Molecular Genetic Diagnosis of Spinal Muscular Atrophy: Clinical Utility, Challenges, and Lessons Learned from Illustrative Cases in a Single Center

Background: Spinal muscular atrophy (SMA) is mainly caused by biallelic SMN1 inactivation. While most patients carry homozygous deletions, 3–5% are compound heterozygotes, making molecular diagnosis challenging. Methods: A tiered diagnostic strategy was applied to 17 pediatric patients, combining copy number analyses (MLPA and targeted long-read sequencing, tLRS), sequence variant detection (RT-PCR cloning and sequencing, allele-specific long-range PCR with nested PCR, and tLRS), and structural variant analysis (ultra-long-read sequencing, Ultra-LRS). Results: Copy numbers were concordant between MLPA and tLRS. MLPA-suggested gene conversions were confirmed by tLRS, while discordant total copy numbers were resolved as large deletions by Ultra-LRS. RT-PCR cloning, and sequencing identified SMN1 variants in 11/12 cases and confirmed aberrant splicing in three cases, but failed for large deletions. AS-LR-PCR with nested PCR characterized the variants in 13/15 but failed in gene conversion cases. tLRS achieved definitive diagnosis in all cases, and Ultra-LRS precisely delineated breakpoint junctions of two large deletions. Conclusions: A hierarchical complementary strategy integrating copy number, sequence, and structural analyses is essential for the accurate diagnosis of compound heterozygous SMA.