP synthase, Hþ transporting, mitochondrial F1 complex
C-type (calcium-dependent, carbohydrate recognition domain) lectin
Protein phosphatase 2 (formerly 2A), catalytic subunit, alpha isoform
Chromosome 14 open-reading frame 108
Cytochrome c oxidase subunit VIIa polypeptide 2 (liver)
Basic leucine zipper and W2 domains 1
Hydroxysteroid (17-beta) dehydrogenase 12
ARP3 actin-related protein 3 homolog (yeast)
CD9 antigen (p24)
Sema domain, immunoglobulin domain (Ig), short basic domain
ATPase, (Naþ)/Kþ transporting, beta 4 polypeptide
creatine kinase, brain
S100 calcium-binding protein A4
Lumican
Fasciculation and elongation protein zeta 2 (zygin II)
Microfibrillar associated protein 5
Lactate dehydrogenase B
Transferrin receptor (p90, CD71)
Uncoupling protein 3 (mitochondrial, proton carrier)
Thioredoxin-interacting protein
KIAA0469 gene product
ATP biosynthesis
Biosynthesis
Glyoxylate cycle
Muscle contraction
Apoptosis
1.491
1.514
1.524
1.528
1.536
1.537
1.548
1.548
1.560
1.568
1.592
1.605
1.638
1.649
1.662
1.694
1.697
1.698
1.702
1.702
1.706
1.711
1.817
1.899
1.903
1.911
2.007
2.011
2.143
2.182
2.557
2.746
22.272
21.785
21.562
0.00206
0.01234
0.00206
0.00206
0.00253
0.01234
0.01234
0.01234
0.01234
0.00030
0.00206
0.01234
0.00206
0.01234
0.01234
0.04923
0.01234
0.00206
0.00206
0.00206
0.01234
0.01234
0.01234
0.00992
0.00206
0.01234
0.02918
0.03133
0.01362
0.01234
0.01234
0.0034
0.00206
0.02575
0.01234
Mutagenesis
Ion transport
Electron transport
Protein folding
ATP biosynthesis
Cell adhesion
Cell cycle regulation
Electron transport
Translation regulation
Metabolism
Cell motility
Cell motility
Immune response
Transport
Cartilage condensation
Signal transduction
Ion transport
Energy pathways
Signal transduction
a
GO annotation.
b
Intersection (TNoMl t-test; Info) P-value.
NFATC4, had been previously related to hypertrophy of
muscle cells (28 – 31). Metabolic signaling pathways
consisting of molecules such as ALDH1A1, MAPK3,
MAOB, PPP2CA, GSTP1 and others were also predicted to
be involved in the disease process (27).
HIBM transcriptome specificity
Several of the individual genes found to be dysregulated in
this study have been implicated in previous studies on other
muscular disorders. To examine whether the transcriptome
signature of HIBM muscle and particularly mitochondria
hyperactivation is a common phenomena also in the
expression profile of other human muscular disorders and to
exclude the possibility that the relatively mild fold change
observed in HIBM represents in fact non-specific secondary
events occurring similarly in many other muscular diseases
as common downstream pathways, we have analyzed the signature set of the 374 genes for specificity. In order to evaluate
the specificity of the transcriptional profile to HIBM, the complete Gene Expression Omnibus (GEO) data sets from nine
different muscular disorders were normalized and reanalyzed
with the same statistical tools as HIBM (intersection of
TNoM, t-test and Info score), and subsequently the resulting
status of the 374 transcripts were looked at specifically. We
applied this new analysis to the following conditions: DMD,
Becker muscular dystrophy (BMD), NM, limb girdle muscular
dystrophy 2I (LGMD2I), all of which are congenital or early
onset muscular disorders; adult onset myopathies such as dysferlinopathy (LGMD2B, limb girdle muscular dystrophy 2B),
limb girdle muscular dystrophy 2A (LGMD2A), facioscapulohumeral dystrophy (FSHD) and inflammatory myopathies
including dermatomyositis (DM) and inclusion body myositis
(IBM). Interestingly, among the genes commonly dysregulated in almost all these conditions were the mitochondrial
UCP3 and S100A4, a calcium-binding protein known to be
involved in the regulation of a number of cellular processes
including cell cycle progression and differentiation. Although
the fold-change expression of S100A4 found in the microarray for HIBM could not be confirmed by real-time PCR, it
maybe worth mentioning that S100A4 was also recently
shown to be increased in hypertrophic rat hearts and to have
a pro-cardiomyogenic effects in embryonic stem cell (32).
