Title: Spinal muscular atrophy diagnostic
States Patent: 7,033,752
Melki; Judith (Paris, FR); Munnich;
Arnold (Paris, FR)
Institut National de la Sante et de la Recherche Medicale
Appl. No.: 109082
Filed: July 2,
The present invention relates to the
discovery of the human survival motor-neuron gene or SMD gene, which is a
chromosome 5-SMA (Spinal Muscular Atrophy) determining gene. The present
invention further relates to the nucleotide sequence encoding the SMN gene
and corresponding amino acid sequence, a vector containing the gene
encoding the SMN protein or a DNA sequence corresponding to the gene and
transformant strains containing the SMN gene or a DNA sequence
corresponding to the gene.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION
As used herein, the term "contig" means overlapping nucleotide sequences.
Previous studies by means of linkages analysis have shown that all three
forms of spinal muscular atrophy map to chromosome 5q11.2-q13.3. (L. M.
Brzustowicz et al, Nature, 344, 540 (1990); J. Melki et al,
Nature. 345, 823 (1990); J. Melki et al, Lancet, 336, 271
(1990). A yeast artificial chromosome (YAC) contig of the 5q13 region
spanning the disease locus was constructed that showed the presence of low
copy-repeats in this region. Allele segregation was analyzed at the
closest genetic loci detected by markers derived from the YAC contig
(C212, C272 and C161) in 201 SMA families. These markers revealed two loci
(C212, C272) or three loci on the 5q13 region (C161). Inherited and de
novo deletions were observed in 9 unrelated SMA patients. Moreover,
deletions were strongly suggested in at least 18% of SMA type I patients
by the observation of marked heterozygosity deficiency for the loci
studied. These results indicated that deletion events are statistically
associated with the severe form of SMA.
By studying all polymorphic DNA markers derived from the YAC contig, it
was observed that the smallest rearrangement occured within a region
bordered by loci detected by C161 and C212-C272 and entirely contained in
a 1.2-Mb YAC clone 903D1. See, for example, French Patent Application No.
9406856 incorporated herein by reference.
The present invention characterized the small nested critical SMA region
of about 140 Kb by a combination of genetic and physical mapping in SMA
patients. This region suggested a precise location for the SMA gene and
therefore, a limited region within which to search for candidate genes.
The present invention identified a duplicated gene from the 5q13 region.
One of them (the telomeric gene) is localized within the critical region.
Moreover, this gene was lacking in 213 out of 230 (92.2%) or interrupted
in 13 out of 230 (5.6%) SMA patients. In patients where the telomeric gene
is not lacking or interrupted, deleterious mutations indicated that this
telomeric gene, termed survival motor-neuron (SMN) gene, is the chromosome
5 SMA-determining gene.
The SMN gene was discovered using a complex system of restriction mapping,
distinguishing the ETel from the ECen by Southern
blot, and the determination of the differences between the ETel
in SMA patients by genetic and physical mapping. After confirming the
location of the SMN gene, a phage contig spanning the critical region of
the telomeric element was constructed to identify specific clones
containing the SMN gene.
Analysis of the SMN gene in SMA patients compared with those of normal
patients revealed either the SMN gene was either lacking or truncated in
98% of SMA patients or had combined mutations not present in normal
To identify a large inverted duplication and a complex genomic
organisation of the 5q13 region, long-range restriction mapping using
pulsed field gel electrophoresis (PFGE) of the YAC contig was performed.
YACs were ordered by comparing their haplotypes with that of the human
donor at the polymorphic loci detected markers C212, C272, C171 and C161
(FIG. 4) (SEQ ID NOS: 14-18)
The restriction enzymes SacII, BssHII, SfiI, EagI and XhoI were used to
digest the YACs containing the telomeric loci detected by markers C212,
C272, C171 and C161 (YAC clone 595C11), the centromeric loci detected by
these markers (YAC clones 121B8, 759A3, 278G7) or both (YAC clones 903D1
and 920C9). Lambda phage libraries of YACs 595C11, 121B8 and 903D1 were
constructed and subclones from phages containing markers C212 (p322), C272
(132SE11), C161(He3), AFM157xd10(131xb4) and CMS1 (p11M1) were used as
probes for PFGE analysis. FIG. 5 shows that probes 132SE11, 11P1 and p322
revealed two loci, and probe He3 revealed 4 loci on the YAC contig,
whereas probe 131xb4 revealed several loci on Sp and 5q13. The restriction
map (FIG. 6) showed that the 5q13 region contained a large inverted
duplication of an element (E) of at least 500 Kb, termed ETel
and ECen for the telomeric and centromeric elements,
The PFGE analysis of SMA and control individuals revealed a high degree of
variability of restriction fragments which hampered the distinghishment of
ETel from the Ecen and the recognition of abnormal
restriction fragments in SMA patients.
