Proximal spinal muscular atrophy 5q (5q-SMA) is autosomal recessive neuromuscular disease, characterized by the loss of motor neurons in the anterior horns of the spinal cord, that leads to progressive muscle weakness and skeletal muscle atrophy. 5q-SMA is caused by the mutation in the survival motor neuron 1 gene (SMN1), situated on the chromosome 5q13. The incidence of SMA is 1 per 6000–10,000 newborns [1]. Rapid and irreversible loss of the motor neurons in SMA begins during the first 3 months of life, and 95% of motor neurons are affected in the patients under the age of 6 months [2]. Delayed molecular genetic diagnosis of SMA leads to untimely initiation of the treatment, which causes a limited clinical effectiveness of therapeutical intervention.

High incidence of the disease is related to the innate genetic instability of 5q13 region due to the presence of an inverted sequence of functional genes SMN1, SERF1A, NAIP, GTF2H2A and their centromeric pseudogenes SMN2, SERF1B, NAIP∆5, GTF2H2B [1]. In 95% of cases, 5q-SMA results from the homozygous loss of the SMN1 gene. The remaining cases of the disease occur in compound heterozygotes when the loss of the SMN1 gene on one allele is combined with point mutations in the SMN1 gene on another allele [3]. Today these forms of SMA are not detected in neonatal testing programs in any country of the world [4]. The prevalence of heterozygous loss of the SMN1 gene in the Russian population is 1 : 36 [5]. Besides, the duplication of the SMN1 is a frequent finding in general population, but it is not associated with SMA [1]. The pseudogene of the SMN1 is the SMN2 gene, which also consists of 9 exons and encodes the same SMN protein. The genes differ in 5 nucleotide in region from the 6-th intron to the 8-th exon. The key difference is exchange of cytosine to thymine in the 7-th exon of the SMN2 gene (c. 840 C > T), which leads to the loss of the binding site for the exon splicing enhancer and the formation of the binding site for the exon silencer. As a result of this nucleotide change, the 7-th exon is excluded in 90% of SMN2 transcripts. The resultant SMN∆7 protein becomes functionally defective and unstable, which causes its rapid degradation by the ubiquitin-proteasome system [1]. Nevertheless 10% of SMN protein, produced from SMN2 gene, retains 7-th exon and can perform its function [6]. Therefore, copy number variation of SMN2 can modify the severity of the disease in persons with SMN1 deletion. SMA type 0 is characterized by absence of SMN1 gene and 1 copy of SMN2 gene. This SMA form presents antenatally with severely decreased fetal movements. SMA type I (Werdnig–Hoffmann disease) is the most frequent type of SMA, accounting for approximately half of patients (SMN2 gene — 2–3 copies) [3, 7]. Affected infants appear normal at birth, but subsequently develop hypotonia, delayed motor milestones, and feeding difficulties within the first 6 months of life. Infants with SMA type I never sit independently.  In the less severe SMA type II (Dubowitz disease), children present between 6 and 18 months of age and are able to sit independently, but never walk. The majority of these patients survive into adulthood (SMN2 gene — 2-4 copes). SMA type III (Kugelberg-Welander disease) includes patients who walk at some point (and for any period of time) during childhood (SMN2 gene — 3–5 copies). SMA type IV is the mildest and least common form of SMA with onset in adulthood [3, 7].

The loss of functional SMN1 genes plays the main role in the pathogenesis of this disease. There are several types of the SMN1 gene loss: complete deletion, conversion of the SMN1 gene into SMN2, the formation of hybrid SMN1/SMN2 structures, and loss of only a few exons of the SMN1 gene (partial deletion). The complete deletion of the SMN1 gene occurs without an increase in the number of copies of the SMN2 gene. Partial deletion is characterized by the isolated loss of several exons, including 7 exon, of the SMN1 gene without changes of the number of copies of these exons in the SMN2 gene [8]. During the conversion, loss of SMN1 gene is accompanied by an increase in the number of copies of the SMN2 gene [9]. The formation of chimeric SMN1/SMN2 genes, in which there are sites belonging to both the SMN1 gene and the SMN2 pseudogene and which are characterized by an homozygous loss SMN1's 7 exons and heterozygous loss SMN1's 8 exon and unequal ratio of 7 and 8 exons in the SMN2 gene, is another mechanism for the loss of the SMN1 gene [10]. It should be noted that in patients with SMA, homozygous loss of the SMN1 gene can be associated with both the individual types of "deletion" described, and with combined cases.

