Study supports combined genetic testing approach in SMA
A combination of genetic testing approaches enabled researchers in Spain to more accurately characterize complex genetic changes that may help explain differences in disease severity in spinal muscular atrophy (SMA), according to a new study.
The findings suggest that “combining multiple genetic diagnostic techniques” enables more accurate genetic characterization and may help inform prognosis and treatment decisions, particularly in complex cases, the researchers wrote.
The study, “Multimodal characterisation of the SMN locus in SMA: copy number quantification and hybrid gene identification,” was published in npj Genomic Medicine.
Researchers examine complex SMA genetics
SMA is mainly caused by the loss of part of the SMN1 gene, which provides instructions for making survival motor neuron (SMN), a protein essential for the health and survival of the nerve cells that control movement. Without enough SMN, these cells become damaged and die, ultimately leading to SMA symptoms.
In addition to SMN1, another gene called SMN2 also provides instructions for making the SMN protein. Although a small difference in its genetic code causes SMN2 to produce mostly shorter and less functional protein, it can still make small amounts of fully functional SMN protein, allowing it to partly compensate for mutations affecting SMN1.
Because of this, SMN2 acts as an important disease modifier in SMA. Usually, patients with more SMN2 copies can produce more SMN protein and often have milder disease. Still, this relationship is not always straightforward, and people carrying the same number of SMN2 copies may experience very different disease severity.
Part of this variability may be explained by certain SMN2 variants. Researchers also increasingly suspect that hybrid or rearranged versions of the SMN1 and SMN2 genes — formed through complex rearrangements and gene conversion events between the two genes — may also influence how much functional SMN protein is produced.
However, the contribution of these hybrid structures to disease severity remains unclear. At the same time, accurately determining SMN2 copy number remains critical for prognosis and treatment decisions.
To this end, researchers evaluated and compared several genetic testing approaches in 78 patients with genetically confirmed SMA to determine which methods most accurately measured SMN2 copy number and identified complex structural changes involving the SMN1 and SMN2 genes.
Testing methods compared for SMN2 copies
Multiplex ligation-dependent probe amplification, or MLPA, is one of the standard methods used in SMA diagnosis. The researchers compared it with newer approaches that have been developed to improve the assessment of SMN2 copy number, detect SMN2 variants, and identify hybrid genes, including digital PCR, AmplideX testing, and long-read sequencing.
Using MLPA, the scientists found that most participants carried three SMN2 copies (59%), while about one-quarter (23.1%) carried four copies, and 16.7% had two copies. One participant carried five SMN2 copies. MLPA also identified genomic rearrangements affecting SMN genes in 10 unrelated participants, including patterns suggestive of hybrid genes.
Digital PCR showed complete agreement with MLPA for SMN2 copy-number measurement, supporting the reliability of both methods. In contrast, some discrepancies emerged with AmplideX testing. While AmplideX correctly measured SMN2 copy number in many participants and identified the SMN2 variant c.859G>C in two cases — a variant linked to greater production of functional SMN protein — discrepancies with MLPA were seen in seven patients on the first run. After repeat testing, two patients still showed discordant values, while five matched MLPA.
Digital PCR also provided additional precision in a particularly complex case. In one participant identified by MLPA as carrying five SMN2 copies, AmplideX could classify the result only as four or more copies, while digital PCR produced an average estimate of 5.46 copies, consistent with five to six copies.
To better characterize the 10 participants with rearrangements identified by MLPA, the researchers next turned to long-read sequencing, a technology that can read longer stretches of DNA and reconstruct the detailed structure of individual genes. This higher-resolution analysis confirmed a more complex genetic picture than standard testing alone could capture.
Nine of the 10 participants carried at least one hybrid copy involving the SMN1 and SMN2 genes, and some carried multiple hybrid copies. Across these patients, the researchers identified nine recurrent hybrid structures, six of which had not been reported before. Despite this detailed analysis, however, no consistent relationship emerged between specific hybrid structures and disease severity.
“Overall, our findings support an integrated diagnostic strategy in which copy-number quantification is complemented by structural characterisation in complex cases,” the investigators wrote.
They added that larger studies will be needed to determine whether specific hybrid structures and genetic patterns within individual SMN copies influence disease severity, progression, or treatment response.
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