Hereditary motor and sensory neuropathy with agenesis of the corpus callosum.

NEUROLOGICAL PROGRESS
Hereditary Motor and Sensory Neuropathy
with Agenesis of the Corpus Callosum
Nicolas Dupré, MD, MSc,1 Heidi C. Howard, PhD,1 Jean Mathieu, MD, MSc,2 George Karpati, MD,3
Michel Vanasse, MD,4 Jean-Pierre Bouchard, MD,5 Stirling Carpenter, MD,6 and Guy A. Rouleau, MD, PhD1
Hereditary motor and sensory neuropathy associated with agenesis of the corpus callosum (OMIM 218000) is an autosomal recessive disease of early onset characterized by a delay in developmental milestones, a severe sensory-motor
polyneuropathy with areflexia, a variable degree of agenesis of the corpus callosum, amyotrophy, hypotonia, and cognitive impairment. Although this disorder has rarely been reported worldwide, it has a high prevalence in the SaguenayLac-St-Jean region of the province of Quebec (Canada) predominantly because of a founder effect. The gene defect
responsible for this disorder recently has been identified, and it is a protein-truncating mutation in the SLC12A6 gene,
which codes for a cotransporter protein known as KCC3. Herein, we provide the first extensive review of this disorder,
covering epidemiological, clinical, and molecular genetic studies.
Ann Neurol 2003;54:9 –18
Hereditary motor and sensory neuropathy associated
with agenesis of the corpus callosum (HMSN/ACC)
(OMIM 218000), also known as peripheral neuropathy associated with agenesis of the corpus callosum
(ACCPN) or Andermann syndrome, is an autosomal
recessive disease of early onset. It is characterized by a
delay in developmental milestones, a severe progressive
sensory-motor neuropathy with areflexia, a variable degree of agenesis of the corpus callosum, amyotrophy,
hypotonia, cognitive impairment, an atypical psychosis,
and occasional dysmorphic features (high arched palate, syndactyly). Howard and colleagues1 recently identified the gene responsible for HMSN/ACC, solute carrier family 12 member 6 (SLC12A6), which codes for
K⫹-Cl⫺ cotransporter 3 (KCC3). In this review, we
present a complete summary on the current state of
knowledge of HMSN/ACC. Although rare, this disorder is particularly interesting because it represents a
unique example of a gene defect causing both developmental and neurodegenerative problems involving both
the central and the peripheral nervous systems. The
callosal agenesis indicates a defect in axonal migration
during embryogenesis, whereas the progressive neuropathy along with the cognitive involvement and the
atypical psychotic occurrences suggest a degenerative
From the 1Centre for Research in Neurosciences and the Department of Neurology and Neurosurgery, McGill University, Montreal; 2Hôtel-Dieu de Chicoution, Chicoutioni; 3Montreal Neurological Hospital and Institute, Department of Neurology and
Neurosurgery, McGill University, Montreal; 4Hôpital Ste-Justine,
Université de Montréal, Montreal; 5Department of Neurology, Hôpital de l’Enfant-Jésus, Laval University, Quebec City, Quebec,
Canada; and 6Hospital São João, Porto, Portugal.
Received Jan 2, 2003, and in revised form Mar 20. Accepted for
publication Mar 20, 2003.
process. The severe sensory-motor neuropathy is striking both for its distinctive pathological features (axonal
swelling) and for the distribution of its abnormalities
(cranial nerves, nerve roots, peripheral nerves). HMSN/
ACC is an ion transporter disorder that is quite different from other diseases caused by dysfunctional ion
channels. The fact that the K-Cl cotransporter 3 protein is at least indirectly involved in early axonal migration across the corpus callosum and in maintenance
of the peripheral nervous system (PNS) throughout life
can lead the way to a generation of new hypotheses on
ion transporter function.