In order to distinguish between the ETel and the ECen,
a Southern blot analysis was then performed. The Southern blot was
performed by the methods described in Sambrook et al, supra.
More specifically, DNA from YAC clones, controls and SMA patients was
digested with restriction enzymes SacI, KpnI, MspI, PstI, PvuII, EcoRI,
HindIII, BgIII and XbaI for Southern blotting and hybridized with clones
132SE11, 11p1, He3, 131xb4 and p322 as probes. None of the probes except
one (He3) detected a difference between the two duplicated elements. Three
HindIII restriction fragments of 12, 11 and 3.7 Kb were detected by probe
He3. A 12 Kb HindIII restriction fragment was detected in YAC clones 754H5
end 759A3, indicating that this fragment corresponded to the most
centromeric locus in the ECen. Conversely, a 11 Kb HindIII
fragment was detected in YACs clones 595C11, 903D1 and 920C9 indicating
that this fragment corresponded to a single locus on the ETel.
Finally, a 3.7 Kb HindIII fragment was noted in non-overlapping YACs
containing either ETel or ECen, indicating that this
fragment corresponded to two different loci. Similar results were obtained
with SacI and KpnI. The three restriction fragments detected by He3 were
observed on the monochromosomal hybrid HHW105 (Carlock, L. R. et al,
Am. J. of Human Genet., 1985, Vol. 37, p. 839) and in 30 unrelated,
healthy individuals, confirming that these fragments were not due to
polymorphisms. The Southern analysis results allowed one to distinguish ETel
from the ECen in both controls and SMA patients.
Thus, once the ETel from the ECen was distinguished,
it was necessary to determine the differences between the ETel
in SMA patients and those of the normal control. This was done by using
genetic and physical mapping. This genetic and physical mapping identified
genomic rearrangements in the telomeric element of ETel of SMA
It was previously shown that 9 out of 201 (9/201) SMA patients displayed
large-scale deletions encompassing either one or the two loci detected by
markers C212 and C272 on one mutant chromosome (J. Melki et al,
Science, 264, 1474 (1994)). On the other hand, 22 out of 30 (22/30)
patients born to consanguineous parents including 13 out of 14 (13/14)
type I and 9 out of 10 (9/10) type III SMA, were homozygous by descent for
the most closely flanking polymorphic markers.
The genomic DNA of the 9 patients harboring large scale deletions and the
22 consanguineous patients displaying homozygosity by descent were
digested with HindIII for Southern blotting and hybridized with probe He3.
The 11 Kb fragment revealed by probe He3 was absent in 12 out of 13
(12/13) consanguineous type I patients. In 2 out of 12 (2/12), the
deletion also involved the 3.7 Kb fragment. By contrast, the 11 Kb
fragment was absent in 1 out of 8 (1/8) consanguineous type III patients
only. Consistently, the 11 Kb HindIII fragment was absent in 4 out of 9
(4/9) patients harboring large scale deletions on one mutant chromosome.
Of particular interest was the absence of the 11 Kb fragment in the
patient harboring a deletion of one of the two loci detected by markers
C212 and C272.
When analyzed together, these observations provided evidence for genomic
rearrangements of ETel in SMA patients and supported the
location of the SMA gene centromeric to the locus revealed by the 11 Kb
HindIII fragment, since all consanguineous type III patients but one were
not deleted for this locus.