Figure 3 summarizes the overall results and divides the
analysis into three subclasses, showing only minor overlap
between HIBM and these other muscular diseases. Interestingly,
Human Molecular Genetics, 2008, Vol. 17, No. 23
3667
Table 3. Mitochondria-related transcripts differently expressed in HIBM
Probe id
Symbol
Gene description
Fold change
P-valuea
208737_at
210149_s_at
217801_at
216591_s_at
204041_at
201931_at
218561_s_at
220495_s_at
201634_s_at
217329_x_at
203609_s_at
201175_at
207328_at
217995_at
221028_at
203067_at
201754_at
201597_at
203613_at
217188_s_at
201568_at
213758_at
218201_at
214241_at
202026_at
204067_at
203028_s_at
212224_at
218671_s_at
219827_at
214214_s_at
200807_s_at
208846_s_at
212604_at
217408_at
203517_at
203095_at
202298_at
216409_at
201816_s_at
201849_at
214126_at
200657_at
213149_at
211569_s_at
218654_s_at
217386_at
215171_s_at
200692_s_at
219133_at
217869_at
208652_at
200884_at
218049_s_at
201193_at
218027_at
ATP6V1G1
ATP5H
ATP5E
SDHC
MAOB
ETFA
C6orf149
C5orf14
CYB5-M
COX7B
ALDH5A1
TMX2
ALOX15
SQRDL
MGC11335
PDHX
COX6C
COX7A2
NDUFB6
C14orf1
QP-C
COX4I1
NDUFB2
NDUFB8
SDHD
SUOX
CYBA
ALDH1A1
ATPIF1
UCP3
C1QBP
HSPD1
VDAC3
MRPS31
MRPS18B
MTX2
MTIF2
NDUFA1
DKFZp547D104
GBAS
BNIP3
MCART1
SLC25A5
DLAT
HADHSC
MRPS33
MRPS11
TIMM17A
HSPA9B
FLJ20604
HSD17B12
PPP2CA
CKB
MRPL13
IDH1
MRPL15
ATPase, Hþ transporting, lysosomal 13 kDa, V1 subunit G isoform 1
ATP synthase, Hþ transporting, mitochondrial F0 complex, subunit d
ATP synthase, Hþ transporting, mitochondrial F1 complex, e subunit
Succinate dehydrogenase complex, subunit C
Monoamine oxidase B
Electron-transfer flavoprotein, alpha polypeptide
Chromosome 6 open-reading frame 149
Chromosome 5 open-reading frame 14
Cytochrome b5 outer mitochondrial membrane precursor
Cytochrome c oxidase subunit VIIb
Aldehyde dehydrogenase 5 family, member A1
Thioredoxin-related transmembrane protein 2
Arachidonate 15-lipoxygenase
Sulfide quinone reductase-like (yeast)
Hypothetical protein MGC11335
Pyruvate dehydrogenase complex, component X
Cytochrome c oxidase subunit Vic
Cytochrome c oxidase subunit VIIa polypeptide 2
NADH dehydrogenase 1 beta subcomplex, 6, 17 kDa
Chromosome 14 open-reading frame 1
Low molecular mass ubiquinone-binding protein (9.5 kDa)
Cytochrome c oxidase subunit IV isoform 1
NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 2, 8 kDa
NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 8, 19 kDa
Succinate dehydrogenase complex, subunit D
Sulfite oxidase
Cytochrome b-245, alpha polypeptide
Aldehyde dehydrogenase 1 family, member A1
ATPase inhibitory factor 1
Uncoupling protein 3
Complement component 1, q subcomponent-binding protein
Heat shock 60 kDa protein 1
Voltage-dependent anion channel 3
Mitochondrial ribosomal protein S31
Mitochondrial ribosomal protein S18B
Metaxin 2
Mitochondrial translational initiation factor 2
NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 1, 7.5 kDa
Hypothetical protein DKFZp547D104
Glioblastoma-amplified sequence
BCL2/adenovirus E1B 19 kDa interacting protein 3
Mitochondrial carrier triple repeat 1
Solute carrier family 25, member 5
Dihydrolipoamide S-acetyltransferase
L-3-hydroxyacyl-coenzyme A dehydrogenase, short chain
Mitochondrial ribosomal protein S33
Mitochondrial ribosomal protein S11
Translocase of inner mitochondrial membrane 17 homolog A (yeast)
Heat shock 70 kDa protein 9B (mortalin-2)
Hypothetical protein FLJ20604
Hydroxysteroid (17-beta) dehydrogenase 12
Protein phosphatase 2 (formerly 2A), catalytic subunit, alpha isoform
Creatine kinase, brain
Mitochondrial ribosomal protein L13