Epidemiological Studies
HMSN/ACC is found mainly in the French Canadian
(FC) population of Quebec, and specifically in two regions of northeastern Quebec, the Saguenay-LacSt-Jean (SLSJ) region, and the Charlevoix County.2,3
In a study based on 101 individuals distributed in 82
families, De Braekeleer and colleagues2 analyzed the
geographical distribution of HMSN/ACC in northeastern Quebec. The overall incidence at birth was 1 in
2,117 live births and the carrier rate was 1 in 23 inhabitants. Genealogical reconstruction was attempted
in 161 obligate carriers, and a set of 22 founders was
Address correspondence to Dr Rouleau, Centre for Research in
Neurosciences and the Department of Neurology and Neurosurgery,
McGill University, 1650 Cedar Avenue, Montreal, Quebec, H3G
IA4 Canada. E-mail: [email protected]
© 2003 American Neurological Association
Published by Wiley-Liss, Inc., through Wiley Subscription Services
9
common to all of them. All these founders originated
from France, including 3 from Poitou, 11 from Perche,
and 4 from unknown regions. The authors concluded
that based on genealogical data, the founders who
came from France in the late 17th century probably
established themselves in the Charlevoix County where
they lived until their descendants moved to SLSJ in the
early 19th century. The Charlevoix County contributed 70% of the immigrants to SLSJ during the first
30 years of settlement, and, because migration was
mainly of familial type,4 many child carriers of the
HMSN/ACC gene may have entered the SLSJ region
with their parents, contributing to the high gene frequency in the region. The most represented founder
couple in SLSJ was found in only 8 of the 75 complete
genealogies, indicating that quite a few carriers must
have introduced the HMSN/ACC gene in this region.
We recently performed an updated survey of the geographical distribution of HMSN/ACC cases across
Quebec, by collecting data from each major pediatric
hospital and rehabilitation center of the province (Fig
1). These results show that despite important migration within the province over the past 20 years, the
disease remains concentrated in the SLSJ region.
Clinical Studies
Clinical Description
In 1966, Leblanc and colleagues5 first reported the existence of HMSN/ACC. He recognized that this “syndrome of callosal agenesis” had a familial component
and was particularly frequent in the SLSJ region of
Quebec. Subsequently, Andermann and colleagues6 described HMSN/ACC as an autosomal recessive disease
characterized by agenesis of the corpus callosum, a motor neuropathy (which they thought was compatible
with anterior horn cell disease), mental retardation,
areflexia, amyotrophy, hypotonia, and dysmorphic features. Subsequent descriptions of the disease recognized
that it involved a sensory-motor neuropathy rather
than an anterior horn cell disease.7 Mathieu and colleagues8 studied 64 patients between the ages of 2 and
34 years old. Forty-five (70%) of the 64 patients from
this phenotypic study were later tested for the HMSN/
ACC gene mutation. They were all found to be homozygous for a single nucleotide deletion in exon 18 of
the SLC12A6 gene (c.2436delG, Thr813fsX813),
thereby providing molecular confirmation of the diagnosis and confirming the genetic homogeneity of this
population (N. Dupré, unpublished data). Mathieu
and colleagues8 found the following clinical characteristics in these patients: ptosis (59%), upper gaze palsy
(30%), facial asymmetry (34%), areflexia (100%), hypotonia (36%), amyotrophy (86%), tremor (25%), seizures (17%), early Achilles’ tendon retraction (47%),
scoliosis (86%). Dysmorphism also was evaluated in a
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systematic way, with the following characteristics
among their 64 patients: brachycephaly (16%), higharched palate (39%), overriding of the first toe (16%),
and partial syndactyly of second to third toes (8%).
Clinical evolution was quantified with the following
measures: average age of onset of walking 3.8 years (47
patients), average age of end of walking capacity 13.8
years (35 patients), average age of appearance of scoliosis
10.4 years (22 patients), and average age of death 24.8
years (6 patients). We recently reviewed 49 FC HMSN/
ACC cases (subjects who are now deceased) and found
the average age of death to be 33 years; this is a longer
survival period than previously estimated (N. Dupré,
unpublished data). The main features of ACCPN in the
FC population can be found in Table 1.
