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Association between centromeric deletions of the SMN gene and sporadic adult-onset lower motor neuron disease.

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Association between Centromeric Deletions
of the SMN Gene and Sporadic Adult-Onset
Lower Motor Neuron Disease
Bruno Moulard, MD, PhD,* FranGois Salachas, MD, PhD,? BtnCdicte Chassande, M D , t Valerie Briolotti,$
Vincent Meininger, MD, PhD,t Alain Malafosse, MD, PhD,§ and William Camu, MD, PhDS
The telomeric copy (r) of the survival motor neuron (SMN) gene is homozygously deleted in more than 90% of patients
with infantile motor neuron disease (MND). In the general population, no homozygous SMNt deletion has been found,
whereas 5% of centromeric SMN (SMNc) deletions can be observed. Although SMNt deletions appear causal for infantile
and at least some adult-onset spinal muscular atrophy (SMA) (type IV), the respective role of SMNdeIetions remains
unclear in adult-onset MNDs. We studied SMN gene in three different groups of patients with adult-onset MNDs. In
sporadic amyotrophic lateral sclerosis (ALS; n = 177) and familial ALS (n = 66), no SMNt deletion had been found, and
the frequency of SMNc deletions was not increased. Conversely, among the 14 patients with sporadic pure lower MND
(LMND), we found 2 patients with homozygous SMNt deletions (14%)and 5 patients with homozygous SMNc deletions
(36%).These data suggest that (1) SMNt deletions do not account for the major part, if any, of adult-onset LMND cases;
and (2) SMNc deletions act as a susceptibility factor for LMNDs in adults. The clinical and genetic heterogeneity of
LMND cases, including SMA type IV,are yet to be unexplained. Further studies on large groups of adult-onset LMND
patients are warranted to refine its nosology.
Moulard B, Salachas F, Chassande B, Briolotti V, Meininger V, Malafosse A, Carnu W.
Association between centromeric deletions of the SMN gene and sporadic adult-onset
lower motor neuron disease. Ann Neurol 1998;43:640-644
Childhood-onset proximal spinal muscular atrophy
(SMA) is the most common and severe infantile motor
neuron disease (MND). The disease is characterized by
a dramatic degeneration of spinal and bulbar motor
neurons.' SMA is inherited with an autosomal recessive
trait and recent studies point out the interest in the
survival motor neuron (SMN) gene on chromosome
5q13, which is deleted on both exons 7 and 8 in more
than 90% of the cases.223SMNis present in two copies
on chromosome 5q13, telomeric (SMNt) and centromeric (SMNc). Only homozygous SMNt deletions are
associated with SMA, and they are not found in the
general population. Homozygous SMNc deletions have
rarely been described in SMA, but are present in 2 to
5% of unaffected subjects.233However, little is known
about the incidence of those deletions in other MNDs,
namely, adult-onset MNDs. Amyotrophic lateral sclerosis (ALS) is the most frequent adult-onset MND,
leading to death in a few months or years after onset.*
In a manner similar to SMA, the disease affects spinal
and bulbar motor neurons, but the degeneration also
involves the corticospinal tract. ALS is mainly sporadic,
but familial cases with an autosomal dominant inheritance account for 10% of the cases.' In a subset of
families, several mutations in the copperhinc superoxide dismutase (SODI) gene on chromosome 21 have
been described.6 However, the cause of this disease remains unknown in the great majority of the cases. The
term adult-onset pure lower motor neuron disease
(LMND) defines a group of diseases characterized by
the degeneration of lower motor neurons, a distal and
asymmetrical onset, a rather disabling course, and the
absence of familial history. Those elements are helpful
for differentiating LMND from the rare adult-onset
SMA, namely, type IV.' The etiopathogenesis of adult
LMND is unknown, but owing to its severity, this disorder is often considered a possible ALS variant. We,
and many other authors,'-''
have described several
families where SMA and adult-onset MNDs coexisted,
suggesting that childhood- and adult-onset MNDs
From the *Laboratoire de Mtdecine Exptrimentale, Institut de Biologie, and $Service de Neurologie B, HBpital Gui de Chauliac and
Laboratoire de Physiopathologie Neuromusculaire, INSERM CJF
37-02, Institut de Biologie, Montpellier; and ?Service de Neurologie, Division Mazarin, Hbpital de la SalpCtri&re,Paris, France; and
§Division de Neuropsychiatrie, HBpital Belle-Idte, GenPve, Switzerland.
