MUTATION IN BRIEF
HUMAN MUTATION Mutation in Brief #723 (2004) Online
© 2004 WILEY-LISS, INC.
Received 8 January 2004; accepted revised manuscript 1 April 2004.
Novel GNE Mutations in Italian Families with
Autosomal Recessive Hereditary Inclusion-Body
Aldobrando Broccolini1*, Enzo Ricci1,2, Denise Cassandrini3, Carla Gliubizzi1, Claudio Bruno3,
Emmanuel Tonoli3, Gabriella Silvestri1, Mario Pescatori1,2, Carmelo Rodolico4, Stefano Sinicropi4,
Serenella Servidei1, Federico Zara3, Carlo Minetti3, Pietro A. Tonali1,5,
and Massimiliano Mirabella1*
1Department of Neuroscience, Catholic University, Rome, Italy;
3 Neuromuscular Disease Unit, Department of Pediatrics, University of Genova, Giannina Gaslini Institute,
Genova, Italy; 4 Department of Neurosciences, Psychiatry and Anaesthesiology, University of Messina, Messina,
Italy; 5 IRCCS “Casa Sollievo della Sofferenza”, San Giovanni Rotondo, Italy
2U.I.L.D.M.-Rome Section, Rome, Italy;
*Correspondence to Aldobrando Broccolini or Massimiliano Mirabella, Department of Neuroscience, Catholic
University, L.go A. Gemelli 8, 00168 Rome, Italy; Tel.: +39-06-30154303; Fax: +39-06-35501909; E-mail:
Grant sponsor: Italian Ministry of Health 2003 (M.M.); Grant sponsor: F.I.R.B. 2001 (P.A.T.)
Communicated by Mark H. Paalman
The most common form of autosomal recessive (AR) hereditary inclusion-body myopathy
(HIBM), originally described in Persian-Jewish families, is characterized by onset in early
adult life with weakness and atrophy of distal lower limb muscles, which progress
proximally and relatively spare the quadriceps. AR HIBM is associated with mutations in
the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene (GNE) on
chromosome 9p12-13. In the present study we have identified seven novel GNE mutations in
patients from five unrelated Italian families with clinical and pathologic features indicative
of AR HIBM. Four were missense mutations (c.1556A>G [p.N519S], c.79C>T [p.P27S],
c.1798G>A [p.A600T] and c.616G>A [p.G206S]), two consisted in a single-base deletion
(c.616delG [p.G206fsX4] and c.1130delT [p.I377fsX16]) and one in an intronic single-base
insertion (c.1070+2dupT). These latter findings further extend the type of GNE mutations
associated with HIBM. Furthermore, in one patient we also identified the c.737G>A
[p.R246Q] missense mutation that corresponds to the one previously reported in a family
from the Bahamas. Interestingly, in two of our families distinct mutations affected
nucleotide c.616 in exon 3 (c.616delG and c.616G>A). The possibility of specific portions of
the gene being more prone to mutations remains to be elucidated. © 2004 Wiley-Liss, Inc.
KEY WORDS: Inclusion-Body Myopathy; HIBM; IBM2; GNE
Autosomal recessive (AR) hereditary inclusion-body myopathy (HIBM; MIM# 600737), originally described in
Persian-Jewish families, is a neuromuscular disorder characterized by onset in the early adult life with weakness
