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Deletion and conversion in spinal muscular atrophy patients Is there a relationship to severity.

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Deletion and Conversion in Spinal Muscular
Atrophv Patients: 1s f i e r e a
Relationshp to Severity?
I
/
Christine J. DiDonato, PhD,* Susan E. Ingraham, BS,t Jerry R. Mendell, M D , t
Thomas W. Prior, PhD,$ Sharon Lenard, MS,' Richard T. Moxley 111, MD,'
Julaine Florence, MHS,** and Arthur H. M. Burghes, PhD*I$
The spinal muscular atrophy-determining gene, survival motor neuron (SMN), is present in two copies, telSMN and
cenSMN, which can be distinguished by base-pair changes in exons 7 and 8. The telSMN gene is often absent in spinal
muscular atrophy patients, which could be due to deletion or sequence conversion (telSMN conversion to cenSMN
giving rise to two cenSMN genes). To test for conversion events in spinal muscular atrophy, we amplified a 1-kb
fragment that spanned exons 7 and 8 of SMN from 5 patients who retained telSMN exon 8 but lacked exon 7. In all
patients, sequence analysis demonstrated that cenSMN exon 7 was adjacent to telSMN exon 8, indicating conversion.
All 5 patients with this mutation had type I1 or I11 spinal muscular atrophy, strongly supporting an association with
chronic spinal muscular atrophy. We also identified 3 families in which 2 siblings had no detectable telSMN but
presented with markedly different phenotypes. We suggest that sequence conversion is a common event in spinal muscular atrophy and is associated with the milder form of the disease. The severity, however, can be modified in either a
positive or negative direction by other factors that influence splicing or expression of the sequence converted SMN gene.
DiDonato CJ, Ingraham SE, Mendell JR, Prior TW, Lenard S, Moxley RT 111, Florence J ,
Burghes AHM. Deletion and conversion in spinal muscular atrophy patients:
is there a relationship to severity? Ann Neurol 1997;41:230-237
Proximal spinal muscular atrophy (SMA) is an autosoma1 recessive neuromuscular disorder that results in loss
of motor neurons in the spinal cord. Affected individuals are classified into three groups depending on the
age at onset, achieved milestones, and life span [I, 21.
Type I SMA is the most severe, with clinical onset
generally occurring before the age of 6 months and
death before the age of 2. Type I1 SMA is of intermediate severity; patients live past the age of 2 and are able
to sit but never gain the ability to walk. Type 111 SMA
corresponds to the mild form of the disease. The onset
of symptoms occurs after the age of I S months and
patients may be ambulatory for long periods in their
lives.
The gene responsible for SMA has been mapped to
an 850-kb interval on 5q12 by linkage analysis [3-111.
Physical maps of the region have been produced but
no consensus map exists. This is in part due to the
complex genomic structure of the region where genes
and markers are present in multiple copies [12-20].
Some multicopy polymorphic markers show a strong
association with SMA [15, 19-21]. This association
can be divided into two categories. Firstly, specific alleles show an association with SMA and secondly, there
is an association of disease severity with the number
of copies (or null alleles) of a marker [20-221. Furthermore, de novo deletions can be observed in some SMA
chromosomes using these markers [ 15, 221.
Three complementary DNAs (cDNAs) that identi@
deletions have been isolated from the SMA region [ 16181. Two of these cDNAs, XS2G3 and neuronal
apoptosis inhibitory protein (NAIP), mark the same
deletion that is detected in approximately 50% of patients with type I SMA [16, 17, 221. The deletion is
less frequent in type I1 and I11 SMA and occurs in 3
to 4% of phenotypically normal carriers [16, 171. The
third transcript is encoded by the survival motor neuron (SMN) gene. These two almost identical genes,
centromeric SMN (cenSMN) and telonieric SMN
(telSMN), are distinguished by two sequence variations
From the "Department of Molecular Genetics, College of Biological
Sciences. the t Deoartments o f Medical Biochemistrv, tNeurolow,
Received Jun 3, 1996, and in revised form Jul 26. Accepted for
Dublication ALIE5 , 1996.
~r
"/
rer, NY; and **Department o f Neurology, Washington University,
St. Louis, MU.