Restitution of SMN protein levels in patients with proximal SMA is valuable treatment option. Experimental models have shown that the time of switching off the SMN protein determined the life expectancy — the earlier the shutdown occurs, the worse observable survival [11]. Moreover, early reconstitution of SMN protein in SMA-models leaded to almost complete recovery [12]. In accordance with this data, presymptomatic therapy in newborns who have a pathological aberration in the SMN1 gene has been investigated. The phase 2 NURTURE study demonstrated substantial clinical benefit of presymptomatic nusinersen therapy in infants with two or three copies of the SMN2 gene [13]. In a multicenter phase III clinical trial for gene replacement therapy (SPR1NT), which was aimed at studying the efficiency and the safety of onasemnogene abeparvovec treatment in children with biallelic deletion of the SMN1 gene and 3 copies of the SMN2 gene (SMA type 2, respectively), recently published results also confirmed high clinical efficiency of presymptomatic treatment of children with SMA [14]. The available therapy and the possibility of effective usage of disease-modifying treatment in presymptomatic stage of disorder currently justify the screening of newborns for 5q-SMA. Australia, Belgium, Canada, Germany, Italy, Japan,

Norway, the Netherlands, Poland and Taiwan, as well as 46 states in the United States have introduced mandatory screening of newborns for SMA. Mandatory screening for SMA for all newborns was launched in 2023 in the Russian Federation [15].

Multiplex ligation probes amplification (MLPA) is the "gold standard" for the diagnosis of SMA, which is able to detect not only homozygous deletions of the SMN1 gene, but also other types of numerical changes in the SMN1 and SMN2 genes. However, this method is not applicable as a screening method due to the complexity and high cost [16, 17]. The most acceptable screening method for the homozygous loss of the SMN1 gene is various modifications of the polymerase chain reaction method with real-time signal detection (RT-PCR), detecting the homozygous loss of the 7-th exon of the SMN1 gene. The benefits of this approach have been confirmed by a number of studies and meta-analyses [18, 19, 20]. Taking into account the high clinical relevance of early diagnosis of SMA, as well as the beginning of mandatory screening for SMA of all newborns in the Russian Federation from 2023, there is a need for diagnostic molecular genetic tests that would allow identifying all possible forms of homozygous loss of the SMN1 gene with high accuracy. It is also should be noted, that one of the most important requirement for neonatal screening test for SMA is possibility to detect homozygous loss of SMN1 gene in dried blood spots (DBS) samples.

Russian company «DNA-Technology» recently issued new PCR kit for molecular screening for SMA in newborn, but extended approbation of this kit for detection of all form of SMN1 gene loss, detected by MLPA test, is highly needed.

The aim of this study is to compare a PCR-based screening test for detection of homozygous SMN1 loss with multiple ligation pro be amplification in patients with spinal muscular atrophy and other numerical changes of SMN1 gene.

METHODS

Sample collection and DNA extraction and purification

DNA samples extracted and purified from the whole blood (Group 1)

To evaluate possibility of detection of different types of SMN1 copy number variation the cohort consisted of 206 samples from Biobank of Centre of Molecular Medicine of Pavlov State Medical University (Saint-Petersburg, Russia) was collected. Patients with clinical suspicion of SMA were referred to laboratories of Pavlov Medical University from 2019 to 2021. Genomic DNA was extracted from peripheral blood using ExtractDNA Blood & Cells (Evrogen; Russia) according to the manufacturer’s instructions, and adjusted to a final concentration of 50 ng/μl. Copy number of SMN1 and SMN2 genes had been routinely evaluated with multiplex ligation-dependent probe amplification (MLPA) assay using SALSA® MLPA® Probemix P060-B2 SMA Carrier (MRC-Holland®; The Netherlands) in accordance with manufacturer’s instructions.

All the samples were divided into 7 subgroups according to SMN1 and SMN2 genotype status. Subgroup 1 «Normal» (54 samples): 2 copies of SMN1 and SMN2 genes — so called «Reference genotype». Subgroup 2 «Deletion» (9 samples): absence of SMN1 gene without increase of SMN2 copy number. Subgroup 3 «Conversion» (47 samples): absence of SMN1 gene with increase of SMN2 copy number. Subgroup 4 «Hybrid» (9 samples): homozygous deletion of exon 7 and heterozygous deletion of exon 8 SMN1 gene with increase of exon 7 SMN2 copy number. Subgroup 5 «Carrier» (51 samples): heterozygous deletion of SMN1 gene. Subgroup 6 «Duplication» (23 samples): 3 copies of SMN1 gene. Subgroup 7 «Other» (13 samples): different combinations of SMN1 and SMN2 copy number that did not meet the criteria of other subgroups. Confidence interval for diagnostic sensitivity and specificity was chosen as 95% according to MedCalc® instrument (https://www.medcalc.org/calc/diagnostic_test.php) (figure).

