CASMA, large-scale screening test, may best detect SMA carriers
Spots gene mutations and changes standard approaches miss, study reports
A new large-scale genetic screening technique for spinal muscular atrophy (SMA) called comprehensive SMA analysis, or CASMA, was more effective at detecting disease-related mutations in carriers than standard approaches, a study reports.
CASMA “is a simple and accurate screening method for SMA, which shows greater clinical efficacy in the large-scale screening of SMA,” the researchers wrote, adding that “carrier screening seems to be the most effective way to prevent SMA birth defects.”
The study, “Evaluating the clinical efficacy of a long-read sequencing-based approach for carrier screening of spinal muscular atrophy,” was published in Human Genomics.
CASMA evaluated in samples from people with standard screening
Most types of SMA are caused by mutations in both copies of the SMN1 gene, one inherited from each biological parent. In nearly all cases, parts of the protein-coding portion of the gene, exon 7 and often exon 8, are missing.
People who inherit one copy of the mutated gene and one healthy copy are carriers. While they won’t develop SMA or disease symptoms, they can pass the condition on to their children.
Cells have a second, almost identical SMN2 gene that can compensate, but only partially, for the loss of SMN1. The number of SMN2 copies, which naturally varies across populations, generally influences SMA severity and progression.
Because early disease treatment improves motor function and survival in SMA patients, carrier screening and prenatal diagnosis are of importance. Clinicians can test the copy number of exon 7 in the SMN1 and SMN2 genes to detect carrier status, confirm an SMA diagnosis, and estimate the disease’s potential severity.
Two standard genetic screening methods to detect SMN1 and SMN2 copy numbers are multiplex ligation probe amplification (MLPA) and fluorescent quantitative PCR (qPCR). While MLPA identifies missing or additional DNA pieces, qPCR quantifies specific DNA sequences.
In this report, researchers in China used MLPA and qPCR tests to determine SMN copy numbers in 1,400 samples from people who underwent carrier screening, and compared the consistency of the results.
Then, for consistent and inconsistent results with these two methods, they applied CASMA to evaluate its detection performance. This technique uses a long-read, sequencing-based approach that enables the analysis of much longer DNA fragments at once.
Negative test samples were defined as those with intact exons 7 and 8 in both the SMN1 and SMN2 genes, while other test results were defined as positive.
CASMA reported to spot SMN1 gene conversions and ‘2+0’ genotypes
From a set of 676 positive samples with consistent MLPA and qPCR results, 44 samples were randomly selected for CASMA analysis. For exon 7 of the SMN1 and SMN2 genes, CASMA results were consistent with those of MLPA and qPCR.
However, the copy numbers of exons 7 and exon 8 in SMN1 were inconsistent in 13 samples. In 12 samples assessed by CASMA, the team detected a conversion between SMN1 and SMN2, in which a mutation converted the SMN1 gene into a less efficient SMN2 gene. In 11 of these samples, the conversion occurred in exon 8.
For 16 samples, inconsistent MLPA and qPCR results were reported for SMN1 exon 7. Upon resampling, the results of both methods were consistent with CASMA data, except for one SMN1 intragenic (within a gene) mutation identified by CASMA in a sample with two SMN1 copies.
A total of 23 SMA carriers were detected, with a prevalence of 1 in 61. Among the carriers, 14 had an SMN1 deletion, with four samples having one SMN1 and SMN2 gene each, and 10 samples having one SMN1 gene and two SMN2 genes. Nine carriers showed an SMN1 to SMN2 conversion, with one SMN1 gene and three SMN2 genes. No samples had one SMN1 and no SMN2 copies, and 66 samples (4.71%) had two SMN2 deletions.
Researchers next examined a family with suspected SMA. Two identical female twins were missing both SMN1 copies. Their mother was a carrier with one SMN1 gene. However, their unaffected father was suspected of having a “2+0” genotype (genetic makeup), meaning he inherited two SMN1 copies from one parent and none from the other.
A person with a “2+0” genotype has a 50% chance of passing the SMA-causing mutation to their children. This is because they have one set of chromosomes with two functional SMN1 gene copies and another set with no functional SMN1 gene copies.
Healthy boy had two SMN1 copies from father, SMN2 copies from mother
Because CASMA can assess the entire length of both SMN1 and SMN2 genes, the team analyzed both parents and one unaffected son. Analysis showed that the son carried two SMN1 copies, both inherited from his father, meaning both were “2+0.” Meanwhile, the boy’s two SMN2 genes came from his mother, with none inherited from his father; thus, he was “2+0” for SMN2.
“Compared with traditional PCR-based genetic testing methods, CASMA can determine the ‘2 + 0’ genotype by genotyping only the parents and one child,” the researchers wrote.
“CASMA can not only quantify the copy number of the SMN gene but also accurately detect intragenic variants and easily determine the ‘2 + 0’ genotype of subjects,” the scientists concluded. “CASMA has great advantages over MLPA and qPCR assays, and could become a powerful technical support for large-scale screening of SMA.”
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