Cognitive Function
The cognitive function of individuals with HMSN/
ACC has been addressed in relatively few studies. In
their clinical study of 64 cases, Mathieu and colleagues8 evaluated 53 subjects for cognitive dysfunction. Using the clinical classification of Taft9 to stratify
the degree of mental retardation, they found that 8%
of cases had normal intelligence, 49% had mild mental
retardation, 40% had moderate mental retardation,
and 4% had severe mental retardation. They did not,
however, indicate the age at which the patients were
evaluated, which, in light of the progressive nature of
the disease, would be relevant information. They also
reported that 39% of patients developed “psychotic episodes” after the age of 15 years. Paranoid delusions,
depressive states, visual hallucinations, auditory hallucinations, or “autistic-like” features characterized these
episodes. Filteau and colleagues10 studied a cohort of
62 patients to look at the relationship between imaging
features and the appearance of “psychotic episodes” in
HMSN/ACC. They identified 20 patients (32%) who
had presented such episodes, with a mean age at the
time of evaluation of 27.6 years and a mean age of
onset of psychosis of 19.5 years. The psychiatric evaluation was performed using a semistructured interview
according to Diagnostic and Statistical Manual III-R
criteria. They found that all 20 identified patients had
paranoid delusions often accompanied by visual or auditory hallucinations described as “monsters” or “persecutors.” Interestingly, it appears that psychosis was
more common in HMSN/ACC patients (32%) than in
adult populations of mentally retarded individuals
(18%).11
Electrophysiology
We recently reviewed the electrophysiological studies of
48 patients (aged 0 to 35 years) evaluated in Chicoutimi by one of the authors (J.M) over the last 20
years (Fig 2). We found that the median motor nerve
conduction velocities ranged from 16 to 57m/sec
Fig 1. Distribution of all hereditary motor and sensory neuropathy associated with agenesis of the corpus callosum cases seen by a
neurologist in the province of Quebec as of July 2002. Each case is represented by a black dot. (A) Distribution throughout the
entire province. (B) Distribution within the Saguenay-Lac-St-Jean region.
(mean, 33m/sec) between 0 and 2 years; from 26 to
45m/sec (mean, 34m/sec) between 2 and 15 years;
from 11 to 39m/sec (mean, 23m/sec) between 15 and
35 years. These conduction velocities show a wide rage
of variability that are unlikely to result from technical
factors because they all were performed by the same
individual using standardized protocols. It is difficult
to classify the neuropathy as primarily “axonal” or primarily “demyelinating,” because the range of conduc-
tion velocities straddles the usual cutoff values used to
make this distinction.12 The sensory nerve action potentials recorded either at the median, ulnar, or sural
nerves were never obtainable even in patients at a
young age. On needle electromyogram, fibrillations
were found in 11 patients (23%) and were, in general,
not abundant. Abnormal motor unit potentials were
found in 12 patients (25%) and ranged from polyphasic to, rarely, giant potentials.
Dupré et al: Motor and Hereditary Polyneuropathy
11
Table 1. Features of HMSN/ACC in the French Canadian
Patients
Clinical features
Dysmorphic traits
Hypertelorism (usually mild)
Syndactyly of second and third toes
Brachycephaly
High-arched palate
Overriding of the first toe
Cognitive Function
Mild to severe mental retardation
“Psychotic episodes” appearing usually during adolescence
Cranial Nerves
Eyelid ptosis (symmetrical or asymmetrical)
Facial plegia (symmetrical or asymmetrical; sometimes
associated with hemifacial atrophy)
Esotropia or exotropia (due to variable combinations of
oculomotor nerve palsies)
Upper gaze palsy
Horizontal nystagmus
Motor
Progressive distal and proximal symmetrical limb weakness with muscle atrophy (the infant is invariably hypotonic in the first year of life, learns to walk with a
device between 2 and 5 yr old and eventually becomes
confined to a wheelchair)
Diffuse limb tremor (probably due to the polyneuropathy)
Reflexes
Invariably absent from infancy
Sensory
All modalities are moderately to severely affected from
infancy
Other
Scoliosis
Pulmonary restrictive syndrome
Seizures (infrequent)
Investigation
Lumbar puncture
Usually mild elevation of proteins
Nerve conductions and EMG in young children
Invariably absent sensory potentials (sural, median, cubital) from infancy
Variable motor nerve conduction velocities (median, cubital, tibial)
Fibrillations
EEG
Usually normal
Imaging
May have no ACC, partial ACC, or complete ACC
May see mild cortical or cerebellar atrophy in older subjects
Sural nerve biopsy and muscle biopsy
Sural nerve biopsies show a lack of large myelinated fibers, signs of axonal loss (ovoids of Wallerian degeneration), and some enlarged axons that on electron microscopy show a decreased density of neurofilaments
Muscle biopsies show nonspecific signs of chronic denervation atrophy
Autopsy
The hallmark of autopsies is the swollen axons demonstrated in cranial nerve samples (third and seventh
especially) as well as in dorsal and ventral nerve roots.
