Charcot-Marie-Tooth disease
Genetic and clinical spectrum in a Spanish clinical series
Rafael Sivera, MD*
Teresa Sevilla, MD, PhD*
Juan Jesús Vílchez, MD,
PhD
Dolores Martínez-Rubio,
MBChB
María José Chumillas,
MD
Juan Francisco Vázquez,
MD
Nuria Muelas, MD, PhD
Luis Bataller, MD, PhD
José María Millán, PhD
Fancesc Palau, MD, PhD
Carmen Espinós, PhD
Correspondence to
Dr. Sevilla:
sevilla_ter@gva.es
ABSTRACT
Objectives: To determine the genetic distribution and the phenotypic correlation of an extensive
series of patients with Charcot-Marie-Tooth disease in a geographically well-defined Mediterranean area.
Methods: A thorough genetic screening, including most of the known genes involved in this
disease, was performed and analyzed in this longitudinal descriptive study. Clinical data were
analyzed and compared among the genetic subgroups.
Results: Molecular diagnosis was accomplished in 365 of 438 patients (83.3%), with a higher
success rate in demyelinating forms of the disease. The CMT1A duplication (PMP22 gene) was
the most frequent genetic diagnosis (50.4%), followed by mutations in the GJB1 gene (15.3%),
and in the GDAP1 gene (11.5%). Mutations in 13 other genes were identified, but were much less
frequent. Sixteen novel mutations were detected and characterized phenotypically.
Conclusions: The relatively high frequency of GDAP1 mutations, coupled with the scarceness of
MFN2 mutations (1.1%) and the high proportion of recessive inheritance (11.6%) in this series
exemplify the particularity of the genetic distribution of Charcot-Marie-Tooth disease in this
region. Neurologyâ 2013;81:1617–1625
GLOSSARY
AD 5 autosomal dominant; AR 5 autosomal recessive; CMT 5 Charcot-Marie-Tooth; MMNCV 5 median motor nerve conduction velocity.
Charcot-Marie-Tooth (CMT) disease refers to the genetically heterogeneous group of hereditary
motor and sensory neuropathies. It is one of the most common inherited neurologic disorders,
with a prevalence of 15.2 to 40 cases per 100,000.1–3 Molecular studies have provided an evergrowing list of more than 40 involved genes and loci (http://www.molgen.ua.ac.be/
CMTMutations/, http://neuromuscular.wustl.edu/, both accessed June 24, 2013). Most of the
patients with CMT disease have autosomal dominant (AD) inheritance, but many have X-linked
or autosomal recessive (AR) inheritance. CMT disease can be classified according to clinical,
electrophysiologic, and nerve pathology findings into demyelinating forms (CMT1, CMT4),
with a median motor nerve conduction velocity (MMNCV) of ,38 m/s and pathologic
evidence of nerve fiber demyelination; and axonal forms (CMT2), with preserved conduction
velocities (MMNCV .38 m/s) and pathologic signs of axonal degeneration and regeneration.4 An
intermediate type (CMT-I) is accepted in which MMNCV lies between 25 and 45 m/s and nerve
pathology shows axonal and/or demyelinating features.5
Clinically, the most frequent CMT phenotype is characterized by progressive distal weakness
and sensory loss appearing toward the second decade, with foot deformities and absent reflexes.
However, other patients develop a much more severe form with onset in infancy or early
*These authors contributed equally to this work.
From the Departments of Neurology (R.S., T.S., J.J.V., J.F.V., N.M., L.B.), Clinical Neurophysiology (M.J.C.), and Genetics (J.M.M.), Hospital
Univesitari i Politècnic La Fe, Valencia; Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (T.S., J.J.V., M.J.C., N.
M., L.B.), Valencia; Departments of Medicine (T.S., J.J.V.) and Genetics (C.E.), University of Valencia; Program in Rare and Genetic Diseases (D.
M.-R., F.P., C.E.), Centro de Investigación Príncipe Felipe (CIPF), Valencia; Centro de Investigación Biomédica en Red de Enfermedades Raras
(D.M.-R., J.M.M., F.P., C.E.), Valencia; IBV-CSIC Associated Unit at CIPF (D.M.-R., F.P., C.E.), Valencia; and School of Medicine (F.P.),
University of Castilla-La Mancha, Ciudad Real, Spain.
Go to Neurology.org for full disclosures. Funding information and disclosures deemed relevant by the authors, if any, are provided at the end of the article.
© 2013 American Academy of Neurology
1617
ª"NFSJDBO"DBEFNZPG/FVSPMPHZ6OBVUIPSJ[FESFQSPEVDUJPOPGUIJTBSUJDMFJTQSPIJCJUFE
childhood and great disability within a few
years, or a milder course with few symptoms
until adulthood. This clinical heterogeneity,
coupled with the expanding genetic diversity,
is the complex scenario of the inherited neuropathies. Comprehensive clinical series, in
which at least the most frequent genes have
been studied, are needed to shed light on
the populational genetic distribution and
genotype-phenotype correlation in CMT disease.6,7 Herein, we present the genetic distribution and phenotypic characterization of an
extensive series of CMT disease after an exhaustive genetic screening in the Region of Valencia, a
geographically well-defined Mediterranean area.
