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Note
Mutation analysis in spinal muscular atrophy characterized by the degeneration of -motor neurons
using allele-specific polymerase chain of anterior horn cells of the spinal cord. SMA
comprises a clinically and genetically heterogeneous
reaction
group of diseases with an estimated incidence of
Akanchha Kesari, Monisha Mukherjee and Balraj Mittal* 1/10,000 live births1 and a carrier frequency of 1/502.
Department of Medical Genetics, Sanjay Gandhi Postgraduate
All three forms of childhood SMA have been mapped
Institute of Medical Sciences, Raebareli Road, to chromosome 5q11.2-q13.3 containing a large
Lucknow 226 014, India inverted duplication consisting of at least four genes,
each of which is present in a telomeric (t) and a
Received 3 September 2003; revised 18 October 2003
centromeric (c) copy2. Survival motor neuron gene
Polymerase chain reaction (PCR), followed by restriction
(SMN1 or SMNt) is the most important gene
digestion is universally used for molecular diagnosis of spinal associated with SMA. It is believed that homozygous
muscular atrophy (SMA). In the present study, we have used a deletion in SMN1 causes the disease in 90-95% of
modified strategy based on amplification refractory mutation classical SMA patients3. Molecular diagnosis of
system-polymerase chain reaction (ARMS-PCR) to develop a
patients with suspected SMA and prenatal diagnosis
rapid and reliable method for mutation detection and prenatal
diagnosis in SMA patients. The telomeric (SMN1) and in families with a history or suspicion of SMA are
centromeric (SMN2) copies of exon 7 of the survival motor based on the detection of homozygous deletion of
neuron (SMN) gene were amplified by ARMS-PCR, using primers exons 7 and 8 of SMN1 gene3. The conventional
specific to SMN1 and SMN2 nucleotide sequence with the exonic protocol involves polymerase chain reaction (PCR),
mismatch G (for SMN1) and A (for SMN2) at the 3 end. The PCR
products were analyzed on agarose gels. All the patients who had
followed by restriction digestion4-5, which is time-
homozygous deletion of exon 7 of SMN1 gene by conventional consuming and prone to diagnostic error due to
PCR-restriction fragment length polymorphism (PCR-RFLP) incomplete digestion. Recently Moutou et al6
method showed the same deletion status by ARMS-PCR. This developed an allele-specific amplification method of
procedure showed a 100% concordance between PCR-RFLP and
fluorescent PCR for the pre-implantation genetic
ARMS-PCR methods for the detection of SMN1/SMN2 status in
patients with SMA. An artifact due to incomplete digestion is not diagnosis (PGD) of SMA from a single cell. Here we
a problem while using ARMS-PCR. The modified protocol is report a strategy with appropriate modifications,
specific, rapid and highly reliable for use in prenatal diagnosis which does not involve the use of fluorescent dyes.
as well. The PCR conditions were also modified which
enabled the use of this method for rapid and reliable
Keywords: spinal muscular atrophy, allele-specific amplification,
DNA diagnosis, prenatal diagnosis, SMN gene, mutation detection as well as prenatal diagnosis of
mutation analysis, polymerase chain reaction. SMA.
as seen in lane 7 (Fig. 2), while 185bp band reports1,3 can be attributed to the presence of point
corresponding to SMN2 gene was present as observed mutations or other genetic alterations that might be
in lane 8 (Fig. 2). The presence of 360bp band for more prevalent in our patients.
connexin-26 gene ruled out any kind of PCR failure in In conclusion, we present here a rapid and highly
all the reactions. The centromeric (SMN2) deletion in reliable method for mutation analysis based on ARMS
a patient reported earlier by us10 was also confirmed strategy for screening of patients suspected to have
by ARMS-PCR (Fig. 2, lane 2). DNA from CVS SMA. This procedure is accurate and less time-
subjected to ARMS-PCR showed bands both in SMN1 consuming and can be used successfully in providing
and SMN2 lanes (Fig. 2, lanes 3 and 4), thereby prenatal diagnosis of SMA.
suggesting that there was no homozygous deletion of
SMN1 or SMN2 gene in the foetus under diagnosis. Acknowledgement
We would like to thank Dr S R Phadke, Genetics
This strategy has already been used in pre-
and Dr S Pradhan, Neurology for providing us the
implantation genetic diagnosis of SMA using
clinical samples. The work was supported from an
fluorescent PCR, followed by analysis on an
intramural grant from Sanjay Gandhi Post Graduate
automated sequencer6. The protocol mentioned here
Institute of Medical Sciences, Lucknow. One of the
requires only PCR, followed by gel analysis, which
authors (MM) acknowledges Council of Scientific
can be completed within 5 hr and it is the first
and Industrial Research (CSIR), New Delhi for
application in SMA diagnosis. The main problem of
providing fellowship under the Pool Scientist Scheme.
SMA diagnosis is the presence of two copies of SMN
gene, the telomeric (SMN1) and its homologue, the
References
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