Swollen axons can also be found scattered in the
white matter
The brain shows either no ACC, partial ACC, or complete ACC with preservation of Probst bundle
HMSN/ACC ⫽ hereditary motor and sensory neuropathy with
agenesis of the corpus callosum; EMG ⫽ electromyogram; EEG ⫽
electroencephalogram.
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Fig 2. Graph representing the median motor nerve conduction
velocity (m/sec) as a function of the age in 48 French Canadian patients with hereditary motor and sensory neuropathy
associated with agenesis of the corpus callosum. Most patients
were evaluated at a young age when they presented with delayed motor development and hypotonia. MNCV ⫽ motor
nerve conduction velocity.
Imaging
In their first report of the disease, Leblanc and colleagues5 had recognized the callosal agenesis based on
pneumoencephalography. This was confirmed in later
studies with the use of computed tomography (CT)
imaging, and then with magnetic resonance imaging
(Fig 3). Unfortunately, because few magnetic resonance
images have been performed on these patients, we
must rely mainly on the data provided by CT imaging
to assess the extent of anatomical abnormalities in the
brain. Mathieu and colleagues8 performed CT imaging
on 64 patients with HMSN/ACC and found no evidence of ACC in 33% of cases, partial ACC in 9%,
and complete ACC in 58%. They also found 4.7% of
patients with diffuse cerebral atrophy and 17% with
cerebellar atrophy. However, CT criteria are not ideal
for assessing the degree of callosal agenesis. Although
signs of complete or near complete ACC are unlikely
to be missed, it is much more difficult to assess partial
ACC. In addition, even in cases where the corpus callosum is anatomically well preserved, axonal loss is seen
on pathology (see autopsy findings below), which suggests that most, if not all cases of HMSN/ACC have
some degree of callosal abnormality.
Pathology
We reviewed five autopsies of patients who died between 26 and 37 years of age, as well as six sural nerves
of patients biopsied between the ages of 2 and 12
years. Two of the cases autopsied have had molecular
testing performed and were found to be homozygous
for the typical FC mutation, c.2436delG mutation, in
exon 18 of SLC12A6.
Fig 3. Brain magnetic resonance image of a 23-year-old
French Canadian patient with hereditary motor and sensory
neuropathy associated with agenesis of the corpus callosum
(ACC), with exon 18 c.2436delG showing complete ACC. (A)
Sagital T1 section. (B) Axial T1 section.
the medial side of the hemisphere corresponded to that
usually associated with callosal agenesis. Probst’s bundles were prominent in these brains. In the last two
cases, the corpus callosum was present in its entirety,
although the posterior portion was thinner than the
anterior (Fig 4A). One of these cases had had molecular testing and was found to have the typical FC mutation. The neocortex appeared grossly normal in all
patients, as did the hemispheric white matter.
Microscopic examination showed scattered small
oval vacuoles in the white matter of the brain in all
cases. They could be mistaken for Buscaino bodies, except that they never contained basophilic or metachromatic material, but were optically empty or contained
an eosinophilic web. The evidence suggested that they
had resulted from axonal swelling. The corpus callosum of the two patients in which it was completely
present appeared normal in its anterior portion except
for occasional vacuoles. In the posterior part, there was
progressively more evidence of axonal loss, with increased packing density of oligodendroglial cells and
proliferation of astrocytes. Where axonal loss was greatest, vacuoles were absent. Occasional pinkish, slightly
enlarged axons could be identified on hematoxylin and
eosin stains.
In contrast with the subtle microscopic changes in
the cerebral white matter, there were striking abnormalities in the spinal nerve roots and cranial nerves.
Massively enlarged axons up to 120␮m in diameter
could be identified on paraffin sections (see Fig 4B).
