Spinal muscular atrophy (SMA) is a group of autosomal recessive neuromuscular disorders characterized by degeneration of the anterior horn cells of the spinal cord, leading to symmetrical muscle weakness and atrophy. The SMA disorder is usually subdivided into four clinical groups.  Patients with type I SMA disease (MIM# 253300) show onset at birth or before six months and typically die of respiratory insufficiency within two years. They are never able to sit or walk. Patients with type II SMA (MIM# 253550) show onset after six months. They can sit but are never able to walk unaided, and life expectancy is significantly reduced. Type III SMA (MIM# 253400) patients show first symptoms after 18 months and can stand and walk but often become wheelchair-bound during youth or adulthood. Type IV SMA is adult-onset, in which patients present with a milder form of muscle weakness (PubMed ID: 20301526).

SMN1 and SMN2 are two (highly similar) genes playing a pivotal role in SMA. The SMN1 and SMN2 genes are located in an inverted repeat area on chromosome 5q13. These two genes differ by only five nucleotides in exons 7 and 8, which includes a single variant (c.840C>T) in the coding sequence of SMN2 that affects the splicing of SMN2. SMN2 is much less efficient in making the SMN protein; therefore, it is the SMN1 gene that is the determinant factor in SMA. Someone lacking functional copies of SMN1 is always a patient, whereas SMA carriers (carrying a single copy of the SMN1 gene) are symptom-free. Establishing the SMN2 copy number is essential in SMA patients only; the more SMN2 copies present, the better the patient will be able to make up for the loss of SMN1 (PMID: 20301526, 29290580).

Approximately 2% of SMA patients have de novo pathogenic variants. About 95-98% of individuals with SMA have a homozygous deletion of exon 7 of the SMN1 gene, which encodes the complete length survival motor neuron protein (PubMed ID: 20301526). The remaining 2% to 5% percent of individuals with SMA are compound heterozygotes for the SMN1 exon 7 deletion and an intragenic missense, nonsense, or frameshift variant in SMN1 (PubMed ID: 14711346).

A small percentage of individuals who are carriers or have a diagnosis of spinal muscular atrophy may have a variant that is not identified by this method (e.g., large genomic deletions, promoter alterations). Rare alterations could lead to false-negative or false-positive results. If the results obtained do not match the clinical findings, additional testing should be considered.

Genomic DNA is extracted from the patient specimen before amplification and sequencing. Long-range polymerase chain reaction (LR-PCR) is performed first to exclude highly homologous gene SMN2, followed by nested PCR for Sanger sequencing to accurately detect variants in the SMN1 gene. Cycle sequencing is carried out after purification of the PCR products using the ABI Big Dye Terminator v.3.0 kit. PCR products are resolved by electrophoresis on an ABI 3730xl capillary sequencer. In nearly all cases, cycle sequencing is performed separately in forward and reverse directions.