In order to characterize the centromeric boundary of the genomic
rearrangement in the disease, the allele segregation at loci detected by
marker C272 in consanguineous SMA patients was analyzed. All
consanguineous SMA type I patients had one single PCR amplification
product, compared with 0 out of 60 controls. This marked heterozygosity
deficiency was due to deletion of one of the two loci detected by C272, as
indicated by the marked decrease of gene dosage with probe 132SE11,
mapping close to this marker. By contrast, 7 out of 9 (7/9) consanguineous
type III SMA patients had two C272 amplification products inherited from
both parents, indicating homozygosity at each locus detected by marker
C272. Moreover, no gene dosage effect was observed with probe 132SE11
indicating the absence of deletion involving the locus detected by C272 in
type III consanguineous patients.
Assuming that the same locus is involved in all three types of SMA, these
results indicate that the disease causing gene is distal to the telomeric
locus detected by C272.
These studies place the SMA gene within the telomeric element ETel,
between the telomeric loci detected by markers C272 and He3 (11 kb HindIII
fragment). Based on long-range restriction mapping using PGFE of the YAC
contig, this critical region is entirely contained In a 140 Kb SacII
fragment of YAC clone 903D1 (or 150 Kb SacII fragment of YAC clone 920D9).
After confirming that the SMN gene was located on a 140 Kb SacII fragment
a phage contig spanning the critical region of the telomeric element was
constructed in order to identify and characterize the SMN gene.
Phage clones containing markers C212, C272, C171 and C161 were isolated
from the λ phage libraries constructed from YAC clones 595C11 and 903D1
and used as a starting point for bidirectional walking. A phage contig (60
Kb) surrounding markers C212, C272 and C171 was constructed based on the
restriction map of the phage clones (FIG. 6).
To identify genes in the contig, the following three stategies were used
1) a search for interspecies-conserved sequences was conducted;
2) exon trapping method was performed; and
3) direct cDNA selection was performed. The genomic probe 132SE11, derived
from the phage containing the marker C272, gave positive hybridization
signals with hamster DNA indicating the presence of interspecies-conserved
sequences. The screening of a λgt10 human fetal brain cDNA library with
probe 132SE11 resulted in the selection of 7 overlapping λ clones spanning
1.6 kbp. Sequence analysis of the clones revealed a 882 bp open-reading
frame (ORF) and a 580 bp noncoding region. A 1.5 kbp clone (BCD541)
contained the entire coding sequence and most of the 3′ non-coding region.
The 3′ end of the cDNA along with its poly(A)+ tail was
obtained by PCR-amplification of a lymphoblastoid cell line cDNA library.
Two cDNA clones lacked nucleotides 661 to 755, suggesting that an
alternative splicing might have occured. Northern blot analysis of poly(A)+
RNA from various tissues including heart, brain, liver, muscle, lung,
kidney and pancreas, revealed the presence of a widely expressed 1.7 kb
transcript. The ORF encodes a putative protein of 294 amino acids with a
predicted molecular weight of approximately 32 Kd.
A homology search using the FASTA and BLAST networks failed to detect any
homology at either the nucleotide or the amino acid level.
To further distinguish whether there was any duplication of the BCD541
gene in the 5q13 region, BCD541 cDNA was used as a probe for Southern blot
and PFGE analysis of YAC clones spanning the disease locus.
Specific hybridization with non-overlapping YACs containing either the ECen
only (YAC clones 759A3, 121B8 and 278G7), or containing the ETel
only (YAC clone 595C11) provided evidence for duplication of the
BCD541 gene. Each gene encompassed approximately 20 kb and displayed an
identical restriction pattern. Evidence for head to head orientation of
the two genes was derived from the location of the SacII and EagI
restriction sites of the non-overlapping YAC clones containing either ECen
or ETel, following hybridization experiments with probes
BCD541 and p322 which flank the SacII and EagI sites of each element
In order to look for divergences in the two copies of the BCD541 gene, the
organization of the telomeric gene was characterized and compared to that
of the centromeric counterpart. Genomic sequence analysis revealed that
the telomeric BCD541 gene is composed of 8 exons (FIG. 3). However, it is
now known that the previously known exon 2 is composed of 2 exons
separated by an additional intron as set forth in FIG. 10, therefore the
SMN gene is composed of 9 exons.