On stains for axons, such as Bodian’s (see Fig 4C),
these immense axons were a pale gray, but numerous
axons with a lesser degree of enlargement showed
darker staining that approached that of normal axons.
In some sections of roots, there were numerous tiny
axons, sometimes with a random or recurrent course
that suggested aberrant regeneration. Moderate axonal
loss per unit area was noted. No myelin could be
stained around the largest axons, whereas moderately
enlarged ones had abnormally thin sheaths. Epoxy resin
sections confirmed the findings of paraffin sections on
the roots and showed numerous onion bulb formations. Teased fibers from motor roots showed the
greatest degree of abnormality at approximately 1cm
from the spinal cord. Axonal enlargement tended to
persist through several internodes, although focal accentuation of enlargement occurred. Balls of Schwann
cells adhered to teased fiber bundles.
FINDINGS IN SURAL NERVES. All biopsies
showed an almost total lack of large myelinated fibers.
Small myelinated fibers from the three oldest patients
(6, 7, and 12 years old) were somewhat reduced in
numbers, whereas in two patients (2 and 3 years old)
small myelinated fibers were increased compared with
controls (see Fig 4D). A few Wallerian ovoids were
BIOPSY
In two cases, the corpus callosum
was entirely absent. One of these tested positive for the
c.2436delG mutation in SLC12A6; the other was not
tested. In a third case, only a small rounded portion of
corpus callosum was present anteriorly and was in contact with a thin lamina terminalis. The gyral pattern on
AUTOPSY FINDINGS.
Dupré et al: Motor and Hereditary Polyneuropathy
13
Fig 4. (A) Medial view of one hemisphere of a hereditary motor and sensory neuropathy associated with agenesis of the corpus callosum patient who has a corpus callosum. The anterior portion of the corpus callosum is thicker and whiter than the posterior part.
(B) Photomicrograph showing the longitudinal view of a lumbar motor root with three large swollen axons. Nuclei, probably of
Schwann cells, border these axons. Hematoxylin and eosin, ⫻560. (C) A swollen axon colored with Bodian’s stain is pale. The
arrow indicates the position where it is connected to a less swollen axon. (D) A sural nerve biopsy at the age of 2 years shows numerous small myelinated fibers and an absence of large fibers. Epoxy resin section, paraphenylene diamine, phase optics, ⫻250.
found in four nerves. Clusters of small myelinated fibers suggestive of axonal regeneration were seen in all
biopsies, and a few thin onion bulbs. Unmyelinated
fibers by phase microscopy and oil immersion appeared
normal. Rare large fibers with an intact sheath but a
crumpled or star-shaped outline of the axon and
slightly dark axoplasm were found in five biopsies.
These were thought to reflect more proximal damage.
In biopsies of two patients (at 2 and 4 years of age),
isolated fibers whose axons were the largest in the nerve
had disproportionately thin myelin sheaths, suggesting
that the axoplasm was swollen and the sheath had un-
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dergone either slippage or partial remyelination. Electron microscopy of one of these unusually large axons
with a thin sheath showed decreased packing density of
neurofilaments without signs of their degradation. This
was interpreted as a sign of increased fluid in the axoplasm.