DNA samples extracted and purified from DBS (Group 2)

To confirm the possibility of detecting a homozygous loss of SMN1 in DNA isolated from DBS samples, a group of 135 patients, whose blood was transported in a dried state on a membrane, was collected from Biobank of Centre of Molecular Medicine of Pavlov State Medical University (Saint-Petersburg, Russia). Patients with clinical suspicion of SMA were referred to laboratories of Pavlov Medical University from 2019 to 2021. Genomic DNA was extracted from DBS using PREP-CITO DBS (DNA-Technology; Russia) according to the manufacturer’s instructions. For all patients copy number of SMN1 and SMN2 genes was evaluated by SALSA® MLPA® Probemix P060-B2 SMA Carrier (MRC-Holland®, Netherlands) in accordance with protocol for DBS membrane.

All the samples were divided into 6 subgroups: «Normal», «Deletion», «Conversion», «Hybrid», «Carrier», «Other» (tab. 1).

Multiplex ligation-dependent probe amplification  (MLPA) assay

The MLPA assay was performed using SALSA® MLPA® Probemix P060-B2 SMA Carrier (MRC-Holland®; The Netherlands) following the manufacturer’s instructions for DNA extracted and purified from whole blood (Group 1) and from DBS membrane (Group 2). The commercial kit contains 17 reference probes and 4 specific probes detecting sequences presented in exon 7 and 8 of either SMN1 and SMN2 genes. MLPA products were analyzed with ABI 3500 genetic analyzer (Thermo Fisher Scientific®; USA). The relative peak height of each sample was calculated and compared with normal controls using MLPA analysis application by GeneMarker® software (SoftGenetics®; USA). The evaluation criteria of deletion/duplication were based on the MLPA kit instructions.

PCR-based screening test

The NeoScreen SMA/TREC/KREC was performed for all patients from Group 1 and Group 2. NeoScreen SMA/TREC/ KREC Real-time PCR Detection Kit (DNA-Technology, Russia) is intended to detect exon 7 of SMN1 gene homozygous deletion and assess levels of T cell receptor excision circles (TREC) and kappa-deleting recombination excision circle (KREC) in newborns’ dried blood spots (DBS) of whole blood for spinal muscular atrophy and primary immunodeficiencies screening by real-time PCR. The method is based on amplification of TREC, KREC, exon 7 of SMN1 gene, and a fragment of the normalizing gene LTC4S (or endogenous internal control (IC), a single-copy genomic locus of Leukotriene C4 Synthase gene) by multiplex polymerase chain reaction (PCR). The use of several fluorescent dyes allows to simultaneously register the results of different amplification reactions taking place in the same tube. Table 2 (tab. 2) shows detection channels of amplification products.

NeoScreen SMA/TREC/KREC assay was carried out using DTprime Real-time Detection Thermal Cycler (DNA-Technology; Protvino, Russia) by following the manufacturer’s instructions (tab. 3). To assess the performance of the assay a positive control (included in PCR Detection Kit) and a negative control were included in each PCR-run. Positive control C+ № 1, containing DNA plasmids with specified targets — TREC, KREC, SMN1, IC, in equal concentration, is intended to evaluate PCR efficiency. Positive control C+ № 2 with plasmid equivalent exon 7 of SMN2 gene allows to assess blocking of SMN2 amplification (control of the SMN1 amplification specificity).

Analysis of SMN1 exon 7 deletion is based on the estimation of the indicator cycle difference (∆ Cp) between the Cp of SMN1 (Hex channel) and IC (Су5 channel) (∆Ср = Ср (Hex) – Cp (Cy5)).

Evaluation of homozygous deletion of exon 7 of SMN1 gene was performed according to the PCR Detection Kit instruction for result’s analysis algorithm (performed automatically by the thermocycler software).

RESULTS

The PCR-based screening method was used on 341 samples with known SMN1 and SMN2 copy numbers (DNA extracted and purified from whole blood — Group 1, 206 samples; DNA extracted and purified from DBS membrane — Group 2, 135 samples). The MLPA assay was used as a reference method (tab. 4, tab. 5). The PCR-based test results was considered true positive if homozygous deletion of exon 7 SMN1 gene is detected for samples with 0 copies of exon 7 SMN1 gene, established by MLPA assay. Presence of at least one exon 7 of SMN1 gene identified by MLPA assay and detection of exon 7 SMN1 signal by test was considered true negative.

