Ethics statement

This study was approved by the Ethics Review Committee of Nagoya University Hospital (approval number:2023–0038), and was conducted in compliance with all of the provisions of the Declaration of Helsinki and the Ethical Guidelines for Medical and Health Research Involving Human Subjects endorsed by the Japanese government.

Prospective clinical study design

From August 3, 2023, to September 21, 2023, five patients with SMA attending the Department of Neurology, Nagoya University Hospital, and two of their parents were enrolled in this study. All saliva specimens were collected at Nagoya University Hospital after obtaining written informed consent from the patients. The collected saliva was mixed with a preservative solution (Biocomma, Japan), or soaked in medical-grade sponge sheets (Cenefom, Taiwan), anonymized, and transported to the Craif, Inc. laboratory. After performing the SMN1 copy number (CN) evaluation tests using saliva samples (raw saliva in sponge sheets for the novel assay and extracted DNA from saliva for the multiple ligation-dependent probe amplification (MLPA) assay), the test results were linked to patient information by collaborators at the Department of Neurology, Nagoya University Hospital, to evaluate the consistency between the results of the novel assay developed in this study and those of the MLPA method, the current gold standard for SMA diagnosis.

Sample sources

Genomic DNA (gDNA) specimens derived from patients with SMA and carriers were purchased from Coriell Institute (NJ, USA). Saliva specimens were collected from SMA patients attending the Department of Neurology, Nagoya University Hospital, and their parents (SMA carriers), placed on sponge sheets or mixed with a saliva preservation solution, and then transported to the Craif Inc. laboratory, as described above.

As positive control samples, commercially available genomic DNA from a single healthy human adult was purchased from BioChain Institute Inc. (CA, USA). Saliva samples were collected from healthy volunteers.

DNA isolation from saliva specimens from SMA patients

Genomic DNA was isolated from saliva specimens collected from healthy adults, patients with SMA, and SMA carriers using the QIAmp DNA Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions.

Specific primer sets for SMN1 and real-time qPCR

For SMN1-specific amplification, we first designed new primer sets, such that one of the five different sites between the SMN1 and SMN2 exon 7 peri-regions was included within the third base from the 3′ end of the forward and reverse primers, yielding over 100 combinations in total. Next, we evaluated SMN1 specificity and amplification efficiency compared with that of the control gene (RPPH1) for each primer combination by real-time qPCR. The combination with the best SMN1 specificity and amplification efficiency in our study was as follows: forward primer: 5′-TTCCTTTATTTTCCTTACAGGGTTCCAG-3′ and reverse primer: 5′-TTGTTTTACATTAACCTTTCAACTTTT-3 (S1 Fig). ‘ The primer set for RPPH1 was as follows; forward primer: 5′-CTTTGCCGGAGCTTGGA-3′ and reverse primer: 5′-GAGAGTAGTCTGAATTGGGTTATGA-3. ‘ We also used other previously reported primers targeting SMN1 used for SMA screening tests in Germany [13]; forward primer: 5′- TATTTTCCTTACAGGGTTCCAG-3′ and reverse primer: 5′- GCTGGCAGACTTACTCCTTAATTTAA-3 (S1 Fig). ‘

Real-time qPCR was performed using a QuantStudio 3 system (Life Technologies, CA, USA) with TaqMan Fast Advanced Master Mix (Life Technologies) at least in triplicate, with initial denaturation at 50°C for 2 min and 95°C for 10 min, followed by 40 cycles in two steps at 95°C for 15 s, 60°C for 1 min. A single acquisition of fluorescence signals was included in the 60°C step.

