Phenotypic expression of a Pro 87 to Leu mutation in the connexin 32

gene in a large Swiss family with Charcot–Marie–Tooth neuropathy

Thierry Kuntzera,*, Murielle Dunanda, Daniel F. Schorderetb, Jean-Michel. Vallatc,Angelika F. Hahnd, Julien Bogousslavskya

aService de Neurologie, Centre Hospitalier Universitaire Vaudois, BH 7/306, 1011, Lausanne, SwitzerlandbDivision de Genetique Medicale, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland

cService de Neurologie, Centre Hospitalier Universitaire Dupuytren, Limoges, FrancedDepartment of Neurological Sciences, London, Ontario, Canada

Received 25 October 2001; received in revised form 23 September 2002; accepted 15 October 2002

Abstract

Background: The clinical manifestations of CMTX have been well described but the natural history has not yet been studied in detail. We

studied phenotype variability in a family with a Pro 87 to Leu mutation of the connexin 32 (Cx32) gene. Methods: A total of 32 family

members, of which 19 patients were affected, underwent clinical, electrophysiological, and genetic studies. Results: Onset was in the second

decade. Clinical features were similar in both sexes when quantitative scores were compared, but more males had a steppage gait and skeletal

deformities. All adult patients had a predominant involvement of the thenar muscles. The median values of nerve conduction velocities (NCVs)

were not statistically different in men and in women. The correlation coefficients were low between motor NCVs within the same extremities,

indicating nonuniform slowing between nerves, the ulnar nerve being the least affected. When disability was rated, a strong correlation was

seen in male patients between severity of motor axonal loss and duration of the disease. The main pathological features were axonal loss,

clusters of regenerating fibers and paranodal demyelination, the hallmark of a Schwann cell pathology. Conclusions: Our data support the

hypothesis that clinical disability in CMTX is caused by loss of large myelinated axons in men. Furthermore, this study shows that the nerves

are not uniformly affected in terms of axonal loss. Preventing axonal degeneration and promoting axonal regeneration in the most affected

nerves might be the best therapeutic approaches to ameliorate disability in CMTX.

D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Hereditary motor and sensory neuropathies; X-linked Charcot–Marie–Tooth; CMTX; Connexin 32 mutation

1. Introduction

Charcot and Marie [1], Tooth [2], and subsequently, Hoff-

mann [3] and Herringham [4] were the first to study what

came to be known as Charcot–Marie–Tooth (CMT) disease.

They recognized the characteristic clinical phenotype of

distal leg weakness and atrophy, decreased sensation, and

absent reflexes, and appreciated the fact that the disease was

hereditary. Little new was added until the late 1950s, when it

was found that some patients with CMT neuropathy had

slow nerve conduction velocities (NCVs) [5]. In the 1960s

and 1970s, the CMT1 and two subtypes were defined by

NCVs, with CMT1 having markedly slow velocities in the

range of 15–30 m/s and CMT2 having near-normal veloc-

ities over 40 m/s [6]. Some investigators [7,8] recognized

families with ‘‘intermediate’’ NCVs, suggesting a third en-

tity. The conduction changes in CMT1 were further defined

as being uniformly slow in contrast to the multifocal slowing

seen in chronic acquired demyelinating neuropathies [9].

This observation proved clinically useful in distinguishing

familial and acquired disorders, and has subsequently been

shown to hold true for patients with CMT1a [8,10–13].

However, it is less clear whether conduction changes are

uniform in other forms of CMT disease [14]. Electrophysio-

logical studies have provided important information about

the cause of weakness in CMT patients; there is increasing

awareness that weakness and disability are less correlated

with NCVs than with the compound muscle action poten-

tial (CMAP) amplitude, a marker of axonal degeneration

[13,15].

0022-510X/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

PII: S0022 -510X(02 )00394 -5

* Corresponding author.

E-mail address: thierry.kuntzer@chuv.hospvd.ch (T. Kuntzer).

www.elsevier.com/locate/jns

Journal of the Neurological Sciences 207 (2003) 77–86

Since the identification of genetic defects of PMP22,MPZ

(P0), and theGJB1 (connexin 32 or Cx32) in the early 1990s,

it has become apparent that the variant formwith intermediate

conduction slowing represents CMT families with mutations

of the Cx32 gene on Xq13.1, and this entity therefore became

known as CMTX [16,17]. Onset and rate of progression of

weakness and sensory loss may be variable both between and

within families [18–23], but it may be similar in affected

identical twins [24]. While affected males usually have more

severe clinical disease and slower nerve conduction veloc-

ities, females gene carriers manifest disease albeit to a lesser

degree. A large number of mutations involving this gene have

been reported and are listed in the mutation database of

inherited peripheral neuropathies at http://molgen-www.uia.

ac.be/CMTMutations/. Although the genetic cause has been

delineated and the clinical manifestations of CMTX have

been well described, the natural history has not yet been

studied in detail. Such information is essential for the under-

standing of the molecular pathogenesis and for future molec-

ular therapies. In this regard, the detailed assessments of a

large multigeneration kinship can provide valuable insights.