230
Copyright 0 1997 by the American Neurological Association
in exons 7 and 8. These variations do not alter the
encoded amino acids [18, 231.
The telSMN gene is absent in approximately 95%
of SMA patients regardless of phenotype. Additionally,
small deletions or point mutations have been found in
patients in whom telSMN is present [18, 241. Current
diagnostic assays that test for the absence of telSMN
rely on the sequence variations present in exons 7 and
8. Although the absence of telSMN is generally interpreted as a deletion, these assays cannot distinguish between a deletion and a sequence conversion event.
In this article we define sequence conversion as an
event where the telSMN locus and cenSMN locus have
exchanged sequences such that the telSMN locus possesses the nucleotides associated with the cenSMN locus. This could occur by a number of mechanisms including gene conversion and unequal crossing over.
Therefore, the majority of patients with severe SMA
could possess a homozygous deletion of telSMN
whereas the majority of mild SMA patients would have
one allele that is a sequence conversion. This would
be consistent with our association studies in which we
presented a model of SMA in which type I SMA had
two severe alleles and milder SMA had a mild and
severe allele [20, 221.
In this study, we analyzed a well-defined SMA patient population for sequence conversion events. We
demonstrated that sequence conversion and not deletion occurred in 5 type I1 or I11 SMA patients who
lack telSMN exon 7 but have telSMN exon 8. As all
these sequence conversions occurred in type I1 or I11
SMA patients, we suggest that a common mutation of
mild SMA chromosomes is sequence conversion (partially functional SMN) and that a common mutation
of severe chromosomes is deletion (nonfunctional or
null allele). We also identified 3 families in which 2
siblings had no detectable telSMN (due to either conversion or deletion), but displayed markedly different
phenotypes. We propose that these families and the
phenotypic variation seen within other SMA families
could be explained by the expression of a telSMN isoform derived from a sequence converted allele that is
regulated by its genetic background.
Materials and Methods
Family Material
All patients fulfilled the diagnostic criteria for proximal SMA
defined by the International SMA Consortium [ l ] and have
been previously described [7, 201.
Sibships were classified as having type I, 11, or I11 SMA.
Families that had siblings of two types were classified as having the more severe phenotype. Overall, 61 families (27 type
I, 24 type 11, and 10 type 111) were analyzed for deletions
of NAIP exon 5 and SMN exons 7 and 8. All patients in
families with more than 1 affected individual were analyzed,
but only 1 was used in constructing the Table.
SMA 6
47
U
I
I
I
I
I
D5S76
D5S6
D5S125
D55435
D551556
telSMN
D55351
MAP1E
D55112
D5S39
Fig 1. Haplotypes of yinal muscular atrophy (SMA) Pedigree
6 indicating variability of SMA phenolype within a sibship.
Individuals 50 and 51 inherited the same markers that j a n k
the SMA genes and both showed absence for exons 7 and 8
of telSMN. However, Individual 51 was still walking at age
20 and at most showed very mild weakness.
Three families had siblings who inherited two identical
chromosome 5s, but had remarkably divergent clinical phenotypes. Families SMA 6 and 14 were described previously
by Burghes and colleagues [7] prior to SMN gene testing.
For Family 6, Individuals 50 and 50.1 had the typical clinical
features of type I1 SMA and had confirmatory electromyography (EMG) and muscle biopsy evaluation. Symptoms in
Individual 50 began at the age of 1 year and she never
walked. Her brother, Individual 5 1, who inherited the same
chromosomal region containing the SMA gene, was still
walking at age 21 and showed no obvious clinical signs of
SMA (Fig 1). In Family 14, Individual 112 had onset of
symptoms at 17 months and was diagnosed with SMA type
11. The diagnosis was confirmed by EMG and muscle biopsy.
Individual 113 was originally diagnosed as normal, but at
age 13 displayed extremely mild weakness, and an EMG and
muscle biopsy examination revealed changes consistent with
SMA. Family 75 was a recently identified family sent for
SMN gene testing. The affected individual, 377, had onset
of symptoms around the age of 1 year and never gained the
ability to walk. The EMG and muscle biopsy findings were
consistent with SMA. At the time of writing, this patient
was 4 years old and his sister, Individual 376, showed no
clinical signs of SMA at the age of 5. Muscle biopsy and
EMG have not been performed on this individual for ethical
reasons.