The FAM-labeled TaqMan MGB probe (Thermo Fisher Scientific, MA, USA) was used to detect both SMN1 and SMN2 in all qPCR experiments of this study; 5′-FAM-ACAAAATCAAAAAGAAGGAAGGTGCTCACA-NFQ-MGB-3′, which had the same sequence (but different reporter dye) as the probe used in the previous report [13]. We also used the NED-labeled TaqMan MGB probe (Thermo Fisher Scientific) to detect RPPH1; 5′-NED-ACCTCACCTCAGCCATTGAACTCAC-NFQ-MGB-3. ‘ Each reaction mixture (20 μL) contained 1 ng gDNA, 200 nM forward and reverse primers, and 200 nM TaqMan MGB probe. The relative SMN1 amplification efficiency compared to the control gene (RPPH1) for each primer set was calculated using the following formula:

Relative amplification efficiency (SMN1) = 2^{{Ct(RPPH1) − Ct(SMN1)}}

Conventional non-quantitative PCR

We applied a conventional PCR reaction involving the following steps: 30 cycles with denaturation at 98°C for 10 s, annealing at 62°C for 30 s, and extension at 72°C for 60 s. Each reaction mixture (20 μL) comprised 3 ng of gDNA, forward and reverse primers diluted to 1 μM, and 0.1 μL Taq polymerase (TaKaRa, Japan). The newly designed primers for SMN1 described above, as well as the previously reported primers used for SMA screening tests in Germany [13] were used to amplify the target region of SMN1 (target sizes: 200 and 82 bp, respectively). We also used the above-described primers to amplify the target region of RPPH1.

Agarose gel electrophoresis

Each PCR product (10 μL) was dyed with 1 μL of Midori Green (Nippon Genetics, Japan), separated on a 4% agarose gel at 7 V/cm for 60 min with 20 bp DNA ladder (TaKaRa, Japan) or a 2% agarose gel at 7 V/cm for 25 min with Gene Ladder 100 (Nippon Gene, Japan), and then visualized using a FAS-BG LED BOX (Nippon Genetics).

Single-stranded tag hybridization (STH) assay

We used a single-stranded tag hybridization (STH) assay provided by TBA Inc. (Sendai, Japan) to detect the PCR amplification products without electrophoresis [14]. In this assay, PCR amplification of the target genomic region was performed using the above reverse primers labeled with biotin at the 5′ end and the above forward primers labeled with single-tag DNA at the 5′ end (F-1 tag for SMN1 primer, F-4 tag for RPPH1 primer), which were modified by TBA. The PCR amplification product was then poured onto a membrane strip with avidin (biotin-binding protein)-coated latex (blue) and trapped by a strong hybridization reaction between the complementary tagged DNA immobilized in a linear shape on the strip. The trapped latex-labeled PCR amplification products turned blue, enabling visual identification of the presence or absence of the target genomic region in the specimen. All experiments were repeated at least thrice.

Multiple ligation-dependent probe amplification (MLPA) assay

All genomic DNA specimens extracted from the saliva of patients with SMA and carriers were tested using the commercially available SALSA MLPA P021 kit for SMA (MRC Holland, Netherlands), according to the manufacturer’s instructions. The data were analyzed using Coffalyser software provided by the manufacturer.

Statistical analysis

The statistical significance of the differences between two groups was analyzed using a paired Student’s t-test. All reported P values were two-sided, with p <0.05 considered statistically significant.

SMN1 specificity and amplification efficiency of the newly developed primers

First, we assessed the SMN1 specificity and amplification efficiency of the newly developed primer sets using qPCR. gDNA samples (1 ng) from healthy adults (SMN1 CN 2), patients with SMA (SMN1 CN 0), and SMA carriers (SMN1 CN 1) were amplified in the target region of the SMN1 and RPPH1 genes using the different primer pairs, and the combination considered to be the best was used in the following study (S1 File). The gDNA from healthy adults and SMA carriers showed SMN1 amplification, whereas the gDNA from patients with SMA showed no amplification, which was similar to the negative control sample (nuclease-free water, NFW) (Fig 1A). The relative amplification efficiency of SMN1 using gDNA (SMN1 CN 0, 1 and 2) compared to the control gene RPPH1 (CN 2) was as follows: SMN1 CN 0:0 ± 0 fold, CN 1:0.46 ± 0.06 fold, CN 2:0.92 ± 0.11 fold (mean ± SD). There was no significant difference in amplification efficiency between RPPH1 (CN 2) and SMN1 (CN 2) (Fig 1B).