METHODS Subjects. This is a longitudinal descriptive study,
which includes all of the patients with the diagnosis of CMT disease
and evaluated at the inherited neuropathy clinic of Hospital Universitari i Politècnic La Fe in Valencia from 2000 to 2012. Patients
with sensory-motor neuropathy were considered to have CMT
disease if a) a causative genetic defect was determined, b) family
members with similar characteristics were detected, or c) sporadic
cases were included if their medical history, examination, and neurophysiology were compatible with CMT disease, and other known
causes of acquired neuropathy were reasonably discarded. Patients
with inherited neuropathies with exclusive motor (distal hereditary
motor neuropathies) or sensory and autonomic (hereditary sensory
and autonomic neuropathies) signs were excluded from this study,
as well as those with hereditary neuropathy with liability to pressure
palsies, and those with complex disorders in which neuropathy was
not the most predominant phenotypic feature. Patients were subclassified with demyelinating or axonal CMT disease according to
MMNCVs of the proband, except when the amplitudes of median
compound motor action potentials were reduced .90%. In those
cases, the conduction velocities to nerves innervating proximal
muscles were measured (palmaris longus for the median nerve,
flexor carpi ulnaris for the ulnar nerve, etc.), and occasionally latencies of other proximal nerves such as the axillary nerve, or pathologic evidence were considered.
Standard protocol approvals, registrations, and patient
consents. This study protocol was approved by the Institutional
Review Board of the Hospital Univesitari i Politècnic La Fe.
Written informed consents were obtained from all of the members included in this study.
Clinical assessments. The clinical assessment included strength,
muscular atrophy, sensory loss, reflexes, foot deformities, as well as
a general and neurologic examination. Muscle strength was graded
using the standard Medical Research Council scale. CMT neuropathy score was recorded in all patients followed since 2006,8 and the
Functional Disability Scale score in those after 20009; previous clinical data were extrapolated to CMT neuropathy score and Functional
Disability Scale score when possible. Comprehensive electrophysiologic studies were performed in 401 of 438 patients (91.6%), and
were not performed only when the genetic diagnosis of another
family member was already available. Lower limb muscle MRI and
sural nerve biopsy were performed only when there were reasonable
doubts regarding the clinical diagnosis or for investigational purposes,
and followed the protocols described previously.10
1618
Neurology 81
Mutational analysis. Blood samples were drawn and genomic
DNA was obtained by standard methods from peripheral white
blood cells. In all of the probands, the CMT1A duplication was
analyzed by MLPA (Multiplex Ligation–dependent Probe Amplification, SALSA kit P033 CMT1; MRC-Holland, Amsterdam, the
Netherlands) in a genetic analyzer ABI Prism 3130xl (Applied
Biosystems, Foster City, CA). Once the CMT1A duplication was
discarded, a mutational screening of genes involved in CMT disease
was performed taking into account the ethnicity of the proband and
the phenotype. In patients with Gypsy ethnicity, the genetic testing
strategy was planned as described previously.11 In Caucasian patients, the mutational screening was clinically oriented, and
included the genes detailed in table 1 until the causative mutation
was identified or all of the genes had been analyzed.
The mutational screening was performed by amplification of
all exons and their intronic flanking sequences, except in the
GJB1 gene in which the promoter sequence has also been analyzed. The Gene Runner version 3.05 software was used for
designing primers (available on request). The PCR products were
analyzed by using denaturing high-performance liquid chromatography (WAVE System; Transgenomic Inc., Omaha, NE), and
the anomalous patterns were investigated by Sanger sequencing
(ABI Prism 3130xl). Finally, in both the MPZ and the GJB1
genes, large deletions and/or duplications were investigated by
Table 1
Genes analyzed in the mutational
screening
CMT1
Caucasian
PMP22
Gypsy
CMT2
a
SH3TC2
a
MFN2
GJB1
NDRG1
GJB1
MPZ
HK1a
MPZ
GDAP1
GDAP1
SH3TC2
HSPB1
FGD4
HSPB8
NEFL
LITAF
LITAF
NEFL
GAN1
DNM2b
BSCL2
GARS
FIG4
AARS
ERG2
KARS
PRX
b
YARS
MTMR2
TRPV4
MTMR13
RAB7
PRPS1
MED25a
DNM2b
LMNAa
YARS
LRSAM1
SOX10
Abbreviation: CMT 5 Charcot-Marie-Tooth.
a
Only founder mutations were analyzed: SH3TC2
p.C737_P738delinsX,
SH3TC2
p.R1109X,
NDRG1
p.R148X, HK1 g.9712G.C, MED25 p.A335V, and LMNA
p.R298C.
b
More than one sequence reference was used because of
the presence of isoforms.
October 29, 2013
ª"NFSJDBO"DBEFNZPG/FVSPMPHZ6OBVUIPSJ[FESFQSPEVDUJPOPGUIJTBSUJDMFJTQSPIJCJUFE
MLPA using the SALSA kits P143 and P129 (MRC-Holland) in
an ABI Prism 3130xl autoanalyzer. We did not screen MT-ATP6,
PDK3, DHTKD1, GNB4, or TRIM2 genes because they had not
been described when this project was concluded.12–16
When possible, segregation analyses within the families were
performed, and novel mutations were analyzed in 200 chromosomes from healthy controls of Spanish ancestry. The biological relevance of the amino acid changes was studied using both SIFT
(http://blocks.fhcrc.org/sift/SIFT.html, accessed June 24, 2013)
and PolyPhen (http://genetics.bwh.harvard.edu/pph, accessed
June 24, 2013) programs. When the detected alteration modified
a splicing sequence, we used the NNSPLICE (http://fruitfly.org:
9005/seq_tools/splice.html, accessed June 24, 2013) and the Splice
View (http://zeus2.itb.cnr.it/;webgene/wwwspliceview_ex.html,
accessed June 24, 2013) software.