We here report a large CMT family with no male-to-male

transmission and provide detailed clinical and electrophysio-

logical data for 19 affected individuals. Linkage analysis

confirmed the known Cx32 gene locus on chromosome X,

and a P87L mutation was found in all affected individuals.

The large number of definitely affected persons in this family

allowed us to demonstrate an age-dependent progression of

this disorder characterized by predominant involvement of

the peroneal and median nerves in male patients. We also

investigated whether electrophysiological findings can be

useful in diagnosing the disease, and in predicting disability

in this length-dependent motor and sensory neuropathy.

2. Patients and methods

2.1. Selection criteria

Between December 1991 and September 1993, seven

consecutive patients from the same family were initially

examined; for three patients, the diagnosis was CMT with

‘‘intermediate’’ NCVs, while the clinical examination and

electrophysiological evaluation were normal in the other

four. In April 1994, these patients were screened genetically

for 17p11.2 duplication, the only genetic test available at that

time, and all were negative. In 1996, the hypothesis of X-

linked CMTwas raised and was reinforced in 1998 when an

extensive pedigree of this family showed no male-to-male

transmission. The pedigree, established over seven genera-

tions, includes 398 members discovered by visiting the

council archives of the village from which the family

originated (Fig. 1). Between September 1998 and April

2000, a prospective study was therefore carried out on fur-

ther 25 patients, known either to be at-risk or to be affected

based on information obtained from several older relatives.

In the meantime, a Cx32 point mutation was confirmed in

certain members of the family. In total, 32 patients and at-risk

individuals were recruited after obtaining informed consent

for clinical, electrophysiological, and genetic studies.

2.2. Clinical data

In addition to providing a standard neurological history,

patients were asked to complete a questionnaire designed by

the French CMT group [11]. Among other items, the ques-

tionnaire asked patients about the age at which they first

noted symptoms or signs and disability was rated according

to a functional disability scale (FDS), ranging from 0,

complete independence without impairment, to 8, totally

bedridden and able to perform only basic bodily functions

(1 signified normal, but with cramps and fatigability, 2

inability to run, 3 difficulty walking, but still possible

unaided, 4 able to walk with a cane, 5 able to walk with

crutches, 6 able to walk with a walker, 7 wheelchair-bound).

All patients underwent standard neurological examinations

which was carried out as recommended by the French CMT

group [11], and 31 of the 32 patients were evaluated by a

single examiner (TK) in order to minimize inter-examiner

differences in the grading of the neurological examination:

strength was assessed with the traditional Medical Research

Council scale; muscle weakness and wasting were scored

separately as 0 if normal, 2 if present in the lower limbs, and

4 if present in both the upper and lower limbs; tendon

Fig. 1. Pedigree of the family in which no male-to-male transmission was

observed. A total of 32 affected and at-risk individuals (asterisks) were

recruited for clinical, electrophysiological, and genetic studies, of which 19

harbored the connexin Cx32 mutation.

T. Kuntzer et al. / Journal of the Neurological Sciences 207 (2003) 77–8678

reflexes were scored in the upper extremities as 0 if normal

and 1 if absent, and in the lower extremities as 0 if normal, 1

if the knee jerk or ankle jerk was absent, and 2 if both were

absent. To evaluate sensory loss, sensory grading was scored

as 0 if normal, 1 if the lower limbs were affected, and 2 if the

lower and upper limbs were affected for large fiber modal-

ities (position sense and vibration) and then for small fiber

modalities (pain and temperature sensation). The motor

neurological disability score (NDS), including both muscle

weakness and muscle wasting, was assessed on a scale from

0 to 8. The global NDS, including the motor NDS, sensory

loss (large and small fiber modalities), and areflexia, was

assessed on a scale from 0 to 15. Special attention was paid

to nocturnal visual loss, pupillary abnormality, deafness,

vocal cord paralysis, nerve hypertrophy, ataxia, tremor, and

pyramidal signs, in addition to pes cavus, hammer toes,

scoliosis, and pectus excavatum.

2.3. Electrophysiological study

NCVs were performed by standard techniques on the

right side of patients, using a Nicolet Viking IV machine.