Polymerase Cbain Reaction Analysis of
N A P Exonf 5 and 13
Multiplex polymerase chain reaction (PCR) of NAIP exons
5 (1893, 1863) and 13 (1258, 1343) was performed using
DiDonato et al: Deletion and Conversion in SMA
231
primers described by Roy and coauthors [ 161. All reactions
were carried out in 4 mM magnesium chloride (MgC12)
buffer using the following reaction conditions: an initial denaturation at 94°C for 3 minutes followed by 35 cycles at
94°C for 30 seconds, 58°C for 30 seconds, and 72°C for
30 seconds. There was no final extension. The PCR products
were visualized on ethidium bromide-stained 2% agarose
gels and scored for the presence or absence of exon 5.
cycling conditions were 1 cycle at 94°C for 3 minutes followed by 35 cycles at 94°C for 30 seconds, 56°C for 30
seconds, and 72°C for 45 seconds.
Claning and Sequencing of Polynzeruse Chain
Reuction Products
Direct sequencing was performed using the dscycle sequenc-
ing system (Gibco-BRL, Gaithersburg, MD). The PCRamplified fragments from SMN exon 7 and 8 were cloned
using a T A cloning vector (Invitrogen, San Diego, CAI. The
DeteL-tion o f cenSMN and telSMN Gene Deletions
SINGLh-STRAND CONFORMATION ANALYSIS OF SMN EXON
subclones were sequenced using the dscycle sequencing system or the dideoxy chain termination method and Sequenase
Version 2.0 (Amersham, Arlington Heights, IL) [26].
7. Primers and amplification conditions for SMN exon 7
were as previously described by Lefebvre and coauthors [ 181.
For single-strand conformation analysis (SSCA), 100 ng of
genomic DNA was amplified by PCR using 10 ng of unlabeled primers R111U and 541C770 in 25-1.11 reactions containing 300 mM dNTPs, 1 unit of Taq polymerase (GibcoB E , Gaithersburg, MD) and 0.5 pl of [a-12P]deoxycytidine
triphosphate (10 mCi/ml). The samples were denatured at
94°C for 8 minutes and loaded onto a Hydrolink MDE
(J.T.Baker) gel. The gels were electrophoresed at 6.5 W for
13 to 15 hours at 4"C, dried, and exposed to x-ray film for
6 to 36 hours at room temperature.
Results
RESI'NCTION ENZYME ANALYSIS Ot. SMN EXONS 7 AND
8.
PCR amplification of SMN exon 7 was performed essentially as described above except the primers R111U and X7DRA [25] were used. SMN exon 8 was amplified using
primers 541C960 and 541C1120 [17] under the same conditions as exon 7. T o differentiate centromeric versus telomeric exons 7 and 8, 15 p1 of each PCR product was
digested with 20 units of Dra I (exon 7) or 15 units of Dde
I (exon 8) in the presence of 4 mM spermidine for 3 hours
at 37°C. The digestion products were electrophoresed
through a 2.5% ethidium bromide-stained agarose gel at
100 V for 2 hours. The restriction enzymes Dra I and Dde
I only cleave the centromeric copies of SMN exons 7 and
8, respectively.
Polymeruse Chain Reuction Amplification of
SMN Exonr 7 to 8
Thirty nanograms of primers R111 U and 54 1C1120 located
in intron 6 and exon 8, respectively, were used to amplify
a 1,010-bp fragment from 100 ng of genomic DNA in a
5Oyl reaction volume using the buffer described above. The
Correlation of
Phenotype
Type I
Type II'
Type 111'
Carrier
the Absence
The SMA population we collected was analyzed for the
absence of telSMN exons 7 and 8 and the deletion of
NAIP exon 5. The absence of telSMN exon 7 was
detected by both SSCA and the restriction enzyme test.