A total of 1,009 patients were evaluated at
our inherited neuropathy clinic during the timeframe
2000 to 2012; 438 of them were considered to have
CMT disease and met our inclusion criteria. All were
Spanish, and 401 of them (91.6%) were currently living or had ancestral roots in the Region of Valencia, in
the Western Mediterranean area. Initially, 275
(62.8%) were classified as demyelinating CMT, and
163 (37.2%) as axonal CMT. Regarding the inheritance pattern, 242 (55.3%) were considered as AD,
51 (11.6%) were AR, 52 (11.9%) were X-linked,
and 93 (21.2%) were considered sporadic. Genetic
diagnosis was achieved in 365 of 438 patients
(83.3%), with a higher success rate in the demyelinating forms (263/275; 95.6%) over the axonal forms
(102/163; 62.6%). The causative mutations were detected in 214 of 242 patients (88.4%) with AD inheritance, 45 of 51 (88.2%) with AR inheritance, 52 of 52
(100%) with X-linked inheritance, and in only 54 of
93 (58.1%) with a sporadic presentation. In table 2,
the detailed genetic diagnosis can be analyzed and
compared with the latest published data, and in the
figure, the distribution according to CMT subtype is
shown. All of the genetic and clinical information has
also been recorded in a readily accessible mutation
database (http://www.treat-cmt.es/db, accessed June
24, 2013).
Table 2
No. of patients (frequency, %)
RESULTS
Patients with demyelinating CMT disease. Of the 275 patients with demyelinating CMT disease, 241 were of
Caucasian ethnicity and 34 were of Gypsy origin. Of
the Caucasian patients with the demyelinating form,
184 (76.3%) carried the CMT1A duplication, which
is the most frequent cause of CMT disease. In the remaining 57 Caucasian patients, the disease causing
mutation was identified in 45 with the following distribution: 25 mutations in GJB1, 9 in MPZ, 4 in PRX, 2
point mutations in PMP22, 2 in FGD4, 2 in SH3TC2,
and 1 in NEFL. Six novel mutations were detected in
demyelinating CMT (table 3). Once the genetic screening was performed, the causative change remained
unknown in 12 patients (4.9%). No mutations were
Genetic distribution and comparison to
other series
Gene
Present study Saporta et al.6 Murphy et al.7
PMP22a
184 (48.8)
290 (55)
GJB1
56 (14.9)
80 (15.2)
46 (17.3)
GDAP1
42 (11.1)
6 (1.2)
2 (0.8)
SH3TC2
27 (7.2)
3 (0.6)
5 (1.9)
MPZ
19 (5.0)
45 (8.5)
13 (4.9)
NDRG1
7 (1.9)
HSPB1
7 (1.9)
MFN2
6 (1.6)
HK1
5 (1.3)
NEFL
GARS
2 (0.8)
21 (4.0)
12 (4.5)
4 (1.1)
4 (0.8)
2 (0.8)
4 (1.1)
3 (0.6)
PRX
4 (1.1)
1 (0.2)
HSPB8
3 (0.8)
PMP22b
2 (0.5)
FGD4
2 (0.5)
KARS
1 (0.3)
YARS
1 (0.3)
TRPV4
1 (0.3)
LITAF
5 (1.0)
5 (1.0)
4 (1.5)
MTMR2
1 (0.4)
GAN
1 (0.4)
FIG4
b
6 (2.3)
3 (1.1)
1 (0.4)
BSCL2
a
168 (63.2)
2 (0.4)
Carriers of the CMT1A duplication.
Carriers of point mutations in the PMP22 gene.
identified in any of the following genes: LITAF, EGR2,
GDAP1, MTMR2, MTMR13, FIG4, PRPS1, DNM2,
YARS, and SOX10. In the Gypsy population, the disease-causing mutation was identified in all cases, and
consisted exclusively of founder mutations related to
CMT disease in the Gypsy population.11
Table 4 shows the relevant clinical features associated with AR forms of demyelinating CMT disease
(CMT4). These forms have certain common characteristics such as early onset, delayed motor development, and severe disability, but other features differ
between the CMT4 subtypes.
Patients with axonal CMT disease. The mutational
screening detailed in table 1 led to identification of
the disease-causing mutation in 102 of 163 patients
with axonal CMT disease (62.6%). In this set of patients, there is a marked genetic heterogeneity, with
mutations in the GDAP1 and GJB1 genes being the
2 most frequent causes of axonal CMT disease. Mutations in the GDAP1 gene correspond to 24 patients
Neurology 81
October 29, 2013
1619
ª"NFSJDBO"DBEFNZPG/FVSPMPHZ6OBVUIPSJ[FESFQSPEVDUJPOPGUIJTBSUJDMFJTQSPIJCJUFE
Figure
Genetic characterization of CMT disease subtypes
Patients evaluated at the inherited neuropathy clinic during the timeframe 2000–2012. a Carriers of the CMT1A duplication. b Carriers of point mutations in the PMP22 gene. AD 5 autosomal dominant; AR 5 autosomal recessive; CMT 5
Charcot-Marie-Tooth.
(14.7% of CMT2) with AD inheritance (caused by the
p.R120W mutation in all cases except one) and 18
patients (11.0%) with AR inheritance and diverse genotype. All of our patients with GDAP1 mutations were
defined as CMT2 because the neurophysiologic findings were clearly axonal, although the pathology
included both axonal features (fiber loss, axonal degeneration, few regenerative clusters, etc.) and myelin
abnormalities (thin myelin sheaths, abnormal myelin
folding, occasional onion bulb–like formations). Patients with AR inheritance developed a severe phenotype with important disability, vocal cord, and
diaphragmatic palsies whereas patients with dominant GDAP1 mutations presented with a mild to
moderate phenotype with certain clinical and MRI
particularities reported previously.10
Mutations in the GJB1 gene were detected in 31
patients with axonal CMT disease (19.0%). It is interesting to note that although the patients were classified
as having demyelinating or axonal CMT disease according to the MMNCVs of the proband, more than
80% of these families would be classified as having
intermediate forms of CMT disease.