Responses for motor median, ulnar, peroneal, and tibial

nerves were recorded from distal muscles using surface

electrodes. Distal conduction distances between a recording

electrode over the motor point and the site of distal nerve

stimulation were 80 mm for the median and ulnar nerves,

100 mm for the peroneal nerve, and 140 mm for the tibial

nerve. Proximal conduction velocity was determined across

the forearm and arm and across the leg, respectively. In order

to compare terminal and proximal segment conduction, we

used the terminal latency index [TLI, calculated as distal

conduction distance (mm)/proximal conduction velocity

(m/s)� distal motor latency (ms)]. Sensory conduction

velocities were recorded orthodromically using surface elec-

trodes over a fixed distance of 100 mm for median nerve

(from digit I) and 140 mm for the sural nerve. To more readily

compare NCV parameters, results are presented as percent-

age of those in age- and sex-matched controls of the authors.

2.4. Statistics

The distribution of electrophysiological data was analyzed

using the Bartlett and Kruskal–Wallis tests to study non-

parametric tests. Differences between groups were analyzed

using the Mann–Whitney U-test and were considered sig-

nificant when the P value was < 0.05. Correlations studies

between two variables of interest were calculated using SAS

Statistical software; the correlation was considered signifi-

cant when the correlation coefficient was >0.4 and P < 0.001.

2.5. Nerve biopsy

The biopsy specimen was divided into several portions;

Paraffin sections were examined using standard methods;

Epon-embedded semithin and ultrathin sections were pre-

pared according to standard techniques for light and electron

microscopic examination; teased nerve preparations were

examined and rated according to Dyck et al. [25].

2.6. Molecular genetic studies

Blood samples were obtained from patients and in chil-

dren, consent was obtained from the accompanying parent.

DNA was extracted from leukocytes according to standard

method. Cx32 mutation analysis was performed as described

previously [17]. Briefly, exon 2 was amplified by PCR using

the primers described and subsequently sequenced directly

using the same primers as for PCR and the BigDye termi-

nator cycle sequencing ready reaction kits (Perkin-Elmer).

The automated sequencing procedure was performed on an

ABI Prism 310 machine. Mutation was confirmed and

looked for in all family members by SSCP analysis.

3. Results

3.1. Clinical findings

3.1.1. Classification of family members

Thirty-two at-risk individuals underwent complete neuro-

logical examination. The median age at investigation was

37.6 years (range 4–74). At the time of examination, 19

individuals, including two children and one woman who was

admitted to our service for stroke, expressed the disease. All

except for one 33-year-old woman were aware of being

affected. Her two young boys who already had distal weak-

ness of the toe and ankle extensors were unaware of the

disease. The findings for these 19 patients, 9 men and 10

women (male/female [M/F] ratio 9:10), were analyzed in the

present study (Table 1). The remaining 13 at-risk individuals

were screened systematically for CMT disease, but the

Table 1

Clinical features in the 19 patients with definite disease according to sex

CMTX

males

CMTX

females

P

Number of patients 9 10

Age at onset (years) 14.8F 9.6

(3 to 29)

13.1F10.2

(2 to 37)

n.s.

Disease duration (years) 15.9F 12.1 31.2F 20.2 n.s.

Motor NDS

(weakness and wasting) (0–8)

6.2F 2.7 4.8F 1.7 < 0.05

Sensory score (0–4) 2F 1.5 1.6F 1.6 n.s.

Tendon reflex score (0–3) 1.5F 0.7 1.2F 0.6 n.s.

Global NDS (0–15) 9.7F 4 7.6F 3.2 n.s.

Foot deformities 7 (97%) 4 (40%) < 0.05

Kyphoscoliosis 2 (22%) 1 (10%) n.s.

Functional disability score (0–8)

0–1 7 (77%) 7 (70%) n.s.

2–3 2 (22%) 3 (30%) n.s.

NDS= neurological disability score; weakness, wasting, sensation and re-

flexes correspond to a summed score from the upper and lower extremities

(see Patients and methods); n.s. = not significant.

T. Kuntzer et al. / Journal of the Neurological Sciences 207 (2003) 77–86 79

results were negative and these patients were therefore

excluded from the analysis.

3.1.2. Age at onset and presenting features in the 19

patients

The age at onset was taken to be the time when the patient

first noticed gait disturbance or weakness in the legs.

Although this varied widely from 2 to 37 years of age (Table

1), 11 patients (61%) developed symptoms during the second

decade of life. Only the person who considered herself

asymptomatic was unable to state this specifically. The

median age at onset was not significantly different between

the sexes. In 17 cases (94.4%) (M/F ratio 9:8), foot drop with

atrophy of the ankle extensors was the initial perceived

symptom, although, on further questioning, most of the

patients remembered that foot deformity had variably been

present since early childhood. None of the patients reported

hand muscle weakness as the first symptom; amyotrophy of

the hands was described later during the course of the disease

(after the third decade) and affected half of the patients (M/F

ratio 4:5). Nine patients (M/F ratio 4:5) experienced para-

esthesias or numbness in the feet, but these sensations were

not present before the end of the second decade. Muscle

cramps were noticed in the legs in one-third of patients (M/F

ratio 2:5) during the first decade. Cramps in the upper

extremities were rarely reported before the third decade.