2 Broccolini et al.
and atrophy of distal lower limb muscles and relative sparing of the quadriceps (Argov and Yarom, 1984; Askanas
and Engel, 1998). Upper limb muscles involvement is observed in the advanced stages of the disease. HIBM
muscle resembles that of sporadic inclusion-body myositis (sIBM) but lacks inflammation and congophilic
inclusions within muscle fibers (Askanas and Engel, 1998). HIBM is associated with mutations in the UDP-N-
acetylglucosamine 2-epimerase/N-acetylmannosamine kinase gene (GNE; MIM# 603824) on chromosome 9p12-
13. A homozygous p.M712T missense mutation in the GNE gene has been found in all HIBM Middle Eastern
families of both Jewish and non-Jewish descent. Because all these patients share a common recombinant haplotype
a genetic common founder hypothesis has been proposed (Eisenberg et al., 2001; Eisenberg et al., 2003; Argov et
al., 2003). GNE mutations have also been found in Japanese patients previously diagnosed as having distal
myopathy with rimmed vacuoles (DMRV; MIM# 605820), thus confirming that HIBM and DMRV are indeed the
same entity (Kayashima et al., 2002; Tomimitsu et al., 2002; Nishino et al., 2002; Arai et al., 2002). A strong
linkage disequilibrium has also been demonstrated in Japanese patients sharing the homozygous missense mutation
p.V572L (Arai et al., 2002). Conversely, patients of other ethnic origin are usually compound heterozygous for
mutations in different regions of the gene (Eisenberg et al., 2001; Darvish et al., 2002; Vasconcelos et al., 2002;
Broccolini et al, 2002; Del Bo et al., 2003; Krause et al., 2003). UDP-N-acetylglucosamine 2-epimerase/N-
acetylmannosamine kinase is a bifunctional enzyme with independent epimerase and kinase domains, which is
expressed in different tissues and has a critical role in the biosynthesis of N-acetylneuraminic acid (Neu5Ac), the
precursor of sialic acid. (Hinderlich et al., 1997; Effertz et al., 1999). Sialic acid is normally present on the distal
ends of N- and O-glycans and is involved in many biological functions including cellular adhesion, formation or
masking of recognition determinants, stabilization of glycoproteins structure and signal transduction (Keppler et
al., 1999). Differential sialylation of cell surface molecules is crucial for their function in physiological as well as
pathological processes (Keppler et al., 1999). Although previous studies have shown a reduced epimerase activity
in peripheral leukocytes from HIBM patients (Nishino et al., 2002), the pathogenic mechanism, triggered by a
possible disturbance of sialic acid metabolism and leading to muscle fiber degeneration, remains to be elucidated.
In the present study we have identified seven novel GNE mutations in patients from five unrelated Italian
families with clinical and pathological features indicative of AR HIBM.
PATIENTS AND METHODS
All eight patients from five unrelated families were diagnosed as having HIBM based on both clinical findings
and muscle pathology. Clinical criteria included: i) onset in the second-third decade of life, ii) initial weakness and
atrophy of distal lower limb muscles with distal-proximal progression, iii) later involvement of upper limb
muscles, iv) slightly increased creatin-kinase blood levels, and v) electromyographic pattern with mixed myopathic
and neurogenic features. All patients showed sparing of the quadriceps with the exception of patients 7 and 8 from
family 5. In fact, both of them showed a severe phenotype with unusual weakness of the quadriceps (grade 2/5,
Medical Research Council scale, in the brother and 3/5 in the sister), which was already present only a few years
after the onset of the disease. Clinical features of all patients studied are summarized in Table 1. All muscle
biopsies showed morphological abnormalities indicative of HIBM, including i) myopathic changes with increased
scatter of muscle fiber diameter and centralization of myonuclei, ii) absence of inflammation and fiber necrosis, iii)
presence of fibers with rimmed vacuoles, iv) filamentous inclusions within the muscle fibers by electron
microscopy, and v) variable amount of angulated atrophic fibers.
Genomic DNA was extracted from blood leukocytes of all patients and, when possible, from available
unaffected family members with informed consent. All 13 exons of the GNE gene were amplified by PCR using
specific pairs of primers (Eisenberg et al., 2001). PCR products were purified and sequenced using the ABI
DyeDeoxy Terminator Cicle Sequencing Kit on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems,
Foster City, CA). When a deletion or insertion mutation was suspected on standard gene chromatogram (exon 3 of
patients 4 and 5; exon 6 of patient 6; exon 7 of patients 7 and 8), the PCR product was cloned into the pCR2.1-
TOPO plasmid (Invitrogen, Carlsbad, CA). At least 20 different clones for each exon were then grown and
sequenced as above using the M13 forward and reverse primers (Invitrogen). All mutations were further confirmed
Novel GNE Mutations in Italian HIBM Patients 3
by single nucleotide polymorphism (SNP) analysis using the SnaPshot Multiplex System Kit according to the
manufacturer instructions (Applied Biosystems). For patients 6, 7, and 8 total RNA was extracted from blood
leukocytes using Trizol reagent (Invitrogen) and total cDNA was generated using the SuperScript III RNase H−
reverse transcriptase and oligo(dT)12-18 primer (Invitrogen). GNE cDNA was then amplified using specific sets of
primers and sequenced as above. The list of all primers used for GNE cDNA sequencing is available upon request.