Isocitrate dehydrogenase 1 (NADPþ), soluble
Mitochondrial ribosomal protein L15
1.491
1.190
1.662
1.336
1.568
1.475
1.143
1.321
1.109
1.277
1.256
1.329
0.897
0.753
0.858
1.364
1.291
1.702
1.289
1.151
1.266
1.150
1.273
1.262
1.320
1.255
1.284
1.537
1.367
0.449
1.285
1.214
1.329
1.326
1.154
1.388
1.294
1.268
0.894
1.213
1.536
1.077
1.355
1.365
1.416
1.316
1.096
1.291
1.309
1.323
1.706
1.697
1.911
1.427
1.524
1.341
0.002057
0.002057
0.012341
0.000080
0.000296
0.002057
0.012341
0.012341
0.012341
0.019201
0.020496
0.021177
0.012341
0.012341
0.012341
0.012341
0.000217
0.002057
0.003729
0.004065
0.002057
0.012341
0.012341
0.012341
0.000652
0.012341
0.016893
0.012341
0.012341
0.002057
0.012341
0.033861
0.002057
0.002057
0.012341
0.002057
0.012639
0.000975
0.017367
0.040420
0.002527
0.013712
0.023624
0.002432
0.012341
0.002095
0.012341
0.002057
0.012341
0.002057
0.012341
0.012341
0.012341
0.012341
0.002057
0.021037
a
Intersection (TNoM; t-test; Info) P-value.
despite no known phenotypic or pathological similarity, 52
(14%) and 45 (12%) different transcripts were found
to be commonly dysregulated in HIBM and LGMD2A or
LGMD2B, respectively. In the case of IBM, a sporadic inflammatory muscle disorder sharing many pathological features
with HIBM and sometimes considered as the sporadic form
of the hereditary disorder, only a subset of 27 dysregulated
genes were found to overlap with HIBM. For the additional
muscle disorders analyzed, only a small overlap was found
and usually at a much higher fold change (Fig. 3; Supplementary Material, Tables S2a – i). These findings emphasize the
ability of the present expression signature to distinguish
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Human Molecular Genetics, 2008, Vol. 17, No. 23
Figure 1. Hierarchical clustering differentiates HIBM from control individuals. Hierarchical clustering dendogram of the 374 transcripts (rows) showing gene
expression patterns that most reflect differences between muscle samples (columns) from 10 HIBM-affected individuals and 10 unaffected controls (from Supplementary Material, Table S1). Genes were clustered using Cluster software (53). All muscle specimens from affected individuals (blue) fell into one branch of
the dendogram, whereas, with two exceptions, samples from unaffected controls (orange) fell into a separate branch. Muscle type for each sample is shown.
Heatmap tiles show relative intensity signal levels represented by color, with red denoting high expression and green indicating low expression levels.
between HIBM and other muscle disorders and lend further
support to the hypothesis that mitochondria hyperactivation
in HIBM is a unique phenomenon.
Mitochondrial morphology is affected
in HIBM muscle cells
Alterations in mitochondrial metabolism and protein
expression are tightly linked to mitochondrial structure
(33,34), Therefore, since the expression of mitochondriarelated transcripts was remarkably altered, we quantitatively
analyzed mitochondrial shape. To this end, primary cell
cultures were established from three HIBM and three control
individuals’ biopsies. Two muscle cultures of affected
individuals were derived from two of the biopsies analyzed
in the microarray experiment (ms17 from B150 and ms15
from B149) and were matched to two novel controls by age,
gender and muscle type (ms28, 20-year-old male, deltoid,
and ms35, 46-year-old male, deltoid). The third pair was
derived from the tibialis anterior muscle of a 30-year-old
female (ms6; HIBM affected) and from the quadriceps of a
28-year-old control female (ms7).