Starting from either the centromeric or telomeric gene loci (in YAC clones
121B8 and 595C11, respectively), PCR-amplification and sequence of each
exon and their flanking regions revealed five discrepancies between the
centromeric and the telomeric BCD541 genes. The first one is a
conservative substitution in exon 7 (codon 280) specific for the telomeric
(TTC) or the centromeric BCD541 gene (TTT). The second one, located in the
3′ non-coding region (exon 8 nucleotide n° 1155) is specific for the
telomeric (TGG) or the centromeric BCD541 gene (TGA). Three other single
base substitutions were observed in the sixth and seventh introns.
The observation of both versions of each exon (exon 7 and 8) on either YAC
clones containing both gene loci (YAC clone 920C9) or the monochromosomal
hybrid HIIW105 demonstrated that these substitutions are neither allelic
nor due to polymorphisms. Band shifts on SSCP analysis of amplified exons
7 and 8 allowed an easy distinction of the telomeric (T-BCD541) and
centromeric genes (C-BCD541) in both controls and SMA patients. All the
unrelated healthy controls tested (n=75) harbored the T-BCD541 gene as
determined by SSCP analysis of exons 7 and 8 (100%). Most of them (89.3%)
also harbored the C-BCD541 gene but 8 out of 75 (8/75) (10.7%) lacked the
A total of 230 SMA patients were tested for single base substitutions
detected in exons 7 and 8 by SSCP method after PCR-amplification of
genomic DNA Among them, 103 belonged to type I, 91 to type II, and 36 to
type III. Interestingly, 213 out of 230 SMA patients (92.6%) lacked the
T-BCD541 gene on both mutant chromosomes compared with 0 out of 75
controls (0%). Moreover, 13 out of 230 SMA patients (5.6%) lacked the
T-BCD541 gene for exon 7 on both mutant chromosomes but retained the
T-BCD541 gene for exon 8 compared with 0 out of 75 controls (0%). Finally,
only 4 out of 230 SMA patients (1.7%) harbored the T-BCD541 gene as
determined by SSCP analysis of exons 7 and 8.
These results show that the T-BCD541 gene is either lacking or truncated
in 98% of SMA patients. In addition, these data support the view that the
disease gene is located between the telomeric locus detected by C272 and
exon 8 of the T-BCD541 gene. Therefore, according to the overlapping
restriction map of the phage contig, the critical region is entirely
contained in 20 kb, suggesting that the telomeric BCD541 gene is the
chromosome 5 SMA-determining gene.
In order to demonstrate that the T-BCD541 gene is responsible for SMA,
point mutations in the 4 SMA patients in whom no rearrangement of the
T-BCD541 gene had been observed were searched. Direct sequencing of PCR
amplification products of each exon with their flanking regions was
performed in the four patients.
A 7 bp deletion in the 3′ splice acceptor site of intron 6 (polypyrimidine
tract) was found in patient SA. Sequence analysis of exon 7 flanking the
deleted intron, recognized the sequence specific for the T-BCD541 gene.
Moreover, the non-deleted PCR-product corresponding to the same region,
harbored the sequence specific for the C-BCD541 suggesting that the other
mutant allelc lacked the T-BCD541 gene.
In patient BI, a 4 bp deletion in the 5′ consensus splice donor site of
intron 7 was found. This deletion occured on the T-BCD541 gene as
determined by sequence analysis of the flanking exon 7.
In patient HU, a point mutation in codon 272 (TAT→TGT) was found. This
mutation changed a Tyrosine to Cysteine. The patient was heterozygous for
the mutation, presumably carrying a different SMA mutation on the other
allele. All three mutations observed in patients SA, HU and BI were not
detected in 100 normal chromosomes ruling out rare polymorphisms.
A different splicing of exon 7 distinguished the C-BCD541 from the
T-BCD541 gene using reverse transcription-based PCR. Eleven SMA patients
were selected for the analysis of their transcripts by Northern blot or
reverse transcription-based PCR amplification. Eight of them belonged to
type I, 1 to type II and 2 to type III. SSCP analysis of genomic DNA
showed an absence of T-BCD541 gene in 10 patients and one patient (SA) had
C-BCD541 and T-BCD541 genes for both exons 7 and 8. Six unrelated controls
who harbored both C-BCD541 and T-BCD541 genes and 2 controls with only
T-8CD541 gene were included in the present study.
