Comparing MLPA and ddPCR for SMN1/SMN2 Copy Number Testing in Spinal Muscular Atrophy

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Comparing MLPA and ddPCR for SMN1/SMN2 Copy Number Testing in Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is a rare, inherited neuromuscular disease that damages motor neurons—the nerve cells that control muscle movement. It’s the leading genetic cause of infant death, affecting about 1 in 11,000 babies worldwide. SMA is caused by low levels of the survival motor neuron 1 (SMN1) protein: most patients have deletions or mutations in the SMN1 gene. A similar gene, SMN2, produces a small amount of functional SMN protein—and its copy number (1 to 8 copies) directly impacts disease severity: more copies mean milder symptoms.

Accurately measuring SMN1 and SMN2 copy numbers is critical for diagnosing SMA, predicting how severe it will be, and guiding treatment. Two common methods are multiplex ligation-dependent probe amplification (MLPA) and droplet digital polymerase chain reaction (ddPCR)—but which is more reliable? A 2020 study from Fujian Medical University in China set out to answer that question.

The Study: Comparing Two Genetic Testing Methods

Researchers led by Jing-Mei Hong, Miao Zhao, and Jin-Jing Li from the First Affiliated Hospital of Fujian Medical University analyzed 24 SMA patients (from a clinical trial, NCT04010604) and three induced pluripotent stem cell (iPSC) lines from Coriell Cell Repositories (a trusted source for genetic research materials). They used both MLPA and ddPCR to measure SMN1/SMN2 copy numbers, repeating tests to check reproducibility—how often the same result is obtained.

The study followed strict ethical guidelines: all families gave informed consent, and the protocol was approved by the First Affiliated Hospital’s Ethics Committee.

MLPA: High Ambiguity, Low Reproducibility

MLPA is a widely used method that uses probes to bind to SMN1/SMN2 DNA sequences, then amplifies and measures those probes to calculate copy numbers. But the study found major limitations:

  • Ambiguous results: 7 of 27 SMN1 tests and 19 of 27 SMN2 tests had “ratio” values (used to estimate copies) that fell outside the kit’s reliable range.
  • Poor reproducibility: Only 74% of SMN1 results and 29.6% of SMN2 results matched between repeated tests.
  • Copy number limits: MLPA couldn’t reliably measure more than 4 SMN copies—even though some patients have higher numbers.
  • Conflicting iPSC data: MLPA gave inconsistent results for the Coriell iPSC lines (e.g., one line had 2 SMN2 copies in Test 1, 3 in Test 2), which didn’t match Coriell’s verified data.

ddPCR: 100% Reproducibility and Clear Results

DdPCR is a newer method that splits DNA into thousands of tiny droplets, each containing a single DNA molecule. It then counts how many droplets have SMN1/SMN2 sequences—giving a direct, precise copy number. The study found:

  • Perfect reproducibility: Every ddPCR test (27 total) gave identical results—100% consistency.
  • Resolved ambiguous cases: DdPCR clarified all the uncertain MLPA results, including 7 SMN1 cases and 19 SMN2 cases.
  • Matches verified data: The Coriell iPSC lines had SMN2 copy numbers that exactly matched Coriell’s records (e.g., 2 copies for SMA type I, 3 for SMA type II).

Real-World Impact: Solving Confusing Cases

The study included two key examples that show why reliable testing matters:

1. SMA Siblings with Varying Symptoms

One family had two SMA siblings with very different symptoms—one more severe than the other. At first, MLPA tests gave conflicting results for their SMN2 copy numbers (4 vs. 2 copies). But ddPCR showed both siblings had 0 SMN1 copies (the cause of SMA) and 4 SMN2 copies. This meant their symptom differences weren’t due to SMN2—it had to be other genetic or environmental factors, which the team could then investigate further.

2. Rare SMN1 Mutation and Carrier Screening

Another case involved a family with a rare SMN1 mutation (c.844C>T). MLPA correctly identified that one family member had 1 copy of the mutated SMN1 (a carrier—someone with one normal and one mutated copy who has no symptoms) and another had 0 copies (SMA type I). But ddPCR gave wrong numbers—likely because its probes were designed to bind to normal SMN1, not the mutated version. This shows why combining both methods is critical for finding asymptomatic carriers, who can pass the mutated gene to their children.

Conclusion: DdPCR Is More Robust—But Combine with MLPA for Mutations

The study’s key takeaway? DdPCR is a more reliable method for measuring SMN1/SMN2 copy numbers—it’s faster, more reproducible, and handles high copy numbers better than MLPA. For most SMA patients, ddPCR alone can provide clear, actionable results.

But there’s a catch: for rare SMN1 mutations (like c.844C>T), MLPA is still valuable. The researchers recommend combining both methods to:

  • Accurately diagnose SMA.
  • Predict disease severity using SMN2 copy numbers.
  • Screen for asymptomatic carriers (who have one normal and one mutated SMN1 copy).

Funding

This work was supported by grants from the National Natural Science Foundation of China (Nos. 81771230, U1905210), the Joint Funds for the Innovation of Science and Technology of Fujian Province (Nos. 2017Y9094 and 2018Y9082), the National Key Clinical Specialty Discipline Construction Program, the Key Clinical Specialty Discipline Construction Program of Fujian, and the Startup Fund for Scientific Research of Fujian Medical University (Nos. 2018QH1050 and 2018QH2035).

References

  1. Cao YY, Zhang WH, Qu YJ, et al. Diagnosis of spinal muscular atrophy: a simple method for quantifying the relative amount of survival motor neuron gene 1/2 using Sanger DNA sequencing. Chin Med J 2018;131:2921–2929. doi:10.4103/0366-6999.247198
  2. Lefebvre S, Burlet PL, Bertrandy S, et al. Correlation between severity and SMN protein level in spinal muscular atrophy. Nat Genet 1997;16:265–269. doi:10.1038/ng0797-265
  3. Passon N, Dubsky de Wittenau G, Jurman I, et al. Quick MLPA test for quantification of SMN1 and SMN2 copy numbers. Mol Cell Probes 2010;24:310–314. doi:10.1016/j.mcp.2010.07.001
  4. Vidal-Folch N, Gavrilov D, Raymond K, et al. Multiplex droplet digital PCR method applicable to newborn screening, carrier status, and assessment of spinal muscular atrophy. Clin Chem 2018;64:1753–1761. doi:10.1373/clinchem.2018.293712
  5. Cusco I, Barcelo MJ, Rojas-Garcia R, et al. SMN2 copy number predicts acute or chronic spinal muscular atrophy but does not account for intrafamilial variability in siblings. J Neurol 2006;253:21–25. doi:10.1007/s00415-005-0912-y
  6. Hong JM, Zhao M, He J, et al. 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. Chin Med J 2020;133:2510–2511. doi:10.1097/CM9.0000000000001102

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