The remaining mutations were actually quite rare,
accounting for only 29 cases (17.8%), and are distributed
1620
Neurology 81
among several genes: 10 patients with mutations in
MPZ, 7 in HSPB1, 4 in MFN2, 3 in HSPB8, 3 in
NEFL, 1 in GARS, and 1 in KARS. In the aggregate,
25 different mutations were identified in the CMT2
series and 10 of them were novel (table 3). Once
the mutational screening was performed, the diseasecausing mutation remained unknown in 61 patients
(37.4%). No change was identified in the following
genes: RAB7, DNM2, YARS, AARS, LRSAM1, and
TRPV4, nor the founder mutations MED25 p.A335V
or LMNA p.R298C.
DISCUSSION A thorough genetic screening has been
performed in an extensive clinical series of patients with
CMT disease in a Western Mediterranean area. Overall,
a molecular diagnosis was achieved in 83.3%, with a
higher success rate in demyelinating than in axonal
CMT disease. In demyelinating patients, these rates
are comparable to the other series in which a comprehensive genetic screening was performed (table 2),6,7,17
suggesting that few genes involved in this form of CMT
disease remain undiscovered. However, in CMT2,
although the success rate is higher than in other series,
37.4% of patients remain without genetic diagnosis.
The mutational distribution described confirms the
October 29, 2013
ª"NFSJDBO"DBEFNZPG/FVSPMPHZ6OBVUIPSJ[FESFQSPEVDUJPOPGUIJTBSUJDMFJTQSPIJCJUFE
Table 3
Novel mutations with detailed assessments and conduction velocities of the probands, and phenotypic peculiarities
Mutation
Gene
Nucleotide
Amino acid (aa)
No. of
Onset, Age at exam,
MMNCV
Presentation patients y
y
CMTNS FDS (m/s)
Phenotypic characteristics
GJB1
c.44_45delinsTT
p.R15L
X-linked
3
20
59
14
3
36.1
Early distal upper limb atrophy
and weakness. Intrafamily
variability regarding severity.
c.529G.A
p.V177M
Sporadic
1
18
34
14
1
30
Early distal upper limb atrophy
and weakness.
c.-540C.G
No aa change
X-linked
10
26
36
15
2
31
Lower limb distal weakness
earlier and more prominent than
upper limb. Includes 2
asymptomatic women.
c.484dupA
p.M162NfsX81
X-linked
2
15
34
13
2
40
Early distal upper limb atrophy
and weakness.
c.141_143dupGAA
p.K48_S49insK
X-linked
4
24
44
12
2
41
Early distal upper limb atrophy
and weakness. Brisk reflexes only
in proband.
SH3TC2
c.3305delA (hom)a
p.H1102LfsX14a
Sporadic
1
9
43
15
3
27
Early sensory ataxia, scoliosis.
Lower . upper limb distal
weakness and atrophy. No
hearing loss.
PRX
c.589G.T;
c.642insC
p.E197X;
p.R215QfsX8
AR
2
2
42
27
8
4
Early onset, sensory ataxia,
scoliosis. Refractory trigeminal
neuralgia in 1/2. Few motor signs.
FGD4
c.1886delGAAA
(hom)
p.K630NfsX5
AR
2
3
34
14
4
11
Early onset but slow progression.
Sensory ataxia. Lower . upper
limb distal weakness and atrophy.
Spinal syringomyelia in 1/2.
GDAP1
c.1031T.G;
c.487C.Tb
p.L344R; p.Q163Xb
Sporadic
1
12
49
12
2
57
Mild phenotype for a recessive
mutation. Distal lower limb
weakness, no vocal cord or
diaphragmatic palsy.
MFN2
c.306dupT
p.G103WfsX41
AD
2
22
40
11
2
52
Classic CMT2 phenotype,
moderate instability.
c.752C.T
p.P251L
Sporadic
1
25
47
13
2
51
Classic CMT2 phenotype.
MPZ
c.21_26dupTGGGGG p.P9_A10dup
AD
2
30
39
9
1
54
Proband with mild phenotype and
his father is mostly
asymptomatic. Upper limb
reflexes are present.
NEFL
c.293A.C
p.N98T
Sporadic
1
3
54
26
8
44
Early onset, severe phenotype.
Hearing loss. Wheelchair bound in
the 4th decade, death at 58 y.
c.1315T.A
p.F439I
Sporadic
1
23
41
8
2
45
Early distal upper limb atrophy
and weakness. Brisk reflexes.
c.1171C.T
p.R391C
Sporadic
1
18
39
10
1
53
Early distal upper limb atrophy
and weakness. Brisk reflexes.
Motor . sensory involvement.
GARS
Abbreviations: AD 5 autosomal dominant; AR 5 autosomal recessive; CMT 5 Charcot-Marie-Tooth; CMTNS 5 CMT neuropathy score; FDS 5 Functional
Disability Scale; hom 5 homozygous; MMNCV 5 median motor nerve conduction velocity (normal values in our laboratory .51.6 m/s).
a
We cited this mutation in Lupo et al.,36 but clinical features were not included.
b
This mutation has been widely described; we have included it because this patient is a compound heterozygote for a novel mutation.
extensive heterogeneity intrinsic to this disease; 56 different mutations have been detected in this series, and
16 had not been described previously. This comprehensive study depicts the genetic distribution of a large
CMT series in the Mediterranean basin, and there are
certain distinctive features compared with other geographic areas.