Genetic Studies
Haplotype Analysis and Gene Mapping
In 1996, Casaubon and colleagues13 performed a
genomewide scan and mapped the HMNS/ACC locus
to a 5cM region on chromosome 15. The FC founder
effect suggested by previous epidemiological studies
subsequently was confirmed by haplotype and linkage
disequilibrium analyses.13,14 Furthermore, Howard and
colleagues1 established that the most common diseaseassociated haplotype covering 4cM on chromosome 15
is present on approximately 64% of disease chromosomes of FC HMSN/ACC patients. In addition,
marker D15S1232, which shared the greatest linkage
disequilibrium with the HMNS/ACC status, was
present in more than 97% of affected chromosomes.1
Howard and colleagues1 reduced the HMNS/ACC
critical region to approximately 1,000kb and proceeded
to test genes within this interval. The SLC12A6 gene,
which codes for a potassium chloride cotransporter
(KCC3) protein, was found to have a guanine deletion
in exon 18 (c.2436delG, Thr813fsX813) on both homologs in all but one FC HMNS/ACC patient.1 This
FC HMNS/ACC patient was found to be a compound
heterozygote with one chromosome 15 homolog bearing the c.2436delG mutation, whereas the other homolog has adjacent cytosine and thymine deleted
with a guanine insertion (c.1584_1585delCTinsG,
Phe529fs532 mutation) in exon 11 of the SLC12A6
gene.1 This patient’s phenotype does not differ significantly from the classic HMNS/ACC phenotype. Furthermore, as expected, all patients homozygous for
c.2436delG mutation in SLC12A6 were homozygous
for at least a portion of the founder haplotype, whereas
the compound heterozygote had one founder haplotype and one nonfounder haplotype at flanking markers. A total of 110 unaffected individuals from the
SLSJ region were screened for the exon 18
(c.2436delG) mutation. Four individuals (3.6%) were
found to be carriers of the mutation, therefore confirming the carrier rate reported in this region by previous epidemiological studies.2
Although it is clear that a geographical cluster exists
in the SLSJ region of the Province of Quebec, very few
similar cases have been described outside Quebec (Table 2). Although the genealogical studies suggest an ancestral couple coming from France in the late 17th
century, no cases of HMSN/ACC have so far been reported in France. Various centers in France have been
contacted, and a few cases have been identified with
atypical yet mildly comparable phenotypes, but no mutations in SLC12A6 were detected in these individuals
suggesting that these are not authentic HMSN/ACC
cases. As for other cases described in the literature
worldwide, we found only six15–17 that have clinical
characteristics completely compatible with those described in our large sample of FC HMSN/ACC patients.8 These reported non-FC cases have yet to be
tested for the HMSN/ACC gene mutation. We have,
however, formed collaborations with clinicians following up patients with typical characteristics of HMSN/
ACC (unreported in the literature) and were able to
identify mutations in the HMSN/ACC gene in two
non-FC families.
A brother (4 years old) and his sister (5 years old)
from the region of Verona (Italy) born from unrelated,
unaffected parents presented with developmental delay,
a sensory-motor axonal polyneuropathy, and callosal
agenesis. Both have the same mutation in exon 15 of
SLC12A6 (c.2023C-T, Arg675X).
Two boys of Turkish origin born from unaffected
parents who are second-degree cousins presented with
developmental delay, areflexia, hypotonia, a sensorymotor polyneuropathy, and complete callosal agenesis.
Both have the same nonsense mutation in exon 22 of
SLC12A6 (c.3031C-T, Arg1011X).
The finding of non-FC cases is significant because it
confirms the existence of HSMN/ACC outside of FC
and predicts the existence of other non-FC cases.
Table 2. Published Cases with a Phenotype Similar to That of French Canadian HMSN/ACC
Ethnic
Origin
No. of
Siblings
Italian
(Veneto)
1
Austrian
2
Tanzanian
(Mwanza)
3
Italian
(Veneto)
Turkish
2
2
NCS/EMG
Nerve Biopsy
Inheritance
(SLC12A6
mutation)
Reference
Clinical Features
Imaging
Developmental delay,
hypotonia,
areflexia, amyotrophy, bilateral ptosis, strabismus
Developmental delay,
areflexia, amyotrophy
Developmental delay,
hypertelorism,
areflexia, amyotrophy
Developmental delay
Complete ACC on
CT
Axonal motor polyneuropathy
Fewer myelinated fibers,
“demyelination”
AR (not tested)
15
Complete ACC on
MRI
Axonal sensory-motor
polyneuropathy
AR (not tested)
16
Complete or partial
ACC on CT
Axonal sensory-motor
polyneuropathy
Fewer myelinated fibers,
thin myelin sheaths,
onion bulbs
Degeneration and vacuolation of axons
AR (not tested)
17
ACC
Not done
Complete ACC on
MRI
AR (exon 15,
c.2023C-T)
AR (exon 22,
c.3031C-T)
1
Developmental delay,
hypotonia,
areflexia
Axonal sensory-motor
polyneuropathy
Sensory-motor polyneuropathy (axonal?)