One patient tested positive for telSMN by SSCA, but
not by restriction digest. Subsequent sequence analysis
showed that this affected individual did not possess
telSMN exon 7 or 8. We detected the homozygous
absence of telSMN exon 7 in 90% of SMA patients,
with exons 7 and 8 being absent in 82% of patients.
One carrier parent with no known symptoms of SMA
showed homozygous absence for exons 7 and 8 of
telSMN. We previously reported that the deletion of
NAIP exon 5 predominates in type I SMA patients
(Table). This deletion in type I SMA is also associated
with the loss of one copy of Agl-CA (D5S1556) [211.
Four carriers with no symptoms of SMA also showed
deletions of exon 5 of NAIP, but none showed homozygous absence for telSMN.
Certain families showed a remarkable discordance of
phenotype between 2 siblings. We identified 3 such
families in which extremely mild or clinically unaffected individuals were haploidentical to their severely
affected sibling across the SMA region of chromosome
5. As expected from the haplotype data, these individuals also showed homozygous absence for telSMN exons
7 and 8. This would indicate that other factors affect-
o f telSMN exoni 7 and 8, and N A P exon 5 with Spinal Muscular AtTophy (SMA) Type
Absence of
telSMN exon 7
Absence of
telSMN exons 7, 8
3
2
9a
18
7
Absence of
telSMN exons 7 and 8
and NAIP exon 5
Absence of
NAIP exon 5
1bh
1
Nothing
Absent
2
3
1
4
1
106
'Four of 9 of these type I SMA patients had a 1,2 genotype and 5 had a 1,l genotype with the marker Agl-CA.
bFourteen of 16 of these type I patients were genotyped with the marker Agl-CA, 13 had a 1,I genotype and 1 had a 1,2 genotype
'Of type I1 or I11 patients, more than 80% genotyped with the marker Agl-CA had a 1,2 genotype.
232 Annals of Neurology Vol 41
No 2
February 1997
Total
27
24
10
111
ing the telSMN locus can alter the severity of the phenotype. In all 3 families, 1 individual was diagnosed
with type I1 SMA with onset following a classic course.
In Family 75, the affected child, Individual 377, had
the classic indications of type I1 SMA whereas his sister, Individual 376, showed no clinical signs of SMA
at the age of 5 years. In Family 14, the sister of a
typical type I1 patient at age 13 showed extremely mild
weakness, and examination of a muscle biopsy specimen and EMG revealed changes consistent with SMA.
Family 6 perhaps represents the most dramatic example, with 2 sisters showing classic SMA and their
brother showing no clinical signs of SMA at age 21.
We were unable to perform muscle biopsy or EMG
studies in this individual, but at the very least he represents a large phenotypic variation (see Fig 1).
Five (15%) type I1 or I11 SMA patients were negative for telSMN exon 7 but positive for telSMN exon
8 (see Table). These patients were analyzed in greater
detail by amplifying the region surrounding exons 7
and 8 of the SMN gene. The resulting PCR products
were subcloned and analyzed by Dde I digestion after
PCR amplification of exon 8. Those clones that were
positive for telSMN exon 8 were sequenced and subjected to PCR of exon 7 and Dra I restriction enzyme
analysis. In all patients, it was found that a sequence
conversion had occurred such that the cenSMN gene
up to and including exon 7 was juxtaposed to telomeric
exon 8. Figure 2 shows the sequence data. In all patients, the gene conversion was similar in extent as far
as could be determined. The pedigree and analysis of
a type I11 family with a gene conversion event are
shown in Figure 3. In this family, 2 affected brothers
showed homozygous absence for telSMN exons 7 and
8. Their second cousin had one chromosome that did
not have the sequences associated with telSMN exons
7 and 8 and one chromosome that had a sequence
conversion that placed cenSMN exon 7 adjacent
telSMN exon 8. Therefore, Individual 70 in this family
was a compound heterozygote. Individuals 63, 64, and
70 all had type I11 SMA with a similar clinical presentation. Individuals 63 and 64 could have either deleted
or sequence converted alleles, but in the case of a sequence conversion, the conversion event would extend
through exon 8 and not be detectable. These results
clearly indicate that sequence conversion occurs in
SMA. It is also notable that this event appears more
frequently in type I1 and 111 SMA chromosomes.