Received May 14, 1997, and in revised form Aug 28 and Dec 1.
Accepted for publication Dec 6, 1997.
Address correspondence to Dr Camu, Service de Neurologie B,
HBpital Gui de Chauliac, 34295 Montpellier Cedex 5 , France.
640 Copyright 0 1998 by the American Neurological Association
share a common genetic background (cases are reviewed by de Belleroche and associates5). We then
studied SMN polymorphisms in three different groups
of adult-onset MNDs, that is, sporadic ALS (SALS;
n = 177), familial ALS (FALS; n = 66), and LMND
(n = 14). Here, we show that homozygous SMNc deletions are present in 36% of the LMND cases, and
the frequency of those deletions in SALS and FALS is
comparable with that of the general population. Furthermore, although SMNt deletions are absent in our
SALS and FALS cases, we describe 2 LMND patients
with this deletion. These data suggest that (1) homozygous SMNt deletions do not comprise or explain the
occurrence of LMND in adults; and (2) the occurrence
of homozygous SMNc deletions may be a genetic risk
factor for adult-onset LMND.
Patients and Methods
FALS (n = 66) and SALS patients (n = 177) had probable
or definite ALS according to international criteria requiring
unequivocal upper and lower motor neuron signs in two or
three regions (eg, bulbar level, upper limbs, and/or lower
limbs), respectively.”,’2 The third group was composed of
14 subjects suffering from late adult-onset LMND, in whom
motor neuropathies with or without conduction blocks,
Kennedy’s disease (absence of abnormal CAG e~pansion),’~
lymphoma, and malignant plasma cell dyscrasia have been
excluded. For those patients, careful attention was given to
the criteria of entry in the study, to ensure that they did not
fulfill criteria for the rare form of adult-onset proximal spinal
muscular atrophy (SMA), the so-called type IV.’” In brief,
they all had an asymmetrical and distal onset of weakness,
with an unequivocal progression throughout the years and
no familial history of MND. For all the patients enrolled in
this study, classification has been done prospectively, and the
subjects have been followed longitudinally to ensure, after
years or at the time of death, that criteria for diagnosis were
still fulfilled.
The following clinical data were obtained for all subjects
(Table 1): (1) age at onset, defined as the time at which the
first symptoms of muscle weakness were noted by the patient; (2) duration of the disease, defined as the interval between onset and death, and expressed as the median survival
for each group, because a significant number of patients were
still alive; and (3) sex.
Analysis of SMN Polymorphisms
Homozygous deletions for SMNt and SMNc in exons 7 and
8 were studied as previously
In brief, both exons were first amplified by polymerase chain reaction (PCR),
using R1 11 and X7DRA as primers for exon 7 and 541C960
and 541C1120 for exon 8. Each product was then digested
at 37°C overnight, with 5 U DraI for exon 7 and 5 U DdeI
for exon 8.14 After digestion, the pattern was analyzed by gel
electrophoresis on 4% Nu Sieve GTG agarose (Biogel, Vista,
CA). Each abnormal pattern was further confirmed by
single-strand conformation polymorphism according to the
technique of Lefebvre and colleagues; slightly modified.
In brief, both exons were amplified by radioactive PCR
([a-33P]dCTP, Isotopchim, Ganogobie, France) with R1 11
and 541C770 for exon 7 and 541C960 and 541C1120 for
exon 8. The PCR product ran, at 4 W, on electrophoresis gel
(Hydrolink MDE, FMC Bioproducts, Rockland, ME) at
room temperature for 14 hours for exon 8 and 15 hours for
Table 1. Characteristics of the Patient5
Age at Onset
Sporadic ALS
Whole group
SMNt deletion
SMNc deletion
No deletion
11 (6.2%)
Familial ALS
Whole group
SMNt deletion
SMNc deletion
No deletion
Whole group
SMNt deletion
SMNc deletion
No deletion
6 2 2 11
Sex Ratio
1.21 (97180)
63 k 9
6 2 & 11
1.2 (615)
1.21 (91175)
52 -+ 12
1 (1.5%)
52 t 12
2 (14%)
5“ (36%)
46-+ 15
40 ? 8
5 6 ? 13
1.16 (35130)
Centromeric (c) and telomeric (t)survival motor neuron (SMN)gene deletions on exons 7 and 8 were determined on agarose gel after a specific
polymerase chain reaction amplification for each exon. The presence of SMN deletion was confirmed by single-strand conformation polymorphism. The percentages for each subgroup are given in reference to the n value for the whole group. The frequency of deletions was compared
between the groups by using
< 0.001.