The expression of this gene in lymphoblasts made it possible to analyze
the BCD541 transcripts in cell lines derived from controls and SMA
patients. Northern blot analysis of RNA from lymphoblastoid cell lines
showed the presence of a 1.7 kb mRNA in all samples. None of the SMA
patients showed a transcript of altered size. It was observed that a
reduced level of transcripts was obtained when compared to the expression
of the β-actine gene in 3 out of 4 type I SMA patients. Normal mRNA level
were found for the other SMA probands.
Since the Northern blot analysis revealed the presence of a transcript in
SMA patients who had the C-BCD541 gene only for both exons 7 and 8 as
determined by SSCP analysis, these results indicated that both C-BCD541
and T-BCD541 genes were expressed. To prove whether both BCD541 genes were
expressed, RT-based PCR amplification of RNA isolated from the
lymphoblastoid cell lines from controls and SMA patients was used. Direct
sequencing of PCR products flanking exons 7 and 8 revealed that patients
who had C-BCD541 only displayed the sequence specific for the C-BCD541
gene. Controls who had both T-BCD541 and C-BCD541 genes, had two types of
transcripts corresponding to both BCD541 genes. These results confirmed
that both genes were expressed. In addition, 2 alternative splicings
involving exon 5 or exon 7 that resulted in different transcripts were
observed. The alternative splicing of exon 5 confirmed previous sequence
data on the cDNA clones.
The analysis of the RT-PCR amplification products encompassing exons 6 to
8 showed that the spliced transcript keeping exon 7, was present in
controls who had both C-BCD541 and T-BCD541 genes or controls who had the
T-BCD541 gene only. Conversely, the alternative spliced transcript lacking
exon 7 was observed in controls who had both genes, but not in controls
who had the T-BCD541 gene only. These results indicated that the
alternative spliced transcript lacking exon 7 was derived from the
C-BCD541 gene only.
The transcript analysis of patient SA harboring a 7 bp deletion of the 3
splice acceptor site of Intron 6 of the T-BCD541 gene revealed the
presence of both spliced transcript keeping exon 7 and alternate spliced
transcript lacking exon 7. Moreover, the sequence analysis of
amplification products from the spliced transcript keeping exon 7, showed
a sequence specific for the C-BCD541 gene (FIG. 2). These results
demonstrated that the 7 bp deletion of intron 6 observed in patient SA was
deleterious for the correct splicing of exon 7 of T-BCD541 gene only. In
addition, because a differential splicing of exon 7 allowed one to
distinguish the 2 BCD541 genes, this difference was analyzed among
controls and SMA patients including patient SA. In controls, the amount of
alternated spliced transcript lacking exon 7 was less abundant than that
of spliced product keeping exon 7. Conversely, in SMA patients, the amount
of alternated spliced transcript lacking exon 7 was equal or more abundant
than that of spliced product keeping exon 7.
These results provide evidence for a difference between controls and SMA
patients at the transcription level of these genes. The alternative
spliced transcript lacking exon 7 resulted in a shorter ORF with a
different C-terminus protein that might have effects on the protein
To further characterize the entire structure and organization of the human
SMN gene, three genomic clones were isolated from a FIX II phage library
derived from YAC clone 595C11 and screened with the full-length BCD541
cDNA (FIG. 2A) as a probe. After selecting several clones that hybridized
to the probe, restriction mapping and Southern blot analysis indicated
that phages L-132, L-5 and L-13 spanned the entire SMN gene.
These three phage clones were further subjected to sequencing using the
Maxam-Gilbert or Sanger et al methods of sequencing disclosed in Sambrook
et al supra.
The nucleotide and amino acid sequence of the entire SMN gene including
exons and introns is set forth in FIG. 10. The human gene is approximately
20 kb in length and consists of nine (9) exons interrupted by 8 introns as
shown in FIG. 10. The human SMN gene has a molecular weight of
approximately 32 kDA.
Although it was thought that only one exon 2 was present in the SMN gene
(see, Lefebvre et al, Cell, 80:155-165 (1995)), the sequencing data
proved otherwise and the previously mentioned exon 2 in Lefebvre et al
supra is in fact composed of 2 exons separated by an additional intron, as
illustrated in FIGS. 9 and 10. To avoid confusion in the renumbering of
exons, the 2 exons in exon 2 are now referred to as exon 2a and exon 2b.
