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

Figures

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  Figure 1. SMA molecular diagnostic procedure.

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  Figure 2. Representative MLPA profiles of SMN1 and SMN2 copy number patterns in SMA patients. (A) common genotype of homozygous E7 and E8 deletion of SMN1, (B) concordant total copy numbers across all probes of SMN1 + 2 in a compound heterozygous SMA patient, (C) an SMN1:SMN2 ratio of 1:3 at E7 and 2:2 at E8, with a total copy number of 4 at both exons, suggesting gene conversion. (D) discordant total copy numbers among different SMN probe regions. Specifically, the total SMN copy number was 3 for E2A-E5, lower than the total copy number of 4 for other SMN regions (e.g., E7/E8), suggestive of a partial SMN1 deletion affecting the E2A-E5. Each MLPA bar graph is enclosed within a red box. The specific probes for SMN1 E7, SMN1 E8, SMN2 E7 and SMN2 E8 are indicated by red arrows, while non-specific SMN probes used for total SMN (SMN1 + 2) copy number determination are indicated by red horizontal brackets.

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  Figure 3. Identification of a novel SMN1 variant, c.280_303delinsTCTTTTGTAG using combined MLPA and AS-LR-PCR approaches. (A), MLPA-P021 analysis shows a single-copy SMN1 pattern. The specific probes for SMN1 E7, SMN1 E8, SMN2 E7 and SMN2 E8 are indicated by red arrows, while non-specific SMN probes used for total SMN (SMN1 + SMN2) copy number determination are indicated by red horizontal brackets. (B) Genomic Sanger sequencing reveals double peaks in both E3 and E7. Subsequent SMN1-AS-LR-PCR identifies the novel variant, c.280_303delinsTCTTTTGTAG in E3, while the E7 region harbors the SMN1-specific base (c.840C). In contrast, SMN2-AS-LR-PCR shows no variant in E3, and the E7 region carries the SMN2-specific base (c.840T). These findings confirm that the novel variant resides on the single functional SMN1 copy. (C), the sequence alignment of SMN1 further illustrates the position of the variant.

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  Figure 4. Identification of a SMN1 c.863G>T variant and a concurrent gene conversion event by integrated sequencing approaches. (A) MLPA P021 reveals a copy number pattern of 1:2:3:2 for SMN1 E7, SMN1 E8, SMN2 E7 and SMN2 E8 (red arrows), suggestive of a gene conversion event involving the E8 PSV (c. *239A>G) in this patient. (B) SMN1-AS-LR-PCR combined with nested PCR fails to clearly determine the allelic origin of the c.840 (the PSV site) and c.863 (the variant site) loci (red arrows), which is subsequently confirmed by genomic cloning and Sanger sequencing. (C) One SMN1 haplotype and three distinct SMN2 haplotypes (SMN2-Hap1, Hap2 and Hap3) are all captured by tLRS. The red boxes highlight the allelic distribution of three key loci—c.840 (the PSV site distinguishing SMN1 from SMN2), c.863 (the variant site), and c.*239 (the E8 PSV)—across SMN1 and the three SMN2 haplotypes. Integrative Genomics Viewer (IGV) shows that the c.863G>T resides on the SMN1 allele. In addition, the E8 PSV of one SMN2 copy is converted from the SMN2-specific base (adenine, A) to the SMN1-specific base (guanine, G), corresponding to c.*239A>G, thereby confirming the gene conversion event.