For mitochondrial morphology analysis, cells were stained
with the mitochondria-specific fluorescent cation rhodamine
123 (R123) and analyzed in three independent experiments
(Fig. 4). We previously validated this approach and demonstrated its suitability for morphology analysis in living cells
(34 –37). Parameters investigated were the number of
mitochondria (Nc), their aspect ratio (AR) as well as their
form-factor (F), reflecting the degree of mitochondrial branching. No significant differences were observed for Nc between
the individual control and HIBM cell cultures (Supplementary
Material, Figs S2 and 4B; gray bars). This demonstrates
that the mitochondrial network is not fragmented in HIBM
Human Molecular Genetics, 2008, Vol. 17, No. 23
3669
Figure 2. Validation of differential gene expression by quantitative real-time PCR. Eight genes identified as dysregulated in HIBM patients were analyzed using
quantitative real-time PCR: COX7A2, cytochrome c oxidase subunit VIIa polypeptide 2; BNIP3, BCL2/adenovirus E1B 19 kDa interacting protein 3; ATP5PE,
ATP synthase, Hþ transporting, mitochondrial F1 complex, epsilon subunit; TFRC, transferrin receptor; LDHB, lactate dehydrogenase B; UCP3, uncoupling
protein 3; TXNIP, thioredoxin-interacting protein; S100A4, S100 calcium-binding protein A4. Relative expression levels of genes are indicated by the fold
changes in expression levels for HIBM patients and control-unaffected individuals. The expression level of a specific gene in the different samples is represented
as the fold value of its expression relative to sample B144, which has been assigned a value of 1 (RQ, relative quantity). PCR reactions were performed in
duplicate using RNA samples from two HIBM-affected and two control individuals previously analyzed on the microarray platform.
cells. Although AR was higher in HIBM cells in one pair (ms6
versus ms7), lower for HIBM cells in another pair (ms17
versus ms28) and similar for the third pair (ms15 versus
ms35) (Supplementary Material, Fig. S2), the differences in
average AR were not statistically significant (Fig. 4B; open
bars). In contrast, the quantification of F revealed a statistically significantly higher value in patient cells, indicating
that mitochondria are more branched in cells of HIBM patients
than in controls (Supplementary Material, Fig. S2; Fig. 4A and
B; black bars).
DISCUSSION
We have identified a transcriptional signature in HIBM consisting of 374 genes, most of them displaying a low fold
change differential expression. This finding could reflect the
mild myopathic changes examined or the adult onset and
slowly progressive course of HIBM. Alternatively, the
summation of different cell types including connective and
endothelial tissue cells, as well as affected and spared
muscle cells, may result in averaging down the expression
differences specific to affected muscle cells.
The major group of differentially expressed transcripts
in HIBM muscle compared with healthy tissue represents
processes taking place in mitochondria. Mitochondria are
the essential organelles for energy supply of cells and it is
now well established that mitochondria are not only the
major suppliers of ATP, but also that they can respond to
multiple physiological stress via signaling processes, cell
growth and differentiation events. In particular, they play a
major role in cell death as sensors of apoptotic signals,
by releasing various pro-apoptotic molecules into the cell
cytoplasm. Mitochondrial intrinsic apoptosis pathway signaling cascade can be induced by several stimuli causing cell
damage, oxidative stress, ER stress and other insults. Interestingly, the transcriptome signature of HIBM muscle includes
transcripts involved in all these pathways, electron transport,
signal transduction, cytoskeleton and cell organization, as
well as in cell death. The precise events upstream to mitochondria apoptotic involvement remain to be fully characterized,
however it is well accepted that Akt is a key factor in this
apoptotic/cell survival process. Notably, our previous findings
of a primary impairment of apoptotic events and survival
defects in HIBM cells, as shown by the lack of activated
Akt response to apoptotic stimuli (38), are in line with the
major mitochondrial involvement reported in these studies.
