Saliva Samples Can Help Diagnose SMA, Study Demonstrates

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by Steve Bryson PhD |

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Genetic analysis of dried saliva spots identified people with spinal muscular atrophy (SMA), a study demonstrated and possibly for the first time.

The scientists noted that this noninvasive and low-cost, alternate method of genetic screening may be suitable for programs held in schools or workplaces without the need for blood collection, or samples can be sent by courier to enable remote diagnosis and eliminate clinical visits.

The study, “Detection of Spinal Muscular Atrophy Patients Using Dried Saliva Spots,” was published in the journal Genes.

Nearly all cases of SMA are caused by mutations in the SMN1 gene, resulting in the impairment of motor neurons, which are nerve cells that control voluntary movements. The main symptoms of SMA are muscle weakness and atrophy (shrinkage).

Genetic screening is the most accurate way to diagnose SMA, which involves collecting a blood sample, letting it dry as a spot, then checking for mutations in the SMN1 gene. Early diagnosis and treatment can significantly alter the disease course and be lifesaving in some cases.

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However, extracting blood from children and adults can be moderately invasive and may require a visit to the clinic and/or blood collected by a healthcare professional.

A less invasive, alternate screening method using dried saliva spots has been developed, tested, and now described by scientists based at the Kobe University Graduate School of Medicine in Japan.

“To our knowledge, there has been no reported study employing [dried saliva spots] as genetic material to screen for SMA,” the team wrote.

While newborn screening for SMA “has begun in many countries, there remain many older children or adults with SMA in the population who were not detected in early infancy … [and] may be diagnosed late or may not be diagnosed,” the researchers added, delaying or denying them access to treatment.

Saliva was collected from 40 SMA patients and 21 healthy controls and air-dried as spots on a special filter paper. All patients had been genetically diagnosed with SMA using dried blood spot analysis.  

The center of the dried saliva spot was punched out, added to liquid, and then put through polymerase chain reaction or PCR, which copied and amplified a selected segment of the SMN1 gene known as exon 7 — commonly deleted or mutated in people with SMA. A second gene called CFTR was also amplified by PCR from the sample to serve as a reference.

A control sample contained segments of the SMN1 and CFTR genes in a preliminary analysis, whereas in a patient sample, the exon 7 segment was missing.

Samples were then subjected to melting curve analysis, in which DNA segments were gradually heated until the two strands of DNA in the double helix separated, which was detected by a fluorescence technique.

The control sample showed a melting peak at 77 C (about 171 F) for the SNM1 segment and another at 82 C (180 F) for CFTR. The patient sample had one melting peak for CFTR, “indicating the absence of SMN1 amplification,” the researchers wrote.

Calculated from the values at the top of the melting peak and reflecting the amount of amplified DNA, the SMN1/CFTR peak ratio (SCR) value was determined. The SCR value for the control sample remained stable for at least one month after saliva collection but decreased slightly beyond that time, “suggesting that SMN1 degraded more easily than CFTR,” the researchers noted.

Based on these findings, all samples were then tested within two weeks of saliva collected.

All of the control samples showed two melting peaks for SMN1 and CFTR, whereas all 40 samples from SMA patients had one melting peak for CFTR, which was consistent with the results of blood tests, the scientists said.

The SCR value of healthy controls was 0.59 while the SCR value of SMA patients was 0.13, which was a statistically significant difference. An SCR cutoff value of 0.2 was established to identify those with and without SMA from these data.

“We demonstrated the potential use of [dried saliva spot] sampling as a good alternative source for SMA detection using [PCR],” the scientists concluded. “The sample collection procedure is non-invasive, easy to handle, and requires no hospital visitation.”