Mutation nomenclature is according to the guidelines of Antonarakis et al. (1998), den Dunnen and Antonarakis
(2000) and den Dunnen and Paalman (2003). Nucleotide numbering refers to the GNE cDNA (accession#
NM_005476 version 2) and starts with the A of the ATG translation initiation codon.
RESULTS AND DISCUSSION
In patients 1 and 2 (family 1), originating from a small village in central Italy, a homozygous A to G
substitution was found at nucleotide (nt) c.1556 in exon 9 (c.1556A>G), converting asparagine to serine at codon
519 (p.N519S), whereas one of their unaffected brothers carried only one mutated allele. Their parents were not
available for genetic study. Patient 3 (family 2) carried a homozygous C to T transition at nt c.79 in exon 2
(c.79C>T) resulting in a proline to serine shift at codon 27 (p.P27S). Her mother resulted heterozygous for the
same mutation whereas no DNA from the father was available for genetic testing. However, both parents
originated from a small community in central Italy (different from that of family 1) and it is, therefore, likely that
they carried the same mutated allele.
Patients 4 and 5 (family 3) were compound heterozygous for a G to A transition at nt c.1798 in exon 10
(c.1798G>A), converting alanine to threonine at codon 600 (p.A600T), and a single base deletion at nt c.616
(c.616delG) in exon 3 with a frameshift in the open reading frame (ORF) that leads to a premature stop at codon
Patient 6 (family 4) was a compound heterozygous for a G to A change at nt c.737 (c.737G>A), which results in
an arginine to glutamine substitution at codon 246 (p.R246Q), and an insertion of a single nucleotide between
position +2 and +3 of intron 6 (c.1070+2dupT). No parental DNA was available for genetic study. To verify
whether the c.1070+2dupT mutation was indeed pathogenic, GNE mRNA was studied by reverse transcriptase
(RT) PCR with a pair of primers amplifying a 850 base pairs (bp) fragment spanning from exon 4 to exon 9. The
PCR product was then cloned into the pCR2.1-TOPO plasmid (Invitrogen) and forty different clones were
sequenced using the M13 forward and reverse primers (Invitrogen). Two different RNA splice variants, related to
the c.1070+2dupT mutation, were found: one lacking the last 9 bp of exon 6 (r.1062_1070del), which results in an
in-frame deletion of three amino acids (p.Q355_C357del) and one lacking the last 16 bp of exon 6
(r.1055_1070del) and resulting in a frameshift of the ORF with a premature stop codon (p.352GfsX15). In both
transcripts the c.737G>A missense mutation was not found, thus confirming that they originated from the allele
bearing the c.1070+2dupT intronic mutation. Interestingly, we also found an additional shorter transcript lacking
the whole exon 4. This latter finding is in agreement with a previous report showing an alternative GNE mRNA
variant lacking exon 4 also in normal individuals (Nishino et al., 2002). Whether this aspect of GNE mRNA
processing has functional significance remains to be determined.
Patients 7 and 8 (family 5), siblings of two unrelated individuals, were also compound heterozygous for a single
base deletion at nt c.1130 in exon 7 (c.1130delT), resulting in a frameshift in the ORF with a premature stop at
codon 392 (p.I377fsX16), and a G to A transition at nt c.616 in exon 3 (c.616G>A), converting glycine to serine at
codon 206 (p.G206S). Analysis of parental DNA showed that the c.1130delT mutation was inherited from the
father whereas the c.616G>A mutation was inherited from the mother.
SNP analysis further confirmed all nucleotide changes found in the GNE gene of our patients. All genetic data
(reviewed but not shown) are summarized in table 1. None of these mutations was found in 50 unrelated healthy
In three families (3, 4, and 5) the finding of a single-base deletion or insertion broadens the spectrum of GNE
mutations associated with HIBM. The c.1070+2dupT mutation in patient 6 (family 4) generated two different GNE
mRNA variants, possibly due to an aberrant splicing between the 3’ splice acceptor site adjoining exon 7 and
cryptic splice donor sites in exon 6. Interestingly, in patients 7 and 8 (family 5) direct sequencing of the GNE
cDNA showed only the c.616G>A transition whereas the c.1130delT mutation could not be seen, thus suggesting
that this latter mutation results in an instable mRNA transcript. No RNA from patients 4 and 5 (family 3) was
4 Broccolini et al.
available to verify whether the c.616delG mutation also results in an unstable mRNA. It is worth noting that
among the GNE mutations associated with HIBM reported to date, all are substitution mutations except two: one
10 bp deletion in exon 2, resulting in a frameshift of the ORF (Nishino et al., 2002), and a large deletion spanning
exon 1-9 (Del Bo et al., 2003). In our study, out of seven novel mutations, two consisted in a single-base deletion
(c.616delG and c.1130delT) and one in an intronic single-base insertion (c.1070+2dupT). Whether this reflects a
higher occurrence of a particular type of GNE mutations in the Italian population is not known.