Discussion
The SMA gene is located in a region that contains
many repeated sequences including genes and markers
[15-201. The markers CATT1, Agl-CA (C272), and
C212 show strong allele association with SMA, indicating that they lie close to the SMA gene [15, 19-22].
The markers Agl-CA (C272) and C212 are present
on normal chromosomes in 1 to 3 copies [15, 20, 221.
In type I SMA, the presence of 1 copy of these markers
on both chromosomes (I, I) is heavily overrepresented,
whereas the 1,2 genotype is overrepresented in type I1
SMA [20, 221.
This has led us to propose a model where type I
SMA patients have two severe alleles and type I1 SMA
patients have a mild and a severe allele. Three cDNA
clones from the critical region containing the markers
previously described were simultaneously reported to
detect deletions in SMA patients [16-181. The cDNAs
for NAIP and XS2G3 detect the same deletion and lie
in close proximity to the CATT1 marker [16, 171. The
NAIP and XS2G3 cDNAs detect homozygous deletions in about 50% of type I SMA patients and less
frequently in type I1 and 111 SMA patients [16, 171.
These cDNAs also detect deletions in carrier parents
with no apparent symptoms [16, 17, 27-30]. The
data in the Table agree with these previous observations.
The third gene is SMN, and there is substantial evidence that it is the SMA-determining gene. The gene
exists as two almost identical copies, telSMN and
cenSMN, that are distinguished by sequence differences in exon 7 and exon 8 [18]. The telSMN gene
is absent in 90 to 98% of SMA patients as reported
in this and other studies [18, 27-30]. In some of the
remaining patients, small mutations have been found,
in particular a 4-bp deletion in exon 3 of telSMN [ 18,
241. The absence of telSMN exons 7 and 8 is often
referred to as a deletion; however, it is not clear in
most cases whether deletion or sequence conversion has
occurred. Sequence conversion is the exchange of
cenSMN sequences into the telSMN locus, resulting
in the telSMN locus looking identical to the cenSMN
locus (Fig 4).
It is often stated that there is no correlation of
telSMN deletion to phenotype. However, this assumes
that deletion and not sequence conversion has taken
place. The marker Agl-CA (D5S1556)/C272 lies at
the 5’ end of SMN just upstream of exon 1 [23]. It
is clear from studies of Agl-CA that there is a definite
correlation between disease severity and the 1,l genotype [2O, 221. In addition, we previously showed that
the 1,l genotype of Agl-CA in type I SMA strongly
correlates with the loss of NAIP exon 5 [22]. The most
likely explanation for these observations is that a large
deletion removes 1 copy of Agl-CA and NAIP exon
5 on both chromosomes in type I SMA but not in
type I1 or I11 SMA [22]. One could predict that this
large deletion would be a null allele for telSMN, and
thus not produce any telSMN gene product. In type
I1 SMA, the association studies would predict one severe (large deletion or null allele) and one mild allele
DiDonato et al: Deletion and Conversion in SMA
233
cenSMN
telSMN
patients
agactatcaa cttaatttct gatcatattt tgttgaataa aataagtaaa atgtcttgtg aaacaaaatg ctttttaaca
80
cenSMN
telSMN
patients
a
tccatataaa gctatctata tatagctatc tatgtctata tagctatttt ttttaacttc ctttattttc cttacagGGT
a
160
cenSMN
telSMN
patients
T
TTCAGACAAA ATC74AMAGA AGGAAGGTGC TCACATTCCT TAAATTmGG Agtaagtctg ccagcattat gaaagtgaat
T
240
cenSMN
telSMN
patients
9
cttacttttg taaaacttta tggtttgtgg aaaacaaatg tttttgaaca tttaaaaagt tcagatgtta aaaagttgaa
9
32 0
cenSMN
telSMN
patients
aggttaatgt aaaacaatca atattaaaga attttgatgc caaaactatt agataaaagg ttaatctaca tccctactag
400
cenSMN
telSMN
patients
9
aattctcata cttaactggt tggttatgtg gaagaaacat actttcacaa taaagagctt taggatatga tgccatttta
480
cenSMN
telSMN
patients
tatcactagt aggcagacca gcagactttt ttttattgtg atatgggata acctaggcat actgcactgt acactctgac
560
cenSMN
telSMN
patients
atatgaagtg ctctagtcaa gtttaactgg tgtccacaga ggacatggtt taactggaat tcgtcaagcc tctggttcta
640
cenSMN
telSMN
patients
atttctcatt tgcagGAAAT GCTGGCATAG AGCAGCACTA AATGACACCA CTAAAGAAAC GATCAGACAG ATCTGGAATG
720
cenSMN
telSMN
patients
TGAAGCGTTA TAGAAGATAA CTGGCCTCAT TTCTTCAAAA TATCAAGTGT TGGGAAAGAA AAAAGGAAGT GGAATGGGTA
800
cenSMN
telSMN
patients
ACTCTTCTTG A T T M G T T ATGTAATAAC CAAATGCAAT GTGAAATATT TTACTGGACT CTTTTGAAAA ACCATCTGTA
880
cenSMN
telSMN
patients
A
AAAGACTGGG GTGGGGGTGG
G
4
.