The CMT1A duplication is by far the most common mutation detected, and all patients were classified
as demyelinating CMT; in fact, none had MMNCV
.30 m/s. CMT1A accounts for 66.9% of the demyelinating forms, which is somewhat lower than other
series that report slightly more than 70%.18 Actually,
these results are biased by the presence of 34 Gypsy
patients affected by demyelinating CMT disease who
harbored the previously described founder mutations
associated with the Gypsy population as we have previously reported.11,19 These 34 Gypsy patients and 8
others of Caucasian ethnicity (4 with mutations in
PRX, 2 in SH3TC2, and 2 in FGD4; table 4) comprise
the 11.6% of demyelinating CMT with an AR inheritance (CMT4). The percentage of patients with AR or
sporadic presentation is in fact greater than in other
series6 and may reflect certain populational peculiarities,
Neurology 81
October 29, 2013
1621
ª"NFSJDBO"DBEFNZPG/FVSPMPHZ6OBVUIPSJ[FESFQSPEVDUJPOPGUIJTBSUJDMFJTQSPIJCJUFE
1622
Neurology 81
October 29, 2013
ª"NFSJDBO"DBEFNZPG/FVSPMPHZ6OBVUIPSJ[FESFQSPEVDUJPOPGUIJTBSUJDMFJTQSPIJCJUFE
Table 4
Genotype-phenotype correlation of the series of patients with autosomal recessive demyelinating CMT disease
Foot
deformity,
%
Scoliosis,
%
Cranial nervesa
CMTNS; MMNCV, m/s;
CMAP, mVb
FDS
SNAP, mV
Prominent; vibratory 5 pinprick; 100
ataxia 100%
91
V-trigeminal neuralgia
(5%); VIII (48%)
16.8; 3.7 24.6; 4.2
0.7; NR 52%
Same
100
100
VIII (40%)
15.6; 4.1 22.7; 4.8
0.3; NR 80%
LL . UL; distal . proximal Same
Yes
Yes
No
15; 2
18; 8.7
NR
Yes
Gene
Mutations
Patients/
families
Onset, Age at
y
exam, y
Weakness
Sensory loss
SH3TC2
p.R1109X (hom)
21/11
3.2
23.4
LL . UL; proximal 38%
p.R1109X/
p.C737_P738delinsX
5/3
4.1
20.3
LL . UL; proximal 40%
p.H1102LfsX14
(hom)
1/1
9
30
p.R529Q (hom)
1/1
8
43
LL . UL; only distal
Same
Yes, mild
VIII
10; 2
28; 9.6
NR
HK1
g.9712G.C (hom)
6/3
4.8
24.2
LL . UL; proximal 33%
Prominent; vibratory . pinprick; 100
ataxia 100%
50
VIII (33%)
14.1; 3
26.3; 5.1
1.9; NR 17%
NDRG1
p.R148X (hom)
2/2
3.8
18.1
LL . UL; proximal 50%
Prominent; vibratory . pinprick; 100
ataxia 100%
100
VIII (50%)
16.3; 3.1 16.7; 6.2
0.9; NR 50%
PRX
p.E197X/
p.R215QfsX8
3/1
2.7
25.7
LL . UL; only distal
Prominent; vibratory . pinprick; 100
ataxia 100%
100
V-trigeminal neuralgia
(33%)
22.7; 5.3 4.9; 1.2
NR 100%
p.E113fsX3 (hom)
1/1
1
12
LL . UL; only distal
Prominent; vibratory . pinprick; Yes
ataxia
Yes
No
18; 3
5.8; 0.5
NR
p.K630NfsX5 (hom)
2/1
2.5
32
LL . UL; only distal
Prominent; vibratory . pinprick; Yes
ataxia 100%
Yes
No
12; 3
11.5; 5.2
NR 100%
FGD4c
Abbreviations: CMAP 5 compound muscle action potential of the median nerve (normal values .9.3 mV); CMT 5 Charcot-Marie-Tooth; CMTNS 5 CMT neuropathy score; FDS 5 Functional Disability Scale; hom 5
homozygous; LL 5 lower limbs; MMNCV 5 median motor nerve conduction velocity (normal values in our laboratory .51.6 m/s); NR 5 not recordable (expressed in % of the patients); SNAP 5 sensory nerve action
potential in median nerve (normal values .16.5 mV); UL 5 upper limbs.
If more than one case, the numeric values are means and the percentages, are relative frequency of a characteristic.
a
VIII nerve was considered affected when the patient reported relevant hypoacusia or the hearing loss was confirmed with audiometry.
b
Nerve conduction studies of median nerve nearest to the moment of physical examination.
c
The 2 patients with mutations in the FGD4 gene had an early onset and moderate disability from infancy, but very slow progression thereafter.
as the Region of Valencia hosts a numerous Gypsy
population (more than 50,000), and certain isolated
areas have a high consanguinity rate.
Mutations in GJB1 were the second most common
genetic diagnosis after CMT1A, accounting for 12.8%
of the CMT series. These patients were classified according to the MMNCV of the proband, but clinically
all patients had a consistent phenotype that was not so
much influenced by conduction velocities, as by sex.20,21
Only 5 patients (9%) had signs of CNS involvement
(brisk reflexes and Babinski sign in 2 of them) with
normal encephalic and spinal MRI. It is worth noting
that in 2 of these patients after a long follow-up (.20
years), the pyramidal signs became less prominent as the
neuropathy progressed, becoming overshadowed by the
neuropathic signs. More than 300 mutations have been
described in the GJB1 gene, throughout the coding
region and exceptionally, in the 5’-UTR (untranslated
region). A very extensive family of our series was found
to be carriers of a novel c.-540C.G mutation in this
region. Its pathogenicity was demonstrated by a luciferase assay (data not shown).