Download:

• PNG
  larger image
• TIFF
  original image

Fig 1. SMN1 specificity and amplification efficiency of the newly designed primer pairs.

(a) Exemplar normalized FAM fluorescence versus cycle number resulting from SMN1 amplification of the gDNA of healthy adults, patients with spinal muscular atrophy (SMA), and carriers, using the newly designed primers. A negative control sample (nuclease-free water, NFW) was tested simultaneously. (b) The relative amplification efficiency of SMN1 (copy number, CN 0, 1, and 2) using the newly designed primers (n = 4, respectively). The fold changes relative to RPPH1 (CN 2) are shown. (c) Relative amplification efficiency of SMN1 (CN 2) using the newly designed primers and the previously reported SMA screening primers (n = 4). The fold changes relative to the previously reported primers are shown. Error bars represent ± standard deviation. *p <0.05 (t-test).

https://doi.org/10.1371/journal.pone.0308179.g001

Furthermore, we compared the amplification efficiency of the SMN1 target region between our primers (200 bp) and the previously reported primers (82 bp) used for SMA screening in Germany [13, 15, 16]. Although neither primer showed nonspecific SMN1 amplification, our new primers showed a significantly higher SMN1 amplification efficiency than the previously reported primers (28.1 ± 9.2 fold, p = 0.0049) (Fig 1C).

Visibility of PCR products by agarose gel electrophoresis

To develop a simple screening assay, we evaluated the visibility of PCR products by agarose gel electrophoresis. In the SMN1 amplification assay, neither the primers used in this study, nor the previously reported primers showed any amplified bands with gDNA (SMN1 CN 0). However, compared with the band of the control gene RPPH1, which was simultaneously amplified with the same amount of gDNA in a separate well, the SMN1 amplification efficiency of the previously reported primers was markedly lower, as suggested by the qPCR results described above (Fig 2A). In the simultaneous SMN1 and RPPH1 amplification assays performed using the newly developed primers, no SMN1 amplification bands were observed for gDNA (SMN1 CN 0). With other gDNA (SMN1 CN 1 and 2), both SMN1 and RPPH1 were sufficiently amplified. In contrast, in the same assay using previously reported primers, the amplified SMN1 bands were not detectable, unlike those for RPPH1 (Fig 2B).

Download:

• PNG
  larger image
• TIFF
  original image

Fig 2. Visibility of PCR products by agarose gel electrophoresis.

Agarose gel electrophoresis of PCR products using gDNA with SMN1 copy numbers (CN) of 0, 1, and 2 (with 20 bp ladder). (a) Target regions of SMN1 (200 or 82 bp) and RPPH1 (102 bp) were amplified separately using the same amount of input gDNA. (b) Target regions of SMN1 (200 or 82 bp) and RPPH1 (102 bp) were simultaneously amplified using the newly designed primers (left) and the previously reported primers (right). (c) Mixtures of gDNA (SMN1 CN 0) and gDNA (SMN1 CN 2) at various ratios were amplified under the same conditions as those described in (b). From left to right, the mixing ratios of gDNA (SMN1 CN 0) and gDNA (SMN1 CN2) were 1:1, 5:1, 10:1, 20:1, 50:1, and 100:1.

https://doi.org/10.1371/journal.pone.0308179.g002

Assuming the contamination of neonatal-derived specimens with maternal tissue, an additional experiment was performed using a mixture of gDNA (SMN1 CN 0) and gDNA (SMN1 CN 2) at various ratios. When there was less than 1% contamination of the normal gDNA (SMN1 CN 2) with gDNA (SMN1 CN 0), one band of the SMN1 became almost undetectable (Fig 2C).