3.1.3. Neurological examination

Proximal muscle strength was normal in both the upper

and lower limbs in all patients whereas they had reduced

muscle power in the distal lower limbs, especially in the toes

and ankle extensors (MRC grades 0 to 4) (Fig. 2). In 17

subjects, all adults, muscle strength was reduced in the small

hand muscles, whereby the thenar muscles were predom-

inantly affected (Fig. 3). Tendon reflexes in the upper and

lower limbs were normal (one male), reduced (12 cases, M/F

ratio 4:8), or absent (six cases, M/F ratio 4:2). No pyramidal

signs were observed. Two male patients had a postural

tremor. No individual had abnormality of the cranial nerves,

although one female patient presented a slowed direct

pupillary reaction. In one male patient, posttraumatic per-

ception deafness was reported, but brainstem auditory

evoked potential (BAEP) interlatency peaks, and brain

MRI were normal. One affected female patient was admitted

to our service for ischemic stroke and had normal BAEPs.

Sensation was normal in six patients (M/F ratio 3:3, age

range 4–63 years), touch and vibration were impaired in five

(M/F ratio 4:1, age range 34–60 years), and all modalities

were affected in eight (M/F ratio 5:3, age range 15–74 years),

with predominant impairment of the distal legs. No selective

involvement of the small fibers was observed.

Foot deformities were frequent in both sexes (63%, M/F

ratio 7:4). Three individuals had scoliosis (M/F ratio 2:1),

three had genu valgus, and one had pectus excavatum. Five

patients (M/F ratio 4:1) required ankle bracing, and three

patients had undergone ankle arthrodesis. Six patients (M/F

ratio 3:3) were receiving invalidity pensions for their neuro-

pathy. Fig. 2 shows the difference in muscle wasting between

female and male patients.

The NDS are summarized in Table 1. The median global

NDS was 8 for all patients combined, 9.7 for men and 7.6 for

women. Seven male patients (77%) and two female patients

(20%) had a high score (global NDS>10). FDS was mild or

absent (0 or 1) in 14 patients (73%), while, in 5 (27%), it was

stage 2 or 3, the patients experiencing difficulty in walking

and running while still being autonomous, even the worst

Fig. 2. Photograph showing the phenotype variation in leg muscle wasting among three female and two male patients in the family. From left to right, patient 1,

the oldest patient encountered in the family, is mildly disabled and is the mother of patient 2, who shows more severe motor involvement. Patient 3 is the most

disabled woman encountered on whom a radial nerve biopsy was performed. Patient 4 presents obvious muscle wasting in the distal muscles of his extremities

with virtual flatness of the calf muscles. Patient 5 considered herself nonaffected before genetic counseling.

T. Kuntzer et al. / Journal of the Neurological Sciences 207 (2003) 77–8680

cases being able to walk unaided. There was no statistically

significant difference between the sexes (Table 1).

3.2. Nerve conduction velocities

We evaluated the frequency distribution of NCVs in 102

motor and sensory nerves in 17 patients, as 2 individuals

refused to be tested. A response could be recorded in 81

nerves (39 in males and 42 in females) and no responses

were evoked in 21 nerves. The median value of NCVs was

38 m/s (range 26–53) in men and 39.2 m/s (range 28–53) in

women, not statistically different. In general, the median

and peroneal nerves were more severely impaired than the

ulnar and tibial nerves, with the ulnar nerve being the least

affected (Table 2 and see correlation study, below). The

values for the motor NCVs and CMAPs amplitude varied

widely from normal to severely reduced (Table 2 and Fig.

4). CMAPs could not be obtained following distal stimula-

tion of the peroneal nerve in five individuals (two women),

of the tibial nerve in two women, and of the median and

ulnar nerves in one woman. Sural nerve sensory nerve

action potentials (SNAPs) were always severely reduced

or absent in adult patients (three men and six women), and

no SNAP could be recorded in the median nerve in three

women. In the two young boys, CMAP and SNAP ampli-

tudes were normal, but motor NCVs were reduced to

between 38 and 42 m/s for the motor nerves.