Mutations in MPZ were detected in only 4.3% of
the series; 9 were classified as demyelinating CMT
and 10 as axonal CMT. In this case, there was important phenotypical variability, as has been reported in
this gene.22,23 Except for one family, demyelinating
patients were more severely affected, with earlier disease onset (first decade), prominent sensory loss, and
moderate to severe disability with progression. One of
these patients, carrier of the MPZ p.S121F mutation,
developed a severe congenital hypomyelinating neuropathy.24 Other genes were actually quite scarcely
affected in our CMT1 series (NEFL, point mutations
in PMP22, PRX, SH3TC2, and FGD4).
There is a great genetic diversity in axonal forms of
CMT disease, as 25 different mutations were detected
in 9 genes. The success rate of our series in these patients
(62.6%) is one of the highest that has been published,
probably because of the ample genetic screening that
has been performed, and the high relative frequency
of GDAP1. The genetic distribution in CMT2 shows
that the 2 most frequent causes of axonal CMT disease
were mutations in the GDAP1 and GJB1 genes, which
combined accounted for 44.8% of patients who had
axonal CMT disease. However, 37.4% of the patients
with CMT2 remained undiagnosed, and this constitutes a great challenge for the near future.
Our series of 42 patients with mutations in the
GDAP1 gene is to date the most extensive one published
and all of them presented neurophysiologic features of
axonal CMT disease. Patients with apparently demyelinating or intermediate nerve conduction studies have
been reported,25,26 but in our patients, the only ones
with slow conduction velocities were those in which
compound motor action potential was ,0.5 mV, and
nerve conduction velocity was clearly normal if measured to nerves innervating proximal muscles. Although
the neurophysiologic findings in these patients were
unequivocally axonal, the pathology included both axonal degeneration and myelin abnormalities.10,27 Eighteen
patients with recessive GDAP1 mutations were detected,
with an early disease onset and rapid progression, and
were wheelchair-bound in the second or third decade in
all cases except 2 (associated with p.L344R/p.Q163X
compound heterozygote, and p.R282C/p.R282C
homozygote genotypes) who had a relatively milder phenotype.27 Twenty-four of 25 patients with dominant
GDAP1 mutations carried the p.R120W substitution,
which is to date the most frequent dominant mutation
detected in the GDAP1 gene. Although this mutation
has been described in families with different geographic
origins,28–30 the GDAP1 p.R120W probably has a
founder effect in our population, and presents with a
mild to moderate phenotype.10
Apart from the high prevalence of GDAP1 mutations, the other notable factor in the axonal CMT
series is the low number of cases with mutations in
the MFN2 gene (2.5%). MFN2 has been identified as
the most common gene in axonal CMT disease in
many series,7,8 accounting consistently for 10% to
33%31–33 of this CMT form, even in other Spanish
Mediterranean areas.34 Certain other European series
have described even lower frequencies35 than our own,
suggesting that the distribution of MFN2 mutations
may be quite heterogeneous within Europe. The remaining mutations identified in axonal patients were
even less frequent, including MPZ, HSPB1, NEFL,
GARS, HSPB8, and YARS genes (15.3% of the
CMT2 series).
The knowledge derived from thoroughly screened
CMT series is essential to comprehend the global picture of this disease, as there may be relevant changes in
the genetic distribution of different areas. A clear example of this is the relatively high prevalence of recessive
forms and the predominance of GDAP1 over MFN2 in
this clinical series. More information about the genetic
distribution in other Spanish or Mediterranean areas is
needed to discern whether this is only a local characteristic, or can be extrapolated to other areas.
AUTHOR CONTRIBUTIONS
Dr. Sivera: acquisition of data, analysis and interpretation, initial manuscript elaboration. Dr. Sevilla: study concept and design, initial manuscript elaboration. Dr. Vílchez: critical revision of the manuscript for
important intellectual content. Ms. Martínez-Rubio: genetic studies,
acquisition of data. Dr. Chumillas: nerve conduction studies, acquisition
of data. Dr. Vázquez: acquisition of data, analysis and interpretation.
Dr. Muelas and Dr. Bataller: critical revision of the manuscript for
important intellectual content. Dr. Millán: genetic studies (CMT1A
duplication), acquisition of data. Dr. Palau: study concept and design,
critical revision of the manuscript for important intellectual content.
Dr. Espinós: study concept and design, study supervision, genetic
screening.
Neurology 81
October 29, 2013
1623
ª"NFSJDBO"DBEFNZPG/FVSPMPHZ6OBVUIPSJ[FESFQSPEVDUJPOPGUIJTBSUJDMFJTQSPIJCJUFE
ACKNOWLEDGMENT
11.
The authors are grateful to Paula Sancho for helping to generate the database of CMT mutations in the Spanish population, to Susana Rovira for
performing the study of the GJB1 gene promoter region, and to Itziar
Llopis for the sample management.
12.
