Genetic Screening Method for Analyzing Survival Motor Neuron Copy Number in Spinal Muscular Atrophy by Multiplex Ligation-Dependent Probe Amplification and Droplet Digital Polymerase Chain Reaction

Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by the degeneration of motor neurons in the anterior horn of the spinal cord, leading to progressive muscle weakness and atrophy. The disease is primarily caused by homozygous deletions or mutations in the survival motor neuron 1 (SMN1) gene, which results in a deficiency of functional SMN protein. A paralogous gene, SMN2, differs from SMN1 by a single nucleotide substitution (C-to-T at Position 6 in exon 7). This transition leads to alternative splicing, producing only 10% of full-length, functional SMN protein. However, SMN2 serves as the sole source of SMN protein in SMA patients, and its copy number (ranging from 1 to 8) is inversely correlated with disease severity. Accurate determination of SMN1 and SMN2 copy numbers is critical for genetic diagnosis, prognostic assessment, and therapeutic decision-making. This study evaluates two genetic screening methods—multiplex ligation-dependent probe amplification (MLPA) and droplet digital polymerase chain reaction (ddPCR)—for their reliability in quantifying SMN1 and SMN2 copy numbers.

Technical Overview of MLPA and ddPCR

MLPA is a semi-quantitative PCR-based method that uses probe hybridization and ligation to detect copy number variations (CNVs). The process involves five steps: DNA denaturation, probe hybridization, ligation, PCR amplification, and capillary electrophoresis. Results are interpreted by comparing peak ratios of target sequences to reference probes. However, MLPA has inherent limitations: it requires a reference DNA sample for normalization, has a maximum interpretable ratio of 2.15 (limiting detection to ≤4 copies), and often produces ambiguous results for samples with copy numbers outside this range.

In contrast, ddPCR partitions DNA samples into thousands of nanoliter-sized droplets, enabling absolute quantification of target DNA without reliance on reference standards. Each droplet undergoes independent PCR amplification, and fluorescent signals are counted to determine the target concentration. This method eliminates the need for normalization and offers high precision, even for samples with high copy numbers (>4).

Comparative Performance in Copy Number Analysis

The study analyzed 27 samples, including 24 SMA patients from a clinical trial (NCT04010604) and three human induced pluripotent stem cell (iPSC) lines (i-1, i-2, i-3) from the Coriell Cell Repositories. The iPSC lines represented SMA type I (2 or 3 SMN2 copies) and type II (3 SMN2 copies).

MLPA Limitations
MLPA produced unreliable results for 7/27 samples in SMN1 and 19/27 samples in SMN2 due to ambiguous ratios or inconsistencies between duplicate tests. For SMN1, MLPA achieved a reproducibility rate of 74.1% (20/27), while for SMN2, the rate dropped to 29.6% (8/27). Notably, MLPA failed to accurately quantify SMN2 copies in iPSC lines: i-1 showed discordant results (2 copies in Test 1 vs. 3 in Test 2), while i-2 and i-3 consistently exceeded the detectable range (>4 copies), contradicting the Coriell-reported values. These findings highlight MLPA’s inability to resolve copy numbers beyond its predefined ratio thresholds.

ddPCR Superiority
ddPCR demonstrated 100% reproducibility (27/27) for both SMN1 and SMN2 copy numbers. It resolved ambiguities in seven samples where MLPA failed to assign definite SMN1 copies (e.g., P-10, F2-II-2, F2-III-4) and provided consistent SMN2 results for all samples, including iPSC lines. For instance, ddPCR confirmed the Coriell-reported SMN2 copies (i-1: 2, i-2: 3, i-3: 3), aligning with clinical phenotypes. Additionally, ddPCR resolved discrepancies in a rare SMA sibling pair: MLPA initially reported 4 SMN2 copies in the proband (F1-II-2) and 2 in his sister (F1-II-1), but a repeat test suggested 4 copies for both. ddPCR analysis of peripheral blood mononuclear cells (PBMCs) and fibroblasts confirmed 4 SMN2 copies in both siblings, indicating that phenotypic variability arose from modifiers unrelated to SMN2.

Case Study: SMN1 c.844C>T Mutation

The study also evaluated a family carrying a rare SMN1 mutation (c.844C>T). MLPA reported 1 SMN1 copy in an SMA type I patient (SMN1 c.844C>T+0) and 2 copies in a carrier (SMN1 c.844C>T+1). However, ddPCR detected 0 and 1 copies, respectively. This discrepancy underscores MLPA’s potential to overestimate SMN1 copies in the presence of point mutations, possibly due to probe binding interference. The case emphasizes the necessity of combining ddPCR with sequencing or MLPA to clarify compound heterozygous mutations.

Clinical and Technical Implications

The robust performance of ddPCR makes it preferable for routine SMN1 and SMN2 copy number analysis, particularly in scenarios requiring high precision, such as newborn screening, carrier testing, and therapeutic monitoring (e.g., pre-post treatment with nusinersen or gene therapy). MLPA remains useful for detecting exon-level deletions or duplications but requires complementary methods to resolve ambiguous cases.

For SMA patients with atypical phenotypes or suspected genetic modifiers, ddPCR provides a reliable foundation for further investigations into secondary modifiers, such as plastin 3 (PLS3) or zinc finger protein 1 (ZPR1). Additionally, the method’s independence from reference DNA simplifies workflow and reduces costs.

Conclusion

This study establishes ddPCR as a superior method for SMN1 and SMN2 copy number quantification, offering unparalleled reproducibility, precision, and ease of use. MLPA’s limitations in resolving high copy numbers and mutation-associated ambiguities necessitate its use in conjunction with ddPCR for comprehensive genetic profiling. The integration of these methods enhances diagnostic accuracy, facilitates personalized treatment strategies, and advances research into SMA pathogenesis and modifier genes.

doi.org/10.1097/CM9.0000000000001102

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