The homozygous p.N519S mutation in family 1, the p.P27S mutation in patient 3 (family 2), the p.A600T
mutation, found in one allele of patients 4 and 5 (family 3), and the p.G206S mutation, found in patients 7 and 8
(family 5), are newly described GNE missense mutations. Interestingly, in patient 6 the G to A change at nt c.737,
converting arginine to glutamine at codon 246 (p.R246Q), corresponds to the one previously found in the affected
individuals of a family from the Bahamas (Eisenberg et al., 2001; Eisenberg et al., 2003). It is noteworthy that a
different mutation in the same codon (p.R246W substitution due to a C to T shift at nt c.736) has been described in
a California family of Irish-English and European background (Darvish et al., 2002). Furthermore, in the present
study different mutations affecting nt c.616 in exon 3 have been found in two unrelated families (c.616delG in
family 3 and c.616 G>A transition in family 5). This is not the first time that GNE mutations affecting the same
codon (either identical or different in terms of aminoacid shift) are described in patients of different ethnic or
geographic origin (Eisenberg et al., 2001; Broccolini et al., 2002; Eisenberg et al., 2003). In addition, in a cohort of
Japanese HIBM patients, completely different haplotypes associated with the GNE gene have been reported in two
individuals carrying a homozygous p.D176V mutation. Although precise information regarding the set of
polymorphic markers used in this study was not available, it was suggested that the same mutation had arisen in
different genetic backgrounds (Nishino et al., 2002). These lines of evidence raise the question whether specific
regions of the GNE gene are more prone to mutations. However, larger population studies will be required to
clarify this issue.
In our group of patients it is difficult to define a precise genotype-phenotype correlation. Patients 7 and 8 from
family 5 showed a severe clinical phenotype with an unusual involvement of the quadriceps just a few years after
the onset of symptoms. In these patients the c.616delG mutation found in one allele results in an instable mRNA
and therefore the residual enzymatic activity relies only on the allele carrying the p.G206S missense mutation. It is
possible that this latter mutation deeply impairs the enzyme function thus resulting in a severe clinical phenotype.
However, we cannot exclude that additional, yet unknown, factors play a role in the modulation of the clinical
symptoms as previously suggested (Nishino et al., 2002). This is particularly evident in the affected members of
family 1. In fact, patients 1 and 2 showed a different clinical course, with the onset of symptoms in the third and
fourth decade of life respectively, although they shared the same homozygous mutation in exon 9 (p.N519S).
Additional studies are required to elucidate the perturbation of muscle metabolism arising from GNE mutations.
This will help clarifying whether mutations in different portions of the gene result in diversified biochemical
defects, possibly providing a tool to better correlate a genetic abnormality with the corresponding clinical
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6 Broccolini et al.
Table 1. Summary of clinical and genetic data of GNE mutations in HIBM patients
nucleotide substitution (c.)2 expected consequence (p.) 3
1/M 25 55 spared
exon 9 [c.1556A>G] +
2/F 37 48 spared
exon 9 [c.1556A>G]
exon 2 [c.79C>T] +
3/F 35 42
spared proximal 2
exon 2 [c.79C>T]
4/M 17 18 distal spared spared
exon 3 [c.616delG] +
5/F 22 25 distal and
exon 10 [c.1798G>A]
exon 4 [c.737G>A] + p.R246Q epimerase
6/F 21 49
intron 6 [c.1070+2dupT]
7/F 25 37 distal and
exon 3 [c.616G>A] +
8/F 23 34 severe
exon 7 [c.1130delT]
1Boldface type displays novel mutations
2Nucleotide position in GNE cDNA, accession# NM_005476 version 2; nucleotide numbering starts with the A of the start codon; 3Protein id. NP_005467.1. 4Predicted protein products of the
two alternatively spliced GNE mRNA