STUDY FUNDING
This collaborative joint project is awarded by IRDiRC and funded by the
Instituto de Salud Carlos III (ISCIII)–Subdirección General de Evaluación y Fomento de la Investigación within the framework of the National
R1D1I Plan (grants IR11/TREAT-CMT, PI08/90857, PI08/0889,
CP08/00053, PI12/00453, and PI12/0946), cofunded with FEDER
funds, and the Generalitat Valenciana (grant Prometeo/2009/051). The
Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas (CIBERNED) and the Centro de Investigación Biomédica en
Red de Enfermedades Raras (CIBERER) are initiatives from the ISCIII.
13.
DISCLOSURE
15.
R. Sivera reports no disclosures. T. Sevilla is funded by grants from the
ISCIII (PI12/0946, PI08/0889) and IRDiRC (IR11/TREAT-CMT).
J. Vílchez received research support from the CIBERNED. D. MartínezRubio, M. Chumillas, J. Vázquez, N. Muelas, L. Bataller, and J. Millán
report no disclosures. F. Palau is funded by grants from Fondo de Investigación Sanitaria (PI08/90857), the Generalitat Valenciana (Prometeo/
2009/051), the IRDiRC (IR11/TREAT-CMT), and the CIBERER.
C. Espinós is funded by a grant from the ISCIII (PI12/00453) and
IRDiRC (IR11/TREAT-CMT). Dr. Espinós has a “Miguel Servet” contract
funded by the ISCIII and the CIBERER (CP08/00053). Go to Neurology.
org for full disclosures.
14.
16.
17.
18.
Received May 10, 2013. Accepted in final form July 30, 2013.
REFERENCES
1. Combarros O, Calleja J, Polo JM, Berciano J. Prevalence
of hereditary motor and sensory neuropathy in Cantabria.
Acta Neurol Scand 1987;75:9–12.
2. Foley C, Schofield I, Eglon G, Bailey G, Chinnery PF,
Horvath R. Charcot-Marie-Tooth disease in Northern England. J Neurol Neurosurg Psychiatry 2012;83:572–573.
3. Skre H. Genetic and clinical aspects of Charcot Marie
Tooth’s disease. Clin Genet 1974;6:98–118.
4. Dyck PJ, Lambert EH. Lower motor and primary sensory
neuron diseases with peroneal muscular atrophy. I. Neurologic, genetic, and electrophysiologic findings in hereditary polyneuropathies. Arch Neurol 1968;18:603–618.
5. Payreson D, Marchesi C. Diagnosis, natural history, and
management of Charcot-Marie-Tooth disease. Lancet
Neurol 2009;8:654–667.
6. Saporta AS, Sottile SL, Miller LJ, Feely SM, Siskind CE,
Shy ME. Charcot-Marie-Tooth disease subtypes and
genetic testing strategies. Ann Neurol 2011;69:22–33.
7. Murphy SM, Laura M, Fawcett K, et al. Charcot-MarieTooth disease: frequency of genetic subtypes and guidelines for genetic testing. J Neurol Neurosurg Psychiatry
2012;83:706–710.
8. Shy ME, Blake J, Krajewski K, et al. Reliability and validity of the CMT neuropathy score as a measure of disability. Neurology 2005;64:1209–1214.
9. Birouk N, Gouider R, Le Guern E, et al. Charcot–Marie–
Tooth disease type 1A with 17p11.2 duplication: clinical and
electrophysiological phenotype study and factors influencing
disease severity in 119 cases. Brain 1997;120:813–823.
10. Sivera R, Espinós C, Vílchez JJ, et al. Phenotypical features of
the p.R120W mutation in the GDAP1 gene causing autosomal dominant Charcot-Marie-Tooth disease. J Peripher
Nerv Syst 2010;15:334–344.
1624
Neurology 81
19.
20.
21.
22.
23.
24.
25.
26.
27.
Sevilla T, Martínez-Rubio D, Márquez C, et al. Genetics
of the Charcot-Marie-Tooth disease in the Spanish Gypsy
population: the hereditary motor and sensory neuropathy–
Russe in depth. Clin Genet 2013;83:565–570.
Pitceathly R, Murphy SM, Cottenie E, et al. Genetic dysfunction of MT-ATP6 causes axonal Charcot-Marie-Tooth
disease. Neurology 2012;79:1145–1154.
Kennerson ML, Yiu EM, Chuang DT, et al. A new locus for
X-linked dominant Charcot-Marie-Tooth disease (CMTX6)
is caused by mutations in the pyruvate dehydrogenase kinase
isoenzyme 3 (PDK3) gene. Hum Mol Genet 2013;22:
1404–1416.
Xu W, Gu MM, Sun LH, et al. A nonsense mutation in
DHTKD1 causes Charcot-Marie-Tooth disease type 2 in
a large Chinese pedigree. Am J Hum Genet 2012;91:
1088–1094.
Soong BW, Huang YH, Tsai PC, et al. Exome sequencing
identifies GNB4 mutations as a cause of dominant intermediate Charcot-Marie-Tooth disease. Am J Hum Genet
2013;92:422–430.
Ylikallio E, Pöyhönen R, Zimon M, et al. Deficiency of
the E3 ubiquitin ligase TRIM2 in early-onset axonal neuropathy. Hum Mol Genet 2013;22:2975–2983.
Lin KP, Soong BW, Yang CC, et al. The mutational spectrum in a cohort of Charcot-Marie-Tooth disease type 2
among the Han Chinese in Taiwan. PLoS One 2011;6:
e29393.
Nelis E, Van Broeckhoven C, De Jonghe P, et al. Estimation of the mutation frequencies in Charcot-Marie-Tooth
disease type 1 and hereditary neuropathy with liability to
pressure palsies: a European collaborative study. Eur J
Hum Genet 1996;4:25–33.