Fig 2. Sequence data o f SMN exons 7 to 8. A 1,010-bp fragment from 5 putative sequence conversion patients wa amplified
and sequenced a described in the text. The sequence is given from the primer R111 U t o the base change in telSMN exon 8. The
three dots after base 900 indicate the continuation of exon 8 to the primer 541 C1120. Patient sequences were compared to those
of renSMN and telSMN, which were obtainedfrom a PAC and P1 clone that contains only the cen or tel SMN locus, respectively. There are only 5-base changes between the cen versus tel S M N loci. In all 5 patients the extent of gene conversion was limited to the sequence between bases 426 und 888. This sequence has been deposited in the gene bank.
(partially functional): the mild alleles could arise by
gene conversion instead of deletion in the majority of
mild SMA chromosomes.
In this study we found that 15% of our type I1 or
I11 population lost telSMN exon 7 but not 8. Analysis
of these patients showed that cenSMN exon 7 had
been placed adjacent to telSMN exon 8, indicating that
sequence conversion had occurred in all of these patients. Two sequence conversion events have been reported previously in SMA patients [18, 241; in 1 patient the conversion event was confined to exon 7 with
no other changes reported [24].Sequence conversion
has also been demonstrated in normal chromosomes
234
Annals of Neurology Vol 41
No 2
February 1997
[30]. However, in this case telSMN exon 7 is placed
adjacent to cenSMN exon 8 and it is not possible to
distinguish which locus the conversion affected.
The frequency of gene conversion in SMA cannot
be determined from this study or previous studies because we only examined those patients in whom
telSMN exon 8 was present (see Fig 4, Table). Comparison of the intensity of telSMN exon 7 and
cenSMN exon 7 on SSCA gels from parents of type
I, 11, or 111 SMA individuals indicates a higher copy
number of cenSMN in both type I1 and I11 SMA families, which is consistent with gene conversion occurring
frequently in type I1 or I11 SMA chromosomes 1301.
A.
05576
d556
D5.5125
D5S435
D5S1556
telSMN €7/€8
D55351
MAPl B
D5S112
d5539
D5576
d556
D5S125
D5S435
D5.51556
telSMNE7/E8
D5S351
MAPl B
D5S112
d5539
8
6.
C.
59 60 61 62 63 64 65 66 70
59 60 61 62 63 64 65 66 70
tel Ex7 b
cen Ex7 b
~~~
tel Ex8
+
cen Ex8
+
~
Fig 3. Haplotypes of spinal muscular atrophy (SMA) Pedigree 8. (A) The pedigree along with the haplotypes for markers linked to
SMA as well as the genotype for exons 7 and 8 of the telSMN gene. Individuals 63 and 64 lack both exons 7 and 8 of telSMN,
whereas their second cousin lacks only exon 7 but has one copy of telSMN exon 8. (B) The polymerase chain reaction (PCR)
results for SMN exon 7 on Individuals 59 to 66 and 70.(C)The PCR results for SMN exon 8 on individuals 59 to 66
and 70.