A L S = amyotrophic lateral sclerosis; LMND = lower motor neuron disease.
Moulard et al: SMN Deletions and Lower Motor Neuron Disease 641
exon 7. For most of the patients, SMN deletions were con-
firmed by Dr J. Melki (Hopital Necker, Paris, France).
StatisticaI Analysis
The frequency of SMN deletions between the groups was
compared by using x2 statistics with Yates’ correction when
needed, using Statgraphics, version 2.6, software.
We determined SMN gene polymorphisms in 257 patients with adult-onset MND-66 FALS patients, 177
SALS, and 14 LMND. The characteristics of the ALS
groups corresponded to those of the literature, with an
earlier age of onset and the predominance of spinal onset in FALS compared with SALS.5 In LMND, onset
was slightly earlier than ALS and median survival was
Homozygous deletions in the SMN gene were observed in 19 patients (Fig). SMNc deletions of both
exons 7 and 8 were found in only 1 FALS patient
(1.5%) and in 11 SALS patients (6.2%) (Table 2). The
type and frequency of homozygous SMN deletions in
A L S (sporadic and familial) did not differ from different groups in the literature (ie, normal individual^,^^'^
SMA parents,16 or other ALS patient^'^).
In the 14 subjects with LMND, seven homozygous
deletions (50%) were noted-five
centromeric in both
exons 7 and 8, one telomeric in exon 7, and one telomeric in both exons 7 and 8 (see Table 2). The com-
Annals of Neurology Vol 43
No 5
May 1998
parison of the frequencies of SMNc deletions between
the three MND groups was highly significant ( p <
0.001; see Table 1). One can consider that the 2 patients with SMNt deletion have in fact adult-onset
SMA such as other already described cases.2’3,18219
was also supported by their clinical profile, which was
different from that of the other LMND patients. Onset
of the disease was before 30 years, and evolution was
the slowest of their group, with a disease duration of
190 and 90 months, respectively, although the handicap was severe.
In SALS and FALS, age of onset, median survival,
and sex ratio did not significantly differ either with or
without SMN deletion. Of the 11 ALS patients with
SMN deletions, 5 were still alive after 14, 22, 31, 92,
and 92 months of disease, respectively. Their clinical
profile was undoubtedly that of ALS, with no atypical
clinical feature even if, in 2 cases, there was unusually
long-lasting ALS (92 months).
In the LMND group, when SMNc deletions are
present, the disease starts earlier (40 vs 56 years without SMNc deletion) and is more severe (25 vs 36
months), and the gender (M/F) ratio is lower (1.5 vs
2.5). This contrasts not only with LMND patients
without any SMN deletion but also with the 2 patients
with SMNt deletions (see Table 1). However, because
of the small number of patients in each subgroup, a
Table 2. Homozygous SMN Deletions
SMNt Deletion
No. of
Exon 7
Exon 7
Exon 8
Exon 7
Sporadic ALS
Familial ALS
Exon 8
Patients and controls from the
Normal individuals’
Normal individuals3
SMA parents”
Exon 7
SMNc Deletion
SMNt = telomeric survival motor neuron ( S M N ) gene; SMNc = centromeric SMN; ALS = amyotrophic lateral sclerosis; LMND = lower
motor neuron disease; SMA = infantile spinal muscular atrophy; MD = missing data.
statistical analysis regarding these parameters was not
possible for the whole LMND group.