All exon-intron bounderies displayed the consensus sequence found in other
human genes and a polyadenylation consensus site is localized 550 bp
downstream from the stop codon (FIG. 10).
Starting from either YAC clones 12188 or 595C11 (which contain the
C-BCD541 and SMN genes respectively, (see, Lefebvre et al, supra) PCR
amplification and sequence analysis of the introns showed three
differences between SMN and C-BCD541 in addition to those previously
described (by Lefebvre et al, supra). These included a base change in
intron 6 (-45 bp/exon 7, atgt, telomeric; atat, centromeric) and two
changes in intron 7 (+100 bp/exon 7, ttaa, telomeric; ttag, centromeric
and at position +214 bp/exon 7, ttat, telomeric; ttgt, centromeric, FIG.
10). The presence of both versions in a YAC clone containing both genes (YAC
920C9), and in the control population demonstrated that these
substitutions are locus-specific rather than due to polymorphism. Band
shifts on single strand confirmation polymorphism (SSCP) analysis of the
PCR amplified intron 7 allowed SMN and its centromeric counterpart
(C-BCD541) to be readily distinguished (see, FIG. 15).
In order to identify sequences potentially important for promoter
function, the organization of the region surrounding exon I of the SMN and
C-BCD541 genes was characterized. Based on restriction mapping, Southern
blot hybridization and PCR amplification, exon 1 and the C272 marker
(D5F150S1, D5F150S2) were located in the same BgIII-EcoRI restriction
fragment of L-132 phage (FIG. 9). PCR amplification using the C272f primer
and a reverse primer chosen in exon 1 was performed and the amplified
product was directly sequenced. Sequence analysis showed that the (CA)
repeat corresponding to the C272 marker are located 463 bp upstream from
the putative ATG translation start site (FIG. 11). Comparative sequence
analyses showed no discrepancy between the 5′ ends of the SMN gene and its
centromeric counterpart (C-BCD541). In addition, sequence analysis showed
the presence of putative binding sites for the following transcription
factors: AP-2, GH-CSE2, DTF-1, E4 μl. HiNF-A, H4TF-1, β-IFN, Sp1 (FIG. 11;
Faisst et al, Nucleic Acids Res., 20;3-26 (1992)).
Besides isolating and characterizing the human SMN gene, the mouse
homologue of the SMN gene was also cloned. Cross-species conservation of
human SMN gene with rodents has been shown in Lefebvre et al, supra and
served to isolate the mouse SMN gene. Screening of a mouse fetal cDNA
library using human SMN cDNA as a probe allowed the isolation of 2
overlapping mouse cDNA clones. Sequence analysis of the clones revealed an
864 bp open-reading frame (ORF) (FIG. 12). The ORF encodes a putative
protein of 288 amino acids (FIG. 12) with an homology of 83% with human
SMN amino acid sequence (FIG. 13).
Either the isolated human or the mouse SMN, the gene can be inserted into
various plasmids such as pUC18, pBr322, pUC100, λgHI, λ18-23, λZAP, λORF8,
and the like. The methods for inserting genes into different plasmid
vectors are described by Sambrook et al supra. Various microorganisms can
be used to transform the vector to produce the SMN gene. For example, host
microorganisms include, but are not limited to, yeast, CHO cells, E.
coli, Bacillus subtilis and the like.
Once recombinantly produced, the human SMN protein or the mouse SMN
protein can be further purified from the host culture by methods known in
Besides recombinantly producing the SMN protein, the present invention
also relates to the production of polyclonal and monoclonal antibodies.
These methods are known in the art as evidenced by Sambrook et al supra.