Not done
1
HMSN/ACC ⫽ hereditary motor and sensory neuropathy with a genesis of the corpus callosum; NCS ⫽ nerve conduction studies; EMG ⫽
electroencephalomyogram; CT ⫽ computed tomography; AR ⫽ autosomal recessive; MRI ⫽ magnetic resonance imaging.
Dupré et al: Motor and Hereditary Polyneuropathy
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Gene Expression, Gene Function, and Animal Models
The gene involved in HMSN/ACC thus is SLC12A6,
which codes for a K⫹-Cl⫺ cotransporter named
KCC3. The data on the expression and function of
this gene are preliminary, and the final data from animal models are yet to come. SLC12A6 initially was
cloned and characterized by both Mount and colleagues18 and Hiki and colleagues.19 By analysis of
multiple tissue Northern blots probed with a KCC3specific sequence, Hiki and colleagues19 showed a
tissue-specific expression pattern in mice with the highest levels observed in kidney, skeletal muscle, heart,
and brain. Race and colleagues20 further characterized
SLC12A6 from human placenta tissue. The predicted
KCC3 protein structure includes (Fig 5) 12 putative
membrane-spanning helices with large NH2 and
COOH termini; a large extracellular loop between
transmembrane domains seven and eight with five potential sites for N-linked glycosylation; two consensus
cAMP-dependent protein kinase phosphorylation sites;
and four consensus protein kinase C phosphorylation
sites in the COOH terminus. They speculated that
KCC3 could be involved in volume regulation, in
transepithelial transport of salt and water, or in regulation of K and Cl concentrations in cells and in the
interstitial space. Shen and colleagues21 studied the
function of KCC3 in mouse fibroblast–derived NIH/
3T3 7-4 cells by comparing KCC3-transfected cells
with mock-transfected cells. They observed that
KCC3-transfected NIH/3T3 cells do not appear to
function in cell volume regulation as is described for
other KCC isoforms, including KCC3 in different cell
types. Instead, they showed disturbances in ion homeostasis (Cl⫺ equilibrium), which they suggested
might have broader roles in phenomena such as cell
proliferation and cell death. Pearson and colleagues22
studied the localization of KCC3 in mouse nervous
system using polyclonal antibodies. They observed that
KCC3 was abundant in all regions of the central nervous system they examined (cerebral cortex, hippocampus, diencephalon, brainstem, cerebellum, white matter
tracts of the internal capsule, corpus callosum, and spinal cord). In contrast, they found relatively low expression of KCC3 in dorsal root ganglia and trace amounts
in sciatic and trigeminal nerves. In the brain and spinal
cord, KCC3 seemed to colocalize with the oligodendrocyte markers CNPase and myeline basic protein,
thereby suggesting expression in white matter tracts.
KCC3 was also present in the cortex, hippocampus,
and in Purkinje neurons. The pattern of KCC3 expression was also shown to change over time, being low at
birth and increasing significantly throughout postnatal
development. Finally, Li and colleagues23 recently
showed that KCC3 mRNA levels are, in general, low
in embryonic rodent brain (mouse and rat) and the
level increases slightly at birth.
Fig 5. Representation of the KCC3 protein. There are 12 transmembrane domains along with large intracellular amino and carboxy termini. Lines perpendicular to the protein represent the different protein-truncating mutations identified in the DNA of hereditary motor and sensory neuropathy associated with agenesis of the corpus callosum patients. The number in brackets indicates the
number of amino acids deleted as a result of each mutation.
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Howard and colleagues1 studied the functional consequence of the predominant FC mutation
(c.2436delG) of SLC12A6 in Xenopus laevis oocytes.