Also consistent with our present observations, our previous
studies pointed to a slow, but accumulating process of apoptosis of HIBM myotubes; indeed this process is in line with the
mild mitochondrial alterations described here, which very
likely stimulate a cascade of events leading eventually to
increased apoptosis, as illustrated by alterations in the cytochrome c and various cell death protein genes such as
DEDD and DAXX. Myotubes and myofibers are long-lasting
structures which demand a very orchestrated combination of
activities to ensure proper maintenance for survival. Mild
defects in even a small number of targets in this cascade,
such as traffic, ubiquitination, cytoskeleton organization or
cell adhesion functions, could impair the subtle functional
balance in the myofiber, causing maintenance difficulties and
eventual apoptosis. Mitochondria can form a more or less
complex functional network within the cytosol, which is
thought to be linked to mitochondrial and cellular metabolism
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Human Molecular Genetics, 2008, Vol. 17, No. 23
Figure 3. HIBM transcriptome specificity. Expression data sets from previous
studies on nine different muscular disorders were collected through GEO database and analyzed using the same statistical approach (Intersection of TNoM,
t-test and Info) as the current HIBM data set. Number of genes dysregulated in
HIBM which are also dysregulated in the nine other muscular diseases are presented in three separated subgroups. (A) Overlapping dysregulated transcripts
in HIBM and inflammatory myopathies (inclusion body myopathy, IBM and
dermatomyositis, DM). (B) Overlapping dysregulated transcripts in HIBM
and adult onset dystrophies LGMD2B, LGMD2A and FSHD. (C) Overlapping
dysregulated transcripts in HIBM and early onset muscular disorders including
DMD/BMD, NM and LGMD2I. Numbers in white represent the number of
overlapping transcripts differentially expressed in two or more conditions,
as labeled, among the 374 transcripts representing the HIBM transcriptome
signature. Numbers in yellow represent the number of differentially expressed
transcripts unique to HIBM. Circle areas are not to scale. The identity of the
genes overlapping with the HIBM transcriptome for each condition is depicted
in Supplementary Material, Tables S2a– i.
both during healthy and pathological conditions (33,34).
Here we show that in HIBM muscle cell cultures, mitochondria are significantly more branched than in control cells,
whereas their length and number are the same. Interestingly,
fibroblasts from complex I-deficient patients containing
highly branched mitochondria displayed a much less severe
biochemical deficiency than patient cells with fragmented
mitochondria (35). This suggests that in HIBM cells,
the increased mitochondrial branching (compatible with
mitochondrial biogenesis) could be part of a compensatory
response. These most likely very slow and accumulating
events could explain why no major changes in mitochondria
had been reported in HIBM biopsies in any previous study
when analyzed by more conventional techniques which
detect more severe changes such as important deformations,
ragged fibers as a parameter of mitochondrial proliferation
or Cox deficiency as mitochondrial functional impairment,
which are well recognized in sporadic IBM (39), but not in
HIBM (40) (Supplementary Material, Fig. S3). Only when
examined by more sensitive tools, very subtle morphological
changes as the branching structure of mitochondria could be
traced. Changes in mitochondrial morphology are observed
in a variety of conditions, including apoptosis (41) and
oxidative stress (35), and have been shown to affect signal
propagation and cell response to apoptotic stimuli (42).
The vast majority of the 374 differential transcripts detected
in this study represents an HIBM-specific, unique mRNA
signature. Only few of these transcripts were also identified
in other muscular disorders, either early onset dystrophies or
myopathies such as DMD, BMD, NM, adult onset myopathies
such as LGMD2B, LGMD2A, or the inflammatory myopathies
DM and even the pathologically close entity IBM, where the
same pathological changes have been described as in HIBM,
such as rimmed vacuoles and inclusion bodies. These findings
point to the fact that although the final muscle pathology of
several myopathies is characterized by loss of muscle mass,
the HIBM-specific transcriptome profile reported reflects a
combination of events occurring most probably upstream of
common pathological pathways and therefore represents
either relatively early events in the mechanism initiated by
the GNE mutation or specific reactions to such events.