The monoclonal antibody can be obtained by the procedure of Kohler and
Milstein, Nature, 256:495 (1975); Eur. J. Immunol, 6;511
(1976) or Harlow and Lane Antibodies, a Laboratory Manual, Cold
Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988), and can be
used, for example, in diagnosing SMA, as well as other motor neuron
Polyclonal rabbit antisera can also be generated against synthetic
peptides corresponding to any part of the SMN amino acid sequence
including the amino terminus and carboxy terminus. More specifically, the
following peptides were synthesized based on the amino acid sequence set
forth in FIG. 1:
N-terminal G G V P E Q E D S V L F R R G T (residues 9-25 of SEQ ID NO: 9)
- S R S P G N K S D N I K P K (residues
173-186 of SEQ ID NO:9)
- F R Q N Q K E G R C S H S L N
(residues 280-291 of SEQ ID NO: 9)
The synthetic peptide may be coupled to a carrier protein such as Keyhole
limpet hemocyanin (KLH) through an amino- or carboxy-artificial cysteine
residue that may be synthetically added to the desired sequence. The
cysteine residue is used as a linker to couple the synthetic peptide to
the carrier protein. The procedure utilized to couple synthetic peptides
to KLH is described by Green et al. Cell, 28:477 (1982).
Approximately, 50-100 μg, preferably 100 μg of synthetic antigen is
dissolved in buffer and emulsified with an equal volume of Freund's
complete adjuvant. About 0.025 ml to 0.5 ml of emulsified antigen-adjuvant
can be injected intramuscularly or intradermaly into a rabbit. Four to six
weeks later, the rabbit is boosted and 20-40 ml of blood is drawn 7-10
days after each booster injection. The serum is then tested for the
presence of antigen using RIA, ELISA or immunoprecipitation. The positive
antibody fractions may then be purified, for example by absorption to
protein A following the method of Goudswaald et al, Scand. J. Immunol.,
More specifically, about 20 to 50 μg of antigen, prepared either by the
recombinant techniques set forth above or synthetically made antigen is
diluted in about 100 μl of buffer and emulsified with an equal amount of
Freund's complete adjuvant. About 30-60, preferably 50 μl of the
emulsified antigen-adjuvant is injected subcutaneously at four sites into
mice. Four to six weeks later, the mice are boosted with an
intraperitoneal injection of about 100 μl containing 5-10 μg of antigen
solubilized in buffer. The mice are bled from the mediam tail vein 7-10
days after the boaster injection and the serum is tested for antibody
using standard methods. Blood is then drawn every 3-4 days until the
antibody titer drops.
Tissue, plasma, serum, cerebral spinal fluid and the like can be used to
detect SMA disease using the above-described monoclonal or polyclonal
antibodies via Western blot (1 or 2 dimensional) or ELISA. These methods
are known in the art as described by Sambrook et al, supra.
A method for detecting SMA as well as in ALS, ACM, and PLS patients who
possibly have these motor neuron disorders, is also encompassed by the
present invention. This method Involves extracting from a patient
suspected of having SMA, DNA from a sample. This sample may include sera,
plasma, cerebral spinal fluid and the like. After extracting the DNA by
known methods in the art, primers that are derived from exons 7 and 8 of
the SMN gene are used to amplify the DNA.
After amplification with the primer, the amplified product is subjected to
SSCP (Single Strand Conformation Polymorphism).
The gels are then subjected to autoradiography to determine if SMA is
present in the sample.
More specifically, it has recently been discovered that in twelve cases of
arthrogryposis multiplex congenita (AMC) associated with SMA, 6 out of 12
patients lacked the SMN gene.
A total of twelve unrelated patients including eight males and four
females of various geographic origins was selected for the study. The
patients were chosen based on the criteria that these patients had:
(1) congenital joint contractures of at least two regions of the body
(see, Stern, JAMA, 81:1507-1510 (1923));
(2) generalized muscle weakness with muscular atrophy and areflexia
without extraocular involvement;
(3) electromyographic studies showed denervation and diminished motor
action potential amplitude; and
(4) muscle biopsies consistent with denervation with no evidence of
storage material or other structured abnormalities (see, Munsat,
Nouromuccular Disorders, 1:81 (1991)).
The study consisted of Dinucleotide Repeat Polymorphism Analysis and SMN
gene analysis (see, Examples) based on DNA extracted from peripheral blood
leukocytes, lymlphoblastoid cell lines or muscle tissue in all twelve
The data from this study is summarized in Table 1 below.