In 14% of the motor nerves (9/59), there was a reduction

in NCVs to < 80% of the lower limit of normal (LLN) when

the CMAP amplitude was >80% of the LLN (Fig. 4). In a

further 22% (13/59), a reduction in conduction velocities to

< 70% of the LLN was recorded when the CMAP amplitude

was < 80% of the LLN (Fig. 4). Therefore, in 36% of the

motor nerves, the reduction in NCVs was in the range of a

demyelinating neuropathy [26]. In 42% of the motor nerves,

the NCVs were normal. Conduction block or pronounced

dispersion of CMAPs was never observed. The TLI in our

patients showed a wide range of values (Table 2). The TLI is

a relative comparison of conduction between the distal and

proximal segments of motor nerves [27]. Taking an index of

V 0.34 as abnormal [28], seven patients (43.5%, sex ratio

3:4) had an abnormal median nerve TLI (Fig. 5C).

3.3. Correlation study

3.3.1. Variability of disease expression between the sexes

The clinical features were similar in both sexes when

global NDS and FDS were compared (Table 1), but moreFig. 3. (A) Hand muscle wasting and predominant involvement of the thenar

muscle in a 33-year-old woman (see Fig. 2, patient 3). She remembered

having had difficulties in running, then in walking, since late childhood. At

the age of 18 from which time she was employed as a secretary, she first

noticed wasting of the thenar and interossei muscles. (B) When the patient

attempts to flex her thumb, contraction of the long flexor pollicis muscle

permits bending of the distal joint, but opposition and contraction of the

flexor pollicis brevis muscle are impossible.

Table 2

Electrophysiological characteristics of the 19 CMTX patients according to

sex

Normal

value

CMTX

males

CMTX

females

P

Median nerve n= 8 n= 9

MNCV (m/s) 48 39.5F 4.6

(35–48)

39F 4.8

(32–47)

n.s.

CMAP (mV) 5 3.6F 2.6

(0.7–5)

4F 1.7

(0.3–7.2)

n.s.

DML (ms) 3.9 5.2F 1.3

(3.8–7.2)

5F 1.8

(3.7–6.2)

n.s.

TLI >0.34 0.36F 0.13

(0.21–0.51)

0.37F 0.05

(0.28–0.42)

n.s.

No response 0 1

Ulnar nerve n= 8 n= 8

MNCV (m/s) 48 37F 6.3

(28–48)

44.6F 8.5

(26–54)

n.s.

CMAP (mV) 5 4.8F 1.3

(2.5–6.2)

6.4F 2.9

(0.2–9.1)

n.s.

DML (ms) 3 3.2F 0.8

(2.8–4.2)

3.1F 0.9

(2.9–4.3)

n.s.

TLI >0.34 0.5F 0.11

(0.33–0.65)

0.49F 0.13

(0.33–0.77)

n.s.

No response 0 1

Peroneal nerve n= 5 n= 7

MNCV (m/s) 40 33F 7.7

(28–49)

33.7F 4.4

(25–36)

n.s.

CMAP (mV) 2.5 1.9F 1.3

(0.1–3.2)

2.8F 4.3

(0.2–5.4)

n.s.

DML (ms) 6.5 7.4F 2.1

(6.3–9.9)

7F 2.2

(6.6–8.1)

n.s.

No response 3 2

Tibial nerve n= 8 n= 7

MNCV (m/s) 40 35.4F 6.7

(31–41)

34.7F 4.6

(26–36)

n.s.

CMAP (mV) 2.5 1.9F 1.4

(0.1–3)

4.1F1.8

(0.1–10.4)

< 0.05

DML (ms) 7 7.6F 2.2

(6.6–9.1)

7.2F 2.4

(6.4–8)

n.s.

No response 0 2

MNCVs=motor nerve conduction velocities; CMAP= compound muscle

action potential; DML= distal motor latency.

T. Kuntzer et al. / Journal of the Neurological Sciences 207 (2003) 77–86 81

males than females had a steppage gait (P= 0.02) and more

males than females showed wasting of leg muscles (P=

0.035; shown in Fig. 2), sensory loss (P= 0.045), and sco-

liosis or foot deformities (P= 0.033).

3.3.2. Correlation between nerves

Within the same extremity, there was no statistical

correlation between the slowing of NCVs in different

nerves, as shown in Fig. 5A and B, in which motor NCVs

Fig. 5. Comparison of ulnar and median motor NCVs (A), tibial and peroneal NCVs (B), and ulnar and median terminal latency indices (C). In order to compare

both nerves, only the results for individuals in whom both nerves in the same extremity were tested are shown. The correlation coefficients are reported at the

upper corner, and all are low. Squares =males; circles = females.