They observed that the truncated mutant was appropriately glycosylated and expressed at the cellular membrane, but that it was nonfunctional. A KCC3 knockout mouse was generated in which exon 3 of SLC12a6
was removed and substituted with a ␤-galactosidase/
neomycin cassette, therefore deleting the remainder of
the protein. KCC3 expression was significantly reduced
in heterozygous mice and was absent in homozygous
mice. Starting at 2 weeks of age, KCC3-null mice
showed an abnormal locomotor function compared
with controls and heterozygotes. They had a low posture with limb weakness and disorganized limb movements. Behavioral tests (exploratory locomotion and
prepulse inhibition) were conducted to assess central
nervous system function: both heterozygous and homozygous mice exhibited a significantly lower level of
exploratory behavior; prepulse inhibition was abnormal
both in heterozygous and in homozygous mice, but
more so in the later. The researchers performed detailed anatomical studies on the brain and spinal cord
of three homozygous mice and three control mice: homozygous mice showed no specific abnormalities from
morphometric analysis (cortex, corpus callosum, cerebellum) and immunostaining. Interestingly, however,
sciatic nerves of homozygous mice showed many very
large axons with thin myelin sheaths, reminiscent of
those seen in HMSN/ACC patient’s biopsies, as well as
extensive Wallerian degeneration.
Discussion
In light of the HMSN/ACC gene discovery, many
questions need to be addressed. What is the role of
KCC3 in the development and maintenance of the
central nervous system and PNS? Is HMSN/ACC primarily a disease of the axon or the myelin sheath? Can
all the features of HMSN/ACC be explained by a single gene defect, or are other genes involved, either
within the same chromosomal region or on other chromosomes as modifier genes? Can environmental factors
influence the phenotypic expression of HMSN/ACC?
Lack of KCC3 in the developing nervous system
may increase the susceptibility of damaging the fibers
migrating across the midline close to the subarachnoid
space to form the corpus callosum. As this structure
forms during embryogenesis, absence of KCC3 must
have phenotypic effects early during neuronal development. One possible mechanism was proposed by Shen
and colleagues,21 who showed that KCC3 may be involved in ion homeostasis (Cl⫺ equilibrium) with a
possible role in cell proliferation via ion-sensitive kinases. The occurrence of cases without callosal agenesis
leaves open the question of interacting genetic and environmental factors, such as prenatal tobacco or alcohol
exposure.24 The findings in the PNS, on the other
hand, are progressive and do not suggest any migratory
abnormality. The site of maximum damage in the PNS
appears to be in the nerve roots, where nerve fibers are
bathed in cerebrospinal fluid. This is where the great
majority of swollen axons are encountered, along with
aberrant regeneration and Schwann cell proliferation.
The presence of some onion bulbs in peripheral
nerve biopsies25 and in nerve roots raises the question
as to whether Schwann cell or axon is the primary site
of damage. ␤-Iminodiproprionitrile is a toxin that leads
to the formation of neurofilamentous masses that focally distend axons.26 Animals treated continuously
with this toxin have shown axonal swellings in nerve
roots along with segments of demyelination and accumulation of onion bulbs.26 Axonal swelling has not
been described as a result of demyelination, and acutely
demyelinated axons tend to show reduced cross sectional area. The pathological evidence in HMSN/ACC
points to the axon as the primary structure damaged
and suggests that watery swelling of the axon is the
earliest morphological abnormality. This would be
consistent with abnormal electrolyte gradients, which
may occur with dysfunction of an electrolyte channel
in the axonal membrane. Acute swelling of axons at
nodes of Ranvier has been reported to occur with toxins that inactivate sodium channels.27
Selective large myelinated fiber loss as seen in the
nerve biopsies could be a useful diagnostic clue in cases
where the corpus callosum is present, although it is
seen also in Friedreich’s ataxia and some children considered to have the neuronal type of hereditary motor
and sensory neuropathy, especially with onset in early
childhood.28 In patients with HMSN/ACC, it almost
certainly reflects greater susceptibility of large axons in
the roots with their more extensive axolemma. Various
secondary changes in axons, such as accumulation of
damaged organelles, probably distal to the site of maximal damage, could be observed, but this could be easily distinguished from the accumulation of neurofilaments in giant axonal neuropathy and of various
abnormal organelles in neuroaxonal dystrophy.
Now that we know the gene defect responsible for
HMSN/ACC, it will be important to continue detailed
clinical studies as well as pursue detailed molecular
studies to better understand the pathophysiology of
this disease. In addition, it will now be possible to determine the prevalence of this disorder outside of Quebec.
This work was supported by “La Fondation des Jumelles Coudées”
and the Canadian Institute of Health Research (#15459, G.A.R.).
Dupré et al: Motor and Hereditary Polyneuropathy
17
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