The diagnosis was made at birth with an uniform phenotype characterized by
a severe hypotonia, absence of movements except extraocular mobility and
contractures of at least two joints. The number of affected joints and the
severity of the postural defects varied from infant to infant, as set
forth in Table 1. Decreased fetal movements were noted in 7 out of 12
(7/12) patients. Neonatal respiratory distress was observed in 9 out of 12
(9/12) patients and facial involvement associated with micrognathia was
noted in 4 out of 12 (4/12) patients. Most of the cases, 8 out of 12
(8/12), died within the first month of life. Four infants are still alive.
No family history was noted except in family 12 in which both the child
and her father were affected suggesting an autosomal dominant form of AMC.
Table 1 shows that the SMN gene was lacking on both mutant chromosomes in
6 out of 12 (6/12) patients (cases 1-6). Among them, 3 out of 6 (3/6)
patients had a large inherited deletion involving both loci detected by
markers C212 and C272 on one parental allele, the other parental carrying
only one locus instead of the expected two, as shown in FIG. 14.
Analysis of SMN exons did not reveal intragenic mutations in the patients
whose SMN gene showed no deletions (cases 7-12). Genetic analysis showed
that the disease gene in a family (case 9) was not linked to chromosome
5q13 as both the affected and healthy siblings carried the same 5q13
haplotype. These data strongly suggest that the patients whose SMN gene
showed no deletions were not linked to the 5q13 SMA locus (cases 7-12).
Hitherto, arthrogryposis was regarded as an exclusion criterion in SMA
(sec, Munsat, supra). But the observation of SMN gene deletion in 6 out of
12 (6/12) patients (50%) strongly indicates that arthrogryposis of
neurogenic origin is related to SMA and that this subgroup and SMA are
allelic disorders. Yet, AMC of neurogenic origin is a genetically
heterogeneous condition since the disease gene was not linked to SMN locus
in 6 out of 12 (6/12) patients. Exclusion of chromosome 5q has also been
shown in one family with two AMC-SMA patients, as described by Lunt et al,
J. Med. Genet., 29:273 (Abstract) (1992).
Thus, by dinucleotide Repeat Polymorphism Analysis and SMN gene analysis,
clinical diagnosis of AMC can be confirmed by the absence or interruption
of the SMN gene. The present invention now provides methods to detect AMC
either in live patients or in utero.
Yet another embodiment of the present invention is the detection of SMA
using specific oligonucleotide probes based on the nucleotide sequence set
forth in FIGS. 3, 10, or for the mouse SMA FIG. 12. If a patient
totally is lacking in the SMN gene, no hybridization to the specific probe
will occur. The hybridization conditions may vary depending upon the type
of sample utilized. It is preferable to conduct such hybridization
analysis under stringent conditions which are known in the art and defined
in Sambrook et al supra. The oligonucleotide probes may be labeled in any
manner such as with enzymes, radioactivity and the like. It is preferable
to use radiolabeled probes.
In another embodiment of the present invention, the human SMN gene can be
utilized in conjunction with a viral or non-viral vector for
administration in vivo directly to the patients suffering from SMA or
related motor neuron diseases or by administration in vitro in bone marrow
cells, epithelial cells fibroplasts, followed by administration to the
patient. See, for example Resenfeld et al, Science (1991) 252, pp.
431 to 434.
The present invention provides a method of detecting SMN gene defects or
the total lack of the SMN gene in a fetus. Amniotic fluid taken from the
pregnant woman is subjected to SSCP analysis according to the methods of
the present invention.
Claim 1 of 22 Claims
1. A method of detecting the
presence in a human patient of an altered Survival Motor Neuron (SMN) gene
associated with Spinal Muscular Atrophy, comprising:
analyzing exon 7 or exon 8 of a gene identified as T-BCD541 (SEQ ID NO:22)
in a biological sample derived from the patient, and
comparing said exon 7 to the corresponding exon from nucleotide position 340
to nucleotide position 401 of SEQ ID NO:12, or exon 8 to the corresponding
exon from nucleotide position 846 to nucleotide position 1408 of SEQ ID
NO:12, which is present in a normal tissue;
wherein an alteration of either exon 7 or exon 8 in said patient sample with
reference to said normal tissue is indicative of the presence of an altered
Survival Motor Neuron (SMN) gene associated with Spinal Muscular Atrophy in
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