Fig. 4. Comparison between median, ulnar, peroneal and tibial NCVs (abscises) and CMAP amplitude (ordinates) in 59 motor nerves of 17 patients expressed as

a percentage of normal values. Motor NCVs correlate with CMAP amplitude but this is not uniform between nerves, as the correlation coefficient was high for

the peroneal and tibial nerves, moderately high for the ulnar nerve, and low for the median nerve. Thirty-six percent of these values were in the range of a

demyelinating neuropathy.

T. Kuntzer et al. / Journal of the Neurological Sciences 207 (2003) 77–8682

in the upper or lower limbs are compared. As can be seen,

the correlation coefficient was low, indicating nonuniform

slowing between different nerves. This is again seen in Fig.

5C, which shows no significant correlation between median

and ulnar nerves TLI in men and women. Median, ulnar,

peroneal, and tibial motor NCVs correlated with CMAP

amplitude (mean r = 0.74, P < 0.001) and age (r = 0.88,

P < 0.001), but this was not uniform between different

nerves (Fig. 4), as the correlation coefficient was high for

the peroneal and tibial nerves, moderately high for the ulnar

nerve, and low for the median nerve.

3.3.3. Correlation between NCV parameters and gender

When supramaximal motor and sensory responses were

pooled, the median value of NCVs was 38 m/s in men and 43

m/s in women (P= 0.12). The median values of NCVs and

CMAP amplitude of the four studied nerves are shown in

Table 2. No significant difference was observed between the

sexes, except for tibial nerve CMAPs amplitude (P= 0.045).

3.3.4. Correlation between NCV parameters, sex, pheno-

type, and disease duration

No correlation was found between reduced NCVs and the

degree of disability, as estimated either by the global NDS or

FDS, in either sex and in any nerve considered in the 17

patients (r < 0.1, P>0.05). However, with longer duration of

disease, there was a tendency to lower median and tibial

nerve NCVs in males (r = 0.82 and 0.98, respectively, P <

0.001), but not in the ulnar and peroneal nerves (r= 0.28 and

0.39, respectively, P>0.05, but no response was evoked in

the peroneal nerve in three men). This tendency was not seen

in females (r < 0.1, P>0.05).

A high correlation coefficient was seen between a high

global NDS value and low CMAPs amplitude with distal

stimulation of the median, peroneal, and tibial nerves in the

pooled data for males (r = 0.78, 0.84 and 0.91, respectively,

P < 0.001), but not for females (r < 0.1, P>0.05). In males,

there was also a close correlation between loss of median,

ulnar, peroneal, and tibial nerve CMAP amplitude and dis-

ease duration (r = 0.80, 0.83, 0.91, and 0.98, respectively, P <

0.001), while, in females, no correlation could be demon-

strated, regardless of the nerve considered (r< 0.3, P>0.05).

There was a global tendency to increased disability, as

estimated either by the global NDS or FDS, with disease

duration (r = 0.29 and 0.25, respectively); this correlation

being greater when only males were considered (Fig. 6)

(males, r = 0.76 and 0.6, respectively, P < 0.001; females,

r = 0.25 and 0.26, respectively, P>0.05).

3.4. Pathology

A dorsal cutaneous radial nerve biopsy was performed in

a 33 year-old severely disabled woman. At low magnifica-

tion, a mild reduction in large myelinated fibers was seen

(6925/mm2; control: 9300/mm2). The fibers seemed to have

Fig. 6. Comparison of changes in disease severity and duration of disease

according to gender. There was a greater tendency to increased disability in

males than in females, as estimated either by the global NDS or FDS (see

text). Squares =males; circles = females, filled symbols and black lines =

NDS; open symbols and grey lines = FDS.

Fig. 7. Electron micrograph of the dorsal cutaneous radial nerve from a

severely disabled young female patient (see Fig. 2, patient 3). (A) The

density of myelinated fibers is decreased. Clusters of regenerating fibers are

indicated by arrows. Onion bulb proliferation of the Schwann cells is seen

around some myelinated fibers (arrowhead). Bar 20 Am. (B) Within clusters

of regenerating fibers, one notices the presence of concentric proliferation of

the Schwann cells onion around a myelinated fiber. Many fibers show

characteristic widening of the adaxonal space. Bar = 10 Am.

T. Kuntzer et al. / Journal of the Neurological Sciences 207 (2003) 77–86 83

appropriate myelin sheaths, but the mean g-ratio (axon

diameter/fiber diameter) was increased (0.7F 0.06; control:

0.52F 0.08). The most prominent lesion consisted of a sig-

nificant number of clusters of regenerating, thinly myeli-

nated nerve fibers, explaining the increase in the g-ratio (Fig.

