Researchers in China report having developed a fast and reliable way for a genetic diagnosis of spinal muscular atrophy (SMA) and to identify carriers, those who do not have the disease but can still pass it to their children.
Called mass spectrometry-copy number variation, or MS-CNV, the new method was found to be as effective as the gold-standard multiplex ligation probe amplification (MLPA) method in two independent sample groups, but it requires significantly less time to provide results .
The findings, “Simultaneous quantification of SMN1 and SMN2 copy numbers by MALDI-TOF mass spectrometry for spinal muscular atrophy genetic testing,” were published in the journal Clinica Chimica Acta.
Nearly all SMA cases are caused by the complete loss of exon 7 in both copies of the SMN1 gene — one inherited from the mother and one from the father — and the simultaneous loss of exons 7 and 8 is also common in these patients. Exons are the sections of a gene that contain the information to generate proteins.
The number of copies of a “backup” SMN gene, SMN2, influences SMA severity, with a higher number typically predicting less severe disease. There is a single nucleotide difference in exons 7 and 8 between SMN1 and SMN2; nucleotides are DNA’s building blocks.
Detecting the loss of exon 7 and exon 8 in both copies of the SMN1 gene is an effective way to screen for and confirm an SMA diagnosis, while the presence of only one mutated gene copy can identify carriers.
These carriers — 1 in every 40 to 50 people across ethnicities — are not affected by the disease, but can pass the faulty gene to their children. Identifying carriers is essential for proper genetic counseling of adults who are planning families.
Several methods of newborn and carrier SMA screening were developed in the last few years.
“Given the availability of approved [SMA therapies] and the tendency of better outcome with earlier diagnosis and treatment, we reasoned a high-throughput method capable of simultaneous quantification of SMN1 and SMN2 Copy numbers at a reasonable cost can greatly facilitate the management of SMA,” the researchers wrote.
With this in mind, a research team largely at Wenzhou Medical University developed a new method for SMA newborn and carrier screening, named MS-CNV, that combines two techniques called competitive PCR and MALDI-TOF mass spectrometry.
This assay allows the simultaneous determination of the number of copies of exons 7 and 8 in both SMN1 and SMN2 genes, as well as exon 5 in the nearby NAIP gene — whose loss has been linked to more severe disease.
MS-CNV was initially found to correctly detect two copies of SMN1, SMN2 and NAIP in 20 DNA samples, with results reproducible when the same samples were re-tested on different dates.
The method was then validated in samples from two independent groups of SMA patients, carriers, and healthy individuals with multiplex ligation-dependent probe amplification, or MLPA, assays running in parallel. The gold-standard method for SMA screening, MLPA assesses the copy number of genes and individual exons.
The first group included 79 blood samples: three from SMA patients, five from known carriers, and 71 from healthy volunteers with unknown SMA genetic information.
Results using both MS-CNV and MLPA were highly concordant for SMN1, SMN2and NAIP gene copy numbers. Both methods correctly identified patients and known carriers, and each method additionally identified three carriers among healthy volunteers.
The second group of 62 blood samples collected from a children’s hospital had been previously tested with MLPA, but the researchers had no information on their genetic status when they the samples with MS-CNV.
Similar to MLPA, the MS-CNV method identified 21 SMA patients, 14 SMA carriers, and 27 non-carriers. Most (81%) patients had lost exons 7 and 8 in both copies of the SMN1 gene. Four (19%) had lost exon 7, while exon 8 remained intact in both gene copies.
Notably, these four patients showed a simultaneous increase for SMN2 exon 7, suggesting that there was a mutation that converted SMN1 exon 7 into SMN2 exon 7 — resulting in a mixed, or hybrid, SMN gene.
Hybrid SMN genes were suggested also in one SMA carrier and in three non-carriers.
“Such additional information may be useful as patients with a hybrid SMN1-SMN2 gene may show a milder [disease]given that a hybrid gene may produce a higher proportion of functional protein,” the wrote.
Both methods resulted in ambiguous results for a small subset of samples that are almost exclusively concerned SMN2. Importantly, an ambiguous value from one method could be corrected by an unambiguous value from the other method, “suggesting an ambiguous test result derived from one method may be resolved by a second method,” the team wrote.
These findings highlight that MS-CNV “can be used for carrier screening and genetic diagnosis of SMA, providing dosages information for both SMN1 and SMN2 given its accuracy and high sample processing throughput,” the researchers wrote.
The total assay time, including laboratory procedures and data analysis, lasts about 6.5 to 7 hours, “a significant improvement over MLPA for both hands-on and overall time,” they added.
“This highly automated method for the quantification of SMN1 and SMN2 gene copy numbers may greatly facilitate carrier screening and early diagnosis of SMA, as well as identifying patients suitable for effective treatment,” the team concluded.