Recently, accumulating evidence points to the role of glycosphingolipids, and especially gangliosides, in mitochondrialmediated regulation of apoptosis events and the development
of pathological processes (43,44). Interestingly, in human
embryonic kidney cells, GNE has been reported to regulate the
levels of GM3 and GD3 synthases, the enzymes responsible for
the incorporation of the first and the second sialic acid structure,
respectively, on glycospingolipids, and subsequently the amounts
of their respective biosynthetic products, the gangliosides GM3
and GD3 (45). In particular, it has been shown that GD3 can
directly interact with mitochondria, recruiting this organelle to
the apoptosis program (46). GD3 ganglioside is thought to
reach mitochondria by the physical continuity occurring
between the endoplasmic reticulum and early Golgi with mitochondrial membranes; this mechanism of recruitment is slower
than the diffusion process of soluble products within the
cytosol and therefore is expected to generate slower kinetics of
apoptosis (45). Furthermore, the specificity of GD3 effects
seems to be due to specific structural features of the two sialic
acid residues because other gangliosides, such as GM3 and
GD1a, do not have any effects on mitochondrial changes (43).
Although GNE transcript and protein levels were found to be
similar in HIBM and control in this study and in an earlier one
(47), respectively, the GNE mutation, by affecting either sialylation or still unrecognized mechanisms, could initiate a slow
process of signal transduction, mostly engaging mitochondrial
pathways and mildly affecting a variety of physiological processes, including remodeling of the cytoskeleton, which would
lead eventually to apoptosis fate. Although mitochondria play
a major role in muscle and indeed various muscular conditions
eventually show mitochondria defects and cell injury, those are
robust events recognized relatively easily (39,48). The rather
subtle involvement of mitochondrial processes identified in
HIBM reveals an unexpected facet of HIBM pathophysiology
which could at least partially explain the slow evolution
of this disorder and give new insights into the disease
mechanism.
Human Molecular Genetics, 2008, Vol. 17, No. 23
3671
Figure 4. Quantification of mitochondrial shape in HIBM myoblasts. (A) Typical example of a control (ms35; top left panel; CT) and HIBM patient myoblast
(ms17; lower left panel) stained with the mitochondria-specific cation rhodamine 123 (RAW). Following image processing, a binary image (BIN) was obtained
from which mitochondrial shape was quantified. (B) Average values of mitochondrial aspect ratio (AR), degree of branching (F) and number of mitochondria per
cell (Nc) in a cohort of three control (CT) and three patient cells.
MATERIALS AND METHODS
Patients and muscle specimens
A total of 20 muscle specimens were available for this
study based on clinical, histological and molecular diagnosis
(Table 1), all in compliance with an approved protocol by
the Institutional Review Board of Wolfson Hospital, Holon,
Israel. All muscle biopsies were collected and immediately
frozen either in liquid nitrogen until used for RNA extraction
or in isopentane for the preparation of frozen sections. An
additional muscle specimen was placed in PBS and used
for the establishment of primary myoblast culture for some
individuals. The 10 normal muscle specimens were collected
from consenting individuals who had undergone either
muscle biopsy which eventually was diagnosed as normal,
or orthopedic surgery. All patients were of Jewish Persian
descent carrying the homozygous M712T mutation in GNE.
In order to detect changes as primary as possible, we selected
only mildly HIBM-affected muscles biopsies when possible.
The pathological changes in the patients’ biopsies were
evaluated by hematoxilin – eosin staining of frozen sections
(Supplementary Material, Fig. S1) and ranged from nonpathological changes in two cases, mild changes in six cases
(including myopathic changes and central nuclei), to moderate
changes presenting atrophic fibers, central nuclei, cytoplasmic
rimmed vacuoles and some extent of fibrosis, in two cases.
Notably, the quadriceps muscle biopsied from a severely
affected patient (B155), already in a wheelchair, was also
mildly affected.
Muscle cell cultures
Primary muscle cultures from HIBM patients carrying the
M712T mutation in GNE and from non-affected control
individuals were established as described previously (38).
Myoblasts were grown in growth medium (GM) until 70%
confluence. To initiate differentiation, GM was replaced by
differentiation medium (DM) containing 2% horse serum
(HS). All studies were carried out on cultures at passage 4 – 8.