7A) and indicating attempts of regeneration. There were very

few myelinated fibers surrounded by Schwann cell prolifer-

ation, resembling onion bulbs (Fig. 7B). Teased fiber anal-

ysis was graded as normal (71%), paranodal demyelination

(24%), segmental demyelination (3%), and Wallerian degen-

eration (2%).

3.5. Genetic analysis

No evidence of consanguinity was found in the family

over seven generations. Direct sequencing of the Cx32 gene

after PCR amplification showed a C to T missense mutation

at position 322 of the cDNA, resulting in the substitution of a

proline at position 87 with leucine. This base change

cosegregated with all affected individuals in the family and

was not seen in 50 normal individuals. Of the 29 family

members, 19 were found to harbor the Pro 87 to Leu

mutation, these being the 19 members who were clinically

affected. Two obligate female carriers found in the pedigree

were not available for DNA testing.

4. Discussion

The present study describes a large number of CMTX

patients from the same family and focuses on the clinical

presentation, neurophysiological, and pathological charac-

teristics, and the genetic background of this disorder. In

principle, it shows that the initial manifestations were pre-

dominantly motor and distal in the lower extremities, as

classically reported in CMTX and in other CMT types. In

this family, however, the most useful distinguishing clinical

feature was the predominantly affected muscles innervated

by the median nerve, which made opposition of the thumb

impossible in adult individuals. This feature has already been

reported [16,21,22] and can be viewed as the clinical hall-

mark of CMTX in adult patients. The electrophysiological

counterpart of this sign is the predominant loss in CMAP

amplitude found by distal stimulation of the median nerve at

the wrist, a feature also observed in other CMTX families, or

predominantly in CMTX women [23], even if this clinical

loss in thumb function has not been emphasized [20].

Onset of symptoms was in the second decade of life in

most patients in this family, but this was preceded by pes

cavus in some patients, indicating an earlier onset with distal

atrophy related to axonal loss. No gender difference in

severity of functional and neurological scores could be

demonstrated statistically, but more men than women had a

steppage gait and men were more impaired than women in

terms of distal leg muscle wasting, sensory loss, and skeletal

deformity, all clinical features that are difficult to quantify.

Associated signs were rarely seen in our family, as postural

hand tremor was only observed twice and no signs of central

nervous system involvement were seen. We considered

incidental the ischemic stroke seen in one woman and the

deafness noticed in one man as posttraumatic. In these two

patients, no central conduction delay or auditory pathway

abnormalities were found by BAEP, contrasting with the

previous reports [29–31], but agreeing with the lack of

central nervous system changes in a CMTX family in which

the entire coding sequence of Cx32 was lost [32].

In women, the clinical signs and electrophysiological

changes were heterogeneous in terms of severity, ranging

from so mild that it was difficult to diagnose obligate at-risk

females to a degree of disability similar to that seen in the

worst affected males. This variability in the clinical pheno-

type in women is thought to be due to Lyonization or X-

inactivation. Lyonization is a mechanism ensuring equal

expression of genes by random inactivation of one of the

two X-chromosomes in females [33]. Females carrying a

mutation on one X-chromosome can therefore express a

range of phenotypes from normal to diseased, depending

on the pattern of X-chromosome inactivation established at

the level of individual Schwann cells [34].

The clinical and electrophysiological data from our study

are similar to those from previously reported studies [16,19–

23,32,35] and from two reports [36,37], with signs of

demyelination and of length-dependent degeneration of the

motor and sensory axons that are more evident in male

patients than in females. Despite the similarity between our

data and those of other investigators, our conclusions from

correlation studies are somewhat different, probably due to

differences in the methodology used for evaluation and

number of patients studied. For example, we compared

electrophysiological parameters with neurological changes

and duration of the disease and found a significant correla-

tion between the degree of axonal loss and the rate of

disability and duration of the disorder in men, whereas

Birouk et al. [20], who compared the average reduction of

NCVs and CMAP amplitude with a functional score meas-

uring the patients’ ability to walk, found no significant

correlation between disease duration and axonal degenera-

tion. Because of their use of averaged data for men and

women combined, significant correlations might have been

obscured.