Claramunt R, Sevilla T, Lupo V, et al. The p.R1109X
mutation in SH3TC2 gene is predominant in Spanish Gypsies with Charcot-Marie-Tooth disease type 4. Clin Genet
2007;71:343–349.
Shy ME, Siskind C, Swan ER, et al. CMT1X phenotypes
represent loss of GJB1 gene function. Neurology 2007;68:
849–855.
Siskind CE, Murphy SM, Ovens R, Polke J, Reilly MM,
Shy ME. Phenotype expression in women with CMT1X.
J Peripher Nerv Syst 2011;16:102–107.
Warner L, Hilz M, Appel S, et al. Clinical phenotypes of
different MPZ (P0) mutations may include Charcot–Marie–
Tooth type 1B, Dejerine–Sottas, and congenital hypomyelination. Neuron 1996;17:451–460.
Shy ME, Jani A, Krajewski K, et al. Phenotypic clustering
in MPZ mutations. Brain 2004;127:371–384.
Sevilla T, Lupo V, Sivera R, et al. Congenital hypomyelinating neuropathy due to a novel MPZ mutation.
J Peripher Nerv Syst 2011;16:347–352.
Baxter RV, Ben Othmane K, Rochelle JM, et al. Gangliosideinduced differentiation-associated protein-1 is mutant in
Charcot-Marie-Tooth disease type 4A/8q21. Nat Genet
2002;30:21–22.
Senderek J, Bergmann C, Ramaekers VT, et al. Mutations
in the ganglioside-induced-differentiation-associated protein-1 (GDAP1) gene in intermediate type autosomal
recessive Charcot-Marie-Tooth neuropathy. Brain 2003;
126:642–649.
Sevilla T, Jaijo T, Nauffal D, et al. Vocal cord paresis and
diaphragmatic dysfunction are severe and frequent symptoms of GDAP1-associated neuropathy. Brain 2008;131:
3051–3061.
October 29, 2013
ª"NFSJDBO"DBEFNZPG/FVSPMPHZ6OBVUIPSJ[FESFQSPEVDUJPOPGUIJTBSUJDMFJTQSPIJCJUFE
28.
29.
30.
31.
32.
Claramunt R, Pedrola L, Sevilla T, et al. Genetics of
Charcot-Marie-Tooth disease type 4A: mutations, inheritance, phenotypic variability, and founder effect. J Med
Genet 2005;42:358–365.
Zimo
n M, Baets J, Fabrizi GM, Jaakkola E, et al. Dominant GDAP1 mutations cause predominantly mild CMT
phenotypes. Neurology 2011;77:540–548.
Ammar N, Nelis E, Merlini L, Barisic N, et al. Identification of novel GDAP mutations causing autosomal recessive Charcot-Marie-Tooth disease. Neuromuscul Disord
2003;13:720–728.
Verhoeven K, Claeys KG, Züchner S, et al. MFN2 mutation distribution and genotype/phenotype correlation in
Charcot-Marie-Tooth type 2. Brain 2006;129:2093–2102.
Calvo J, Funalot B, Ouvrier RA, et al. Genotype-phenotype
correlations in Charcot-Marie-Tooth disease type 2
33.
34.
35.
36.
caused by mitofusin 2 mutations. Arch Neurol 2009;
66:1511–1516.
Auranen M, Ylikallio E, Toppila J, Somer M, Kiuru-Enari S,
Tyynismaa H. Dominant GDAP1 founder mutations is a
common cause of axonal Charcot-Marie-Tooth disease in
Finland. Neurogenetics 2013;14:123–132.
Casasnovas C, Banchs I, Cassereau J, et al. Phenotypic
spectrum of MFN2 mutations in the Spanish population.
J Med Genet 2010;47:249–256.
Braathen GJ. Genetic epidemiology of Charcot-MarieTooth disease. Acta Neurol Scand Suppl 2012;126:1–22.
Lupo V, Galindo MI, Martínez-Rubio D, et al. Missense
mutations in the SH3TC2 protein causing Charcot-MarieTooth disease type 4C affect its localization in the plasma
membrane and endocytic pathway. Hum Mol Genet 2009;
18:4603–4614.
Your Commitment to Neurology Is Needed on
Capitol Hill!
Join us in Washington, DC, to educate Congress about the issues that are affecting you, your
practice, and your patients. The next Neurology on the Hill will take place on March 3 and 4,
2014. Openings are limited, and members of the American Academy of Neurology must apply
online by December 1, 2013. There is no application fee. The Academy will cover travel and hotel
accommodations at the Ritz-Carlton Pentagon City. Tell Congress your personal story. You don’t
need a public policy background, just a passion for neurology and the desire for positive change.
Visit www.aan.com/view/NOH2014 for more information.
Fall AAN Webinars: Help for Your Practice, CME for
Your Career
The American Academy of Neurology offers cost-effective Practice Management Webinars that
can be attended live or through convenient recordings posted online five days after the event.
AAN members can save 25 percent on all regular webinars! Plus, physicians can earn 1.5 valuable
CME credits for each webinar, and administrators receive a certificate of completion. Mark
your calendar for upcoming programs and register today for these and other 2013 webinars at
www.aan.com/view/pmw13:
Online Now
Meaningful Use Stage 2: Prepare Your Practice
October 29
Protecting Your Reputation as a High Quality Physician
November 19
11 Months and Counting: Are You Ready for ICD-10?
Neurology 81
October 29, 2013
1625
ª"NFSJDBO"DBEFNZPG/FVSPMPHZ6OBVUIPSJ[FESFQSPEVDUJPOPGUIJTBSUJDMFJTQSPIJCJUFE