Indian Journal of Biochemistry & Biophysics Vol. 40, December 2003, pp. 439-441 Note Mutation analysis in spinal muscular atrophy using allele-specific polymerase chain reaction Akanchha Kesari, Monisha Mukherjee and Balraj Mittal* Department of Medical Genetics, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Raebareli Road, Lucknow 226 014, India Received 3 September 2003; revised 18 October 2003 Polymerase chain reaction (PCR), followed by restriction digestion is universally used for molecular diagnosis of spinal muscular atrophy (SMA). In the present study, we have used a modified strategy based on amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) to develop a rapid and reliable method for mutation detection and prenatal diagnosis in SMA patients. The telomeric (SMN1) and centromeric (SMN2) copies of exon 7 of the survival motor neuron (SMN) gene were amplified by ARMS-PCR, using primers specific to SMN1 and SMN2 nucleotide sequence with the exonic 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 homozygous deletion of exon 7 of SMN1 gene by conventional PCR-restriction fragment length polymorphism (PCR-RFLP) method showed the same deletion status by ARMS-PCR. This procedure showed a 100% concordance between PCR-RFLP and ARMS-PCR methods for the detection of SMN1/SMN2 status in patients with SMA. An artifact due to incomplete digestion is not a problem while using ARMS-PCR. The modified protocol is specific, rapid and highly reliable for use in prenatal diagnosis as well. Keywords: spinal muscular atrophy, allele-specific amplification, DNA diagnosis, prenatal diagnosis, SMN gene, mutation analysis, polymerase chain reaction. Proximal spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder, characterized by the degeneration of α-motor neurons of anterior horn cells of the spinal cord. SMA comprises a clinically and genetically heterogeneous group of diseases with an estimated incidence of 1/10,000 live births 1 and a carrier frequency of 1/50 2 . All three forms of childhood SMA have been mapped to chromosome 5q11.2-q13.3 containing a large inverted duplication consisting of at least four genes, each of which is present in a telomeric (t) and a centromeric (c) copy 2 . Survival motor neuron gene (SMN1 or SMNt) is the most important gene associated with SMA. It is believed that homozygous deletion in SMN1 causes the disease in 90-95% of classical SMA patients 3 . Molecular diagnosis of patients with suspected SMA and prenatal diagnosis in families with a history or suspicion of SMA are based on the detection of homozygous deletion of exons 7 and 8 of SMN1 gene 3 . The conventional protocol involves polymerase chain reaction (PCR), followed by restriction digestion 4-5 , which is time- consuming and prone to diagnostic error due to incomplete digestion. Recently Moutou et al 6 developed an allele-specific amplification method of fluorescent PCR for the pre-implantation genetic diagnosis (PGD) of SMA from a single cell. Here we report a strategy with appropriate modifications, which does not involve the use of fluorescent dyes. The PCR conditions were also modified which enabled the use of this method for rapid and reliable mutation detection as well as prenatal diagnosis of SMA. Materials and Methods Consecutive SMA patients from January 2000-June 2002 diagnosed in the Genetics and Neurology out patient department (OPD) of the Institute, on the basis of inclusion and exclusion criteria as per International SMA Consortium 7 have been included in the study. The patients belonged to three types of SMA (types I- III). The blood (5 ml) was collected in ethylene diamine tetra acetic acid (EDTA) from 35 patients and 20 controls. Controls had no symptoms of any muscle disease and were apparently healthy. DNA was extracted from blood by the standard phenol- chloroform method 8 . Chorionic villi sampling was done under ultrasonography by expert clinicians from ___________ *Address for correspondence Phone: 091-0522-2668005-08; Extn. 2322 (O), 2329 (Lab) Fax: 91-0522-2668 973/ 2668 078 /2668 017 E-mail: balraj@sgpgi.ac.in bml_pgi@yahoo.com Abbreviations: SMA, spinal muscular atrophy; SMN, survival motor neuron; ARMS-PCR, amplification refractory mutation system-polymerase chain reaction; RFLP, restriction fragment length polymorphism; CVS, chorionic villi sample; PGD, pre- implantation genetic diagnosis; OPD, out-patient department; EDTA, ethylene diamine tetra acetic acid; dNTP, deoxyribo nucleotide triphosphate.