There is increasing electrophysiological evidence that

CMTX is a demyelinating disorder, rather than an axonal

neuropathy [36,37], a mechanism reported in the earlier

studies with phenotypic/genotypic correlation [14]. The

findings of the present study might explain why there is no

consensus on the type of electrophysiological changes found

in CMTX. Electrophysiological evidence in favor of a demy-

elinating process can be difficult to detect if responses are

only recorded from muscles innervated by the median and

peroneal nerves, as stimulation of these two nerves can result

in small or unobtainable responses and therefore suggest se-

vere motor axonal loss without detecting parameters indica-

T. Kuntzer et al. / Journal of the Neurological Sciences 207 (2003) 77–8684

tive of demyelination. This point is exemplified in the two

youngest patients seen in our study who had intermediate

slowing of median and ulnar motor nerve conduction veloc-

ities at a time when the amplitudes were normal. Further-

more, our electrophysiological evaluation of four motor

nerves shows clearly that there is nonuniform slowing of

nerve conduction and low terminal index latencies within the

same limb, and that the ulnar nerve and then the tibial nerve

are the least affected nerves in terms of axonal loss. A

recently published study of a large series of patients has also

found internerve heterogeneity of motor NCVs in CMTX

females [23]. The importance to carry out NCVs in different

nerves is therefore stressed. In cases where no responses

could be recorded from distal upper limb muscles, stimula-

tion of the musculocutaneous nerve and recording responses

over the biceps brachialis muscle may be useful.

Two previous reports [36,37] as well as the current study

demonstrate that CMTX is a nonuniformly demyelinating

neuropathy that produces distal weakness, atrophy and

sensory loss. In our study, we have further demonstrated

that these clinical findings as well as the patients’ disability

are caused by a length-dependent and age-dependent loss of

sensory and motor axons. The biological basis of this

degeneration likely is disruption of the normal interactions

between axons and their Schwann cells, leading to decreased

axonal function. In biopsied nerve studies of different

CMTX families, morphometric analyses have regularly

found loss of large myelinated fibers and the presence of

numerous regenerative clusters of thinly myelinated fibers

[22,38–40]. As shown by the frequent signs of paranodal

demyelination in our teased fiber analysis, a high incidence

of widening of the nodes of Ranvier might also be counted as

a specific feature of CMTX, and this has been recently

demonstrated in a careful study of 14 CMTX nerve biopsy

samples, in which the nerve morphometry of our biopsied

patient is included [38]. Segmental demyelination and

remyelination have also been seen, but were not as prom-

inent as described by others [36]. Widening of the nodes of

Ranvier might be related to a primary pathology of the

Schwann cells, reflecting the consequence of the identified

molecular genetic defect in the Cx32 protein. This protein is

expressed in rodent and human myelinating Schwann cells

within the paranodal myelin loops and Schmidt Lanterman

incisures [34,38] and is thought to form ‘‘reflexive’’ gap

junctions, providing a direct transcytoplasmic diffusion path-

way across the compact myelin sheath [41,42]. The Cx32

gene is widely expressed in a variety of tissues and the

restriction of pathological manifestations to the peripheral

nerve in CMTX is therefore puzzling. It is possible that other

connexins compensate functionally for mutated Cx32 in the

brain, as well as in other tissues. Alternatively, Cx32 may

play a unique functional role in the Schwann cell that is not

complemented by other connexins. The description of the

effects of mutations in almost all regions of the Cx32 gene

indicates that most of the molecule is important in the correct

functioning of gap junction [35,42]. The mechanisms caus-

ing the peripheral nerve damage are unknown, but several

observations indicate that molecular abnormalities of intrin-

sic Schwann cell proteins can result in profound axonal

pathology and in distal accentuated axonal degeneration and

fiber loss. From recent research, it has become evident that at

a time of high metabolic demand such as regeneration-

associated myelination, mutant Schwann cells may fail to

differentiate into a competent state to provide trophic support

for efficient axonal sprouting and to induce myelination

[38,43–45]. Furthermore, the functional consequences of

individual Cx32 mutations have been studied by assessing

the electrophysiological channel properties in transfected

mammalian cells, and it was shown that the transduction of

signals may be variable in individuals or in families depend-

ing on the formation of nonfunctional channels or on the

formation of channels with altered permeability properties

[46]. This could account for the variations in nerve con-

duction velocity seen within different nerves in the same

individual or in different family members. On the other hand,

the paranodal distribution of the mutated Cx32 may make the

axon-Schwann cell unit susceptible to environmental con-

straints, such as repeated microtraumatic lesions at compres-

sion sites in susceptible nerves such as the median and the

peroneal nerves, as we know that the nodal regions being

long known as the most susceptible structure of the periph-

eral nerve to compression [47]. The fact that axonal loss does

correlate with disability in CMTX has important implica-

tions for future treatment of the disease. Correction of the

Schwann cell defect along the entire course of each affected

nerve might be difficult, while correction of the Schwann cell

defect distally in the median and peroneal nerves, the most

affected nerves, might be more feasible, by gene transfer or

by applying exogenous growth factors to degenerating

axons. Decompression of the median nerve at the wrist

might also be considered early in the course of the disease,

but its usefulness remains to be demonstrated.

Acknowledgements

We gratefully acknowledge the cooperation of the family

members. This work was supported by the Association

Suisse Romande et Italienne contre les Myopathies.

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