RNA preparation and microarray hybridization
Following tissue disruption in dry ice and homogeneization,
total RNA was isolated using TriReagent (Molecular Research
Center, Cincinnati, OH, USA) according to the manufacturer’s
recommended protocol. The integrity of the isolated RNA was
analyzed using a standard 1% agarose gel electrophoresis, and
purified total RNA was quantified using Nanodrop ND1000.
Subsequently, RNA was labeled, fragmented and individually
hybridized to Affymetrix HG-U133A oligonucleotide arrays
(Affymetrix Incorporated, Santa Clara, CA, USA). The complete recommended protocol for preparation and microarray
processing as well as detailed annotations of HG-U133A
arrays are available at the Affymetrix URL (http://www.
affymetrix.com). Briefly, 5 mg of mRNA was used to generate
first-strand cDNA by using a T7-linked oligo (dT) primer.
After second-strand synthesis, in vitro transcription was performed with biotinylated UTP and CTP (Enzo Diagnostics,
Farmingdale, NY, USA), resulting in 300-fold amplification
of RNA. The target cDNA generated from each sample was
processed as per the manufacturer’s recommendation using
an Affymetrix Gene Chip Instrument System. Briefly, spike
controls were added to 15 mg fragmented cRNA before
hybridization. Arrays were then washed and stained with
streptavidin – phycoerythrin, before being scanned on an
Affymetrix GeneChip scanner, and the expression value for
each gene was calculated using Affymetrix Microarray
Software Suite 5.0 (MAS5.0).
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Human Molecular Genetics, 2008, Vol. 17, No. 23
Microarray analysis and normalization
For data normalization, all array scans were pre-processed
with RMAExpress software (49), which employs RMA normalization (background adjustment and quantile normalization), to summarize the final values from the scanner calls.
We considered genes to be valid for analysis if the gene
probe was defined as ‘Present’ (P) according to MAS 5.0 for
at least three out of the 20 samples analyzed. This resulted
in 7530 valid genes. For each gene g and each sample i, the
expression value eg,i was divided
p by the geometric mean of
the gene across all samples n (Pi¼1neg,i). Expression ratios
were then transformed to log (base 2). The complete microarray raw and processed data have been deposited in the GEO
database (series number GSE12648).
Statistical analysis and clustering
The general approach to analysis has been previously outlined
(50) and performed using the ScoreGenes package (http://
compbio.cs.huji.ac.il/scoregenes/). To identify probe sets
which are significantly differentially expressed in HIBM
versus normal muscle tissue and to best distinguish between
the groups, three powerful statistical tests were applied to the
database: threshold number of misclassifications, TNoM (a nonparametric test that measures the number of classification errors
committed when using the best simple threshold to distinguish
between two classes, based on the expression levels of the given
gene) (51), Student’s t-test (two-tailed t-test to measure whether
the mean expression of the gene in the two classes are significantly different) and Info (a non-parametric test that estimates
the uncertainty remaining about the class of a sample after
observing the expression of the individual gene). A lower
Info score indicates a higher predictive value for a given gene
(51). In all three methods, we standardize the score by using
P-values that report the probability of getting this score under
the null hypothesis. For the t-test, the hypothesis is that the
gene has the same mean expression in both classes; in the
TNoM and Info, the null hypothesis is that the sample labels
are independent of the expression value. We compute t-test
P-values using Student’s t distribution. The computation of
P-value for TNoM and Info is based on exact computation
(52). Eventually, differentially expressed genes between the
two tagged groups of samples were evaluated by the intersection of the three methods: to select statistically significant
genes, we chose all overlapping genes that displayed a
P-value of at most 0.05 in all three scoring methods.
Biological pathway analysis
Functional annotation clustering was performed by DAVID
(http://david.abcc.ncifcrf.gov) (26) with medium classification
stringency. The co-regulated functional categories among the
differentially expressed genes were ranked according to a
modified Fisher’s exact test and referred to as ‘enrichment
score’. Changes in gene expression between the experimental
groups were tested for enrichment in functional annotations.
Ingenuity Pathway Analysis (IPA) was further used to find
significant pathways related to the genes dysregulated in
HIBM patients. This significance is expressed as P-value
calculated using the right-tailed Fisher’s exact test to
measure the likeliness of genes from the data set file to participate in that biological function or pathway, as defined in IPA.