DiDonato et al: Deletion and Conversion in SMA
235
MODEL OF SMA MUTATIONS
Apart from the mechanisms indicated above, it has
been reported that a 4-bp deletion in exon 3 of
telSMN-can occur in type or I11 SMA chromosomes
[24].This apparently contradictory observation is remarkably similar to the situation seen in cystic fibrosis
(CF) where the missense mutation, R117H, is observed
in three phenotypic variants of CF. The severity of the
phenotype is modulated by intron variants that affect
exon splicing efficiency [33].Given that genetic background (either modifier genes or intron variants) can
affect the severity of phenotype in a positive or negative
manner, one might expect to find families with siblings
showing variant phenotypes. Indeed, in this article, we
report families who show a remarkable variation in
phenotype between siblings. Other investigators published similar pedigrees that are even more extreme,
with affected and normal siblings showing absence of
telSMN [27, 28, 34, 351. This result could be interpreted as evidence against telSMN being the SMA
gene, or that additional factors are necessary for the
presentation of the SMA phenotype. However, the
affected and unaffected individuals reported are haploidentical. Thus, other genes in the area should also
share identical mutations or characteristics unless a de
novo event has occurred, which seems unlikely in these
families. In all cases, these variant families have
been reported for type I1 or I11 SMA and never type
i
TELSMN
CEN SMN
T
A
T
A
lype I1 or Ill SMA
Mild allele
sequence mnversian
p-ip
p--p
T
or
A
T
A
T
G
Fig 4. Diagrammatic representation of deletion and sequence
conversion of the tel SMN gene in spinal nirrscular atrophy
(SMA).
To accurately determine the frequency of gene conversion in SMA chromosomes, it will be necessary to develop precise quantitative assays for telSMN and
cenSMN.
In this study, all the sequence conversions detected
occurred in type I1 or I11 SMA families, implying a
correlation of this mutation to severity. Other studies
showed type I SMA patients who lack telSMN exon
7 but retain telSMN exon 8 [27-301. O n e possible
explanation is that these patients truly do have deletions that remove telSMN exon 7 but not 8, thus disrupting the telSMN locus. Even if gene conversion
does occur in type I SMA chromosomes, this does not
indicate that the alleles in the severe and mild SMA
patients are filnctionally equivalent. Firstly, the extent
of the gene conversion may not be equivalent. Secondly, the conversion event could result in the insertion of an additional mutation into the SMN gene
similar to the situation in Gaucher's disease where gene
conversion inserts the mutated pseudogene sequences
[31, 321. Thirdly, the 5' end of the S M N genes contains a C,,G island that could undergo
" differential
methylation, and thus affect the level of expression
from the
influence the
gene*
genes
Of the mutated gene to
in
the appropriate manner. At present, it is not clear how
the gene
event
disruption Of the
telSMN gene to give rise to sm, or which of the
mechanisms indicated above are operational.
236 Annals of Neurology
Vol 41
No 2
February 1997
I SMA.
We suggest that as sequence conversion occurs in
SMA and appears most common in type I1 or I11 families, it is most likely that they have at least one allele
that is a sequence conversion. This converted gene
could then be influenced by modifier genes that may
affect methylation or the selection of splice sites. Indeed, modifier loci recently were shown to influence
the severity of another autosomal recessive condition,
CF, in mice [36].
In conclusion, we suggest that sequence conversion
is a common event in SMA and is associated with a
milder form of the disease. The severity can, however,
be modified in either a positive or negative direction
by other factors that influence either the splicing or
the expression of the sequence converted SMN gene.
Severe SMA results when there are two null alleles and
no gene product is produced by the telSMN locus,
whereas mild SMA occurs when one allele is partially
functional. Confirmation of this hypothesis awaits detailed RNA and protein studies.
This research was funded by Families of SMA and the Muscular
Dystrophy Association (MDA). The MDA and European Neuromuscular Centre funded the International SMA Consortium meetings.
We are grateful to all SMA families for their kind cooperation and
to all clinicians for their help in providing both blood samples and
clinical details of parients. We would like to thank Susan Coulson-
Burghes and Patricia McAndrew for help in editing the manuscript.
Furthermore, we thank all members of the International SMA Consortium for helpful discussions.
18.
19.
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