Determination of exon 7 SMN1 deletion in Iranian patients and heterozygous carriers by quantitative real-time PCR

Abstract

Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by the degeneration of motor neurons of the spinal cord anterior horns, leading to progressive atrophy of proximal muscles, paralysis, respiratory failure, and even infant death. Patients with SMA are classified into three types, based on the age at onset and clinical severity (Munsat and Davies 1992; Wirth et al. 1995): type I (MIM 253300) is the most severe form, type II (MIM 253550) is the intermediate form, and type III (MIM 253400) is the mildest form. With an incidence of 1/6000 to 1/10000 and a carrier frequency of 1/40 to 1/50, SMA is the second most frequent autosomal recessive disease in Europeans (Pearn 1980). The SMA determining gene called the ‘survival motor neuron’ gene (SMN) is present on 5q13 in two copies, SMN1 and SMN2, which differ by only five nucleotide exchanges within their 3′ ends (Lefebvre et al. 1995). Two of these base-pair exchanges, located in exons 7 and 8 (which allow SMN1 to be distinguished from SMN2) currently are used for direct diagnosis of SMA. Therefore, the first step in this test is to amplify specifically SMN1 exon 7 or exon 8 and later on subjecting the PCR product to restriction digestion, a PCR-RFLP approach (Scheffer et al. 2001). In a study of 1122 patients with type I, type II, or type III SMA it was shown that in about 94% of SMA patients (in all three types) homozygous absence of SMN1 is responsible for this disease (Scheffer et al. 2000). Approximately half of the remaining patients were identified as compound heterozygote (with deletions and intragenic SMN1mutations), whereas the other half, without SMN1 mutations, were considered as distinct genetic entities (Wirth et al. 1999). The carrier test for