Quantitative real-time RT – PCR
To validate the microarray results, we quantified the expression
of several representing transcripts: COX7A2, ATP5E, LDHB,
UCP3, BNIP3, TRFC, S100A4 and TXNIP transcripts in two
affected patients and two control individuals by quantitative realtime PCR (Applied Biosystems, Foster City, CA, USA). One
microgram of total RNA, from the same RNA preparation that
was analyzed in the microarray, was reverse-transcribed using
the Reverse Transcription Reagents Kit (Invitrogen, Carlsbad,
CA, USA), according to the manufacturer’s instructions, using
random primers in a 20 ml reaction. Subsequent PCR reactions
were performed in duplicates, and 2 ml of the cDNA reaction
was used for each assay. The quantitative real-time PCR analysis
was performed with the ABI Prism 7500 Sequence Detection
System (Applied Biosystems) by using TaqMan Universal PCR
Master Mix and Assays-on- Demand Gene Expression probes
(Applied Biosystems) (COX7A2, assay ID: Hs01652418_m1;
ATP5E assay ID: Hs00953807_g1; LDHB, assay ID:
Hs00929956_m1; UCP3, assay ID: Hs01106050_g1; BNIP,
assay ID: Hs00969291_m1; TRFC, assay ID: Hs00174609_m1;
S100A4, assay ID: Hs00243202_m1; TXNIP, assay ID:
Hs01006899_g1). Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Hs99999905_m1) was used as an endogenous
control. Transcripts expression levels and fold changes between
HIBM-affected and control samples were normalized to
GAPDH in each RNA sample.
GEO data sets analysis
In order to identify commonly differentially expressed genes
in various muscle diseases and to assess HIBM transcriptome
specificity, mRNA expression profile data sets of affected
and normal muscle for each of the following diseases were
compiled and analyzed in the same manner as the current
experimental samples (intersection between TNoM, Student’s
t-test and Info). The following data sets were analyzed in
this process: GSE1007, Duchenne muscular dystrophy
(DMD); GSE3307, Becker muscular dystrophy (BMD), limb
girdle muscular dystrophy type2A (LGMD2A), type 2B
(LGMD2B), type 2I (LGMD2I) and Fascioscapulohumeral
dystrophy (FSHD); GSE1551, dermatomyositis (DM) and
inclusion body myopathy (IBM), and nemaline myopathy
(NM) (22). Among the significantly differentially expressed
genes obtained by this analysis for each condition, transcripts
overlapping with the HIBM signature were compiled.
Mitochondrial morphology analysis
Primary myoblasts cultures, derived from affected and control
muscle specimens, were loaded with the mitochondria-specific
fluorescent cation rhodamine 123 and visualized by video-rate
confocal microscopy, as described in detail previously (36).
The parameters examined were the mitochondrial aspect
ratio (AR, reflecting the mitochondrial length/width ratio),
the mitochondrial form-factor (F, which is a measure of
Human Molecular Genetics, 2008, Vol. 17, No. 23
the degree of mitochondrial branching) and the number of
mitochondria per cell (Nc).
SUPPLEMENTARY MATERIAL
Supplementary Material is available at HMG Online.
FUNDING
This work was supported by a grant from the German – Israeli
Foundation for Research and Development (GIF), Jerusalem,
Israel, and in part by grants from the Association Francaise
contre les Myopathies (AFM) and from the Neuromuscular
Disease Foundation (NDF).
ACKNOWLEDGEMENTS
We are grateful to all patients who made this study possible.
We thank Drs Peter Kang and Alvin Kho for PCA analysis
and for useful comments and suggestions. We also thank
Tanya Goltser and Dr Ronnen Segman for great technical
assistance and helpful discussion, and Dr Laura Canetti for
statistical analysis. We are grateful to Jasmine Jacob-Hirsch,
Dr Ninette Amariglio, Dr Gidi Rechavi and Dr Naftali
Kaminski from the Functional Genomics Unit at the Sheba
Medical Center for excellent assistance with array samples
and for array analysis. Special thanks to Professor Zohar
Argov and Dr Ron Dabby for biospy material; to Zippora
Shlomai for the establishment of the myoblast cultures used
in this study and to Sharita Timal for assistance with the
cell mitochondrial assays.
Conflict of Interest statement. None declared.
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