In different groups of adult-onset MNDs (FALS,
SALS, and LMND), we have shown that homozygous
deletions in the centromeric part of the SMN gene are
significantly more frequent in LMND. In SMA, telomeric deletions of SMN exons 7 and 8 are closely associated with the disease, but only a few patients were
found to carry SMNc d e l e t i ~ n .Little
is known about
the role of the centromeric region of the SMN gene,
but the high frequency of SMNc deletions in adultonset LMND, and the difference in clinical profile of
those subjects in our study (earlier onset, more severe
evolution, and different gender ratio) compared with
those without SMNc deletion, suggest that this deletion
may be a genetic factor of susceptibility for developing
The frequency of SMNc deletions in SALS in our
study is similar to that found in a recent study,” 6.4%
compared with 8.4% (see Table 2). However, if additional deletions were described in exons 7 and 8 alone
(3 cases, centromeric) in this last study, no specific data
were available from LMND patients because of the
small number of patients. The study by Jackson and
collaborator^'^ focused on the NdP gene. This gene,
as SMN, is associated with childhood-onset SMA but
to a much lesser extent.2o Homozygous N A P deletion
was found in 1 patient with typical ALS in the study
by Jackson and colleague^.'^ We also searched for
NMP deletions and found 1 ALS patient with a deletion (data not shown). The frequency of NAIP deletions in both studies corresponds to the estimated frequency in the general population. In SMA, the N A P
deletion usually coexists with the SMNt d e l e t i ~ n .Sub~
sequently, the absence of a concomitant SMN deletion
in 2 patients with ALS does not allow supposition that
the N A P deletion plays a role in the etiopathogenesis
of ALS.
In adult-onset SMA, SMNt deletions have been described but less frequently than in the childhood-onset
type.1s919Nevertheless, the strong association between
SMA and SMNt deletions, in the literature, led us to
consider that our 2 patients with both LMND and
SMNt deletions had in fact adult-onset SMA, even
without a family history. O n the other hand, the following are several reasons for arguing that LMND patients with SMNc deletions cannot be considered nosologically as having the same type of MND: (1) The
onset of the disease is distal and asymmetrical; (2) there
is a clear evolution of the disease throughout the years;
(3) there is no family history; (4) SMNc deletions have
not been associated with SMA in any previous study,
even in the less severe cases, namely, adult-onset SMA
type IV; and (5) in the LMND group, those patients
with SMNt deletions are consistently different from
those with SMNc deletions, with an earlier onset and
with a much less severe disease with long-lasting evolution. A recent report strengthens these points, showing that a father with distal SMA and his son with
proximal SMA (SMA type I) had no common abnormality on the SMN gene.2’
Not all the LMND patients had one or the other
SMN deletion. We cannot, however, rule out the possibility that other abnormalities of the SMN genes are
present in those patients, or in ALS. In support of this
hypothesis, mutations have been found in different
areas of the SMN gene for a more restricted number of
patient^.^^-^^ Different murations on the same gene
have already been shown to be responsible for different
phenotypes. In ALS, where more than 60 SOD1 mutations have been described, different point mutations
can lead to strikingly different phenotypes in which the
motor neuron degeneration remains the main clinical
feature although disease duration may vary from 1 to
Moulard et al: SMN Deletions and Lower Motor Neuron Disease
20 years.26 However, the nosology of LMND is much
less precise than that of ALS, and larger studies, necessarily multicentric, and focused on this type of
MND, are then warranted to confirm our data and
help to refine our understanding of this disease.
In summary, the study of the SMN gene on chromosome 5q13 in adult-onset MNDs showed that centromeric deletions were statistically more frequent in
LMND than in sporadic or familial ALS. The hypothesis that the telomeric part of the SMN gene has a major role on motor neuron metabolism is a consequence
of studies on childhood-onset SMA. The high frequency we found for centromeric deletions in adultonset LMND, a disease that is in several respects nosologically distinct from SMA, and the particular clinical
profile of those subjects, led us to hypothesize that
both parts of the SMNgene could be involved in motor neuron metabolism. SMNt would have a major influence leading to severe motor neuron degeneration
when abnormal, and SMMc would exert a less important role, such as that of a susceptibility factor, increasing the risk of having adult-onset lower motor neuron
The Association Franpise pour la Recherche sur la Sclkose Laterale
Amyotrophique and the Association FranCaise contre les Myopathies
provided financial support. This study was also supported by PHRC
and DRC AP-HP grants.
We gratefully acknowledge the contribution of Dr J. Melki for
checking our genetic data.
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