Establishment of an innovative simple screening assay by combining SMN1-specific primers with an STH assay

To convert the non-quantitative conventional PCR assay described above into a system that can be performed in ordinary hospitals or clinics, we employed the STH assay (TBA, Japan). We confirmed that the same pattern of blue bands, indicating the presence of the target region, was detected in the STH assay using the newly developed primers as in agarose gel electrophoresis. However, the same assay using previously reported primers failed to identify blue bands corresponding to SMN1 (Fig 3A). To evaluate the effect of gDNA contamination in patients with SMA, STH assays were performed on the same samples as in Fig 2C, and at least 10% contamination of normal gDNA with gDNA from SMA patients did not affect the test results (S2 Fig).

Download:

• PNG
  larger image
• TIFF
  original image

Fig 3. Establishment of an innovative simple screening assay by combining SMN1-specific primers with STH assay.

(a) STH assay results of the same sample set shown in Fig 2B (with primers modified for STH assay). (b) Evaluation of direct PCR products using healthy saliva samples (left: agarose gel electrophoresis with 100 bp ladder; right: STH assay). The blue bands in the STH assay indicate the presence of the target gene region.

https://doi.org/10.1371/journal.pone.0308179.g003

To further simplify the test system, the possibility of performing direct PCR amplification using body fluids (saliva) was investigated. To add a stable amount of saliva to the reaction system, we first soaked a medical sponge sheet (0.8 mm thick) with saliva, punched out a section with a puncher (1.5 mm diameter), and added it to the PCR solution. PCR was then performed, followed by further evaluation using the STH assay. Consequently, we confirmed that the direct PCR method using saliva yielded the same results as the PCR method using the isolated gDNA (Fig 3B). STH assays were performed in over 50 independent experiments, and reproducibility was confirmed.

A pilot prospective study to evaluate the feasibility of the new SMA screening assay using saliva specimens from SMA patients and carriers

These results provide a framework for innovative SMA-screening assays. In this workflow, the DNA isolation step was omitted, and the evaluation step after PCR was completed in a very short time, requiring only approximately one hour in total (Fig 4A).

Download:

• PNG
  larger image
• TIFF
  original image

Fig 4. A pilot prospective study to evaluate the feasibility of the new SMA screening assay.

(a) This rapid and easy-to-use SMA screening assay was made possible by developing new SMN1-specific primers with high amplification efficiency of SMN1. (b) Clinical study flowchart for evaluating the accuracy of the new SMA screening assay. (c) and (d) Results of the MLPA assay using extracted gDNA (top) and our new assay using the corresponding raw saliva on a sponge sheet (bottom). (c: SMA patient; d: SMA carrier). The area in the yellow box shows the copy number ratio for SMN1 exons 7 and 8. (e) Results of the MLPA assay using extracted gDNA (top), our new assay using the corresponding raw saliva in the sponge sheet (middle), and saliva with preservation solution (bottom). In the middle panel, neither the SMN1 band nor the control RPPH1 band were confirmed, indicating an insufficient sample volume.

https://doi.org/10.1371/journal.pone.0308179.g004

We further conducted a prospective clinical study to validate the accuracy of this assay. Five patients with SMA attending the Department of Neurology, Nagoya University Hospital, and two of their parents who were SMA carriers were enrolled in this study. The collected saliva specimens were anonymously randomized and transported to the laboratory of Craif Inc., where each specimen was analyzed for SMN1 exon 7 status by our new assay using raw saliva and by the MLPA assay using gDNA extracted from saliva. Each result was analyzed and linked to patient information by collaborators at the Department of Neurology, Nagoya University Hospital, to evaluate the consistency between the assays (Fig 4B). In all cases, the results of our new assay were consistent with the results of the MLPA assay, although saliva added with preservation buffer (total 1 μL) was used for our assay in one case as the amount of saliva soaked in a sponge sheet was insufficient (Fig 4C–4E).