The results in Group 1 were as follows. In subgroup 1 «Normal» 54 participants (26%) carried 2 copies of exon 7 SMN1 gene and all samples were true negative by PCR-based screening. In subgroup 2 «Deletion», subgroup 3 «Conversion», subgroup 4 «Hybrid» all samples were true positive (n = 65, 32%). In subgroup 5 «Carrier» all 51 participants (25%) had single copy of searched exon and all of the were true negative. In subgroup 6 «Duplication» all 23 samples (11%) carries 3 copies of exon 7 SMN1 were true negative by PCR protocol. Finally subgroup 7 «Other» consisted of 13 probes with different SMN1 and SMN2 exon count. 6 samples (3%) carried more than 1 copy of exon 7 SMN1 gene and were true negative. 5 samples (2%) in group 8 had single copy of SMN1 gene and also were true negative. And 2 probes did not contain any of exon 7 SMN1 and were true positive (1%). See results in tab. 4.

The results in Group 2 are presented below. In subgroup «Normal» 99 participants (73%) had 2 copies of exon 7 SMN1 gene and all samples were true negative by PCR-based screening. In subgroup «Deletion», subgroup «Conversion», subgroup «Hybrid» all samples were true positive by PCRbased screening (n = 18, 13,3%). Group 2 did not contain samples with SMN1 gene duplication according to MLPA assay. In subgroups «Carrier» all 9 samples (7%)  were true negative. In subgroup «Other» 1 probe (0,7%) did not contain any of exon 7 SMN1 and were true positive. And 8 samples (6%) had single copy of SMN1 gene and were true negative by PCR protocol. See results in tab. 5.

DISCUSSION

Spinal muscular atrophy is an autosomal recessive neurodegenerative disease, characterized by progressive skeletal muscle weakness. A laboratory test for neonatal screening of SMA should detect all types of SMN1 loss in DNA isolated and purified from whole samples and from DBS samples.

There are several methodological approaches for homozygous SMN1 loss, however, different modifications of real-time PCR proved to be the most suitable for neonatal SMA screening [16, 21]. Approaches, based on PCR, are highly robust, characterized by a very low number of false negative and false positive results, simple in handling, have a low cost and compatible with DBS samples [2, 22]. Screening for homozygous SMN1 loss with test is based on the detection of homozygous loss of the 7 exon of SMN1 gene by real-time PCR [6].

SMN1 gene is located in a highly unstable genome region, which is saturated with repeating inverted genes and Alu-sequences. Due to that fact, there is a wide spectrum of genetic aberrations, characterized by variation in copy number of SMN1. In order to assess the specificity of detection of homozygous SMN1 loss with PCR-based kit in Group 1, which consist of  DNA samples extracted and purified from the whole blood, 139 samples with 1–3 copies of SMN1 gene were selected (subgroups «Normal», «Carrier», «Duplication», 11 samples from subgroup «Other»). In all 139 samples screened for homozygous SMN1 loss with PCR-based test results were negative. Predictably, this emphasizes that a benign change in the copy number of the SMN1 genes does not affect the effectiveness of detection of homozygous SMN1 loss.

Detection of different types of homozygous loss of SMN1 is the main task of neonatal SMA screening. In Group 1 PCR-based kit was able to detect homozygous SMN1 loss in all samples (n = 67), included in the study. PCR-based test showed high sensitivity and specificity for detection of SMN1 loss. This is in line with a number of studies, dedicated to the methodological problem of SMA screening [2, 23].

To confirm the effectiveness of the kit to detect a homozygous loss of SMN1 gene in DNA isolated from DBS samples, patients in Group 2 were selected. For all patients DNA was isolated and purified from DBS samples by PREP-CITO DBS kit for subsequent evaluations by PCR-based test. For all patient MLPA analysis was also performed in parallel. In all samples with 1–3 copies of the SMN1 gene (n = 116), the results of the test were negative for homozygous loss of SMN1 gene. This highlights that a benign change in the copy number of SMN1 genes does not affect the specificity of detecting homozygous loss of SMN1 in DNA isolated from DBS samples with PCR-based test. For samples with 0 copies of exon 7 SMN1 gene results of screening of homozygous SMN1 loss by kit were positive for all patients.

A limitation of the PCR-based test is the inability to detect rare forms of 5q-SMA associated with  heterozygous loss of one copy of the SMN1 gene and pathogenic variants on the second copy of the gene (compound heterozygosity). According to research results, the prevalence of this form of 5q-SMA is up to 5% of all cases [3]. However, it should be noted that at the moment this subtype of the disease are not detected in neonatal testing programs in any country of the world [4]. When clinical signs and symptoms of 5q-SMA are identified and screening is negative for homozygous SMN1 gene loss, an extended study is required to detect SMN1 gene loss at one allele in combination with SMN1 gene point mutations on another allele.

CONCLUSIONS

This pilot study showed, that PCR-based screening test is able to detect homozygous SMN1 gene loss in both DNA samples extracted and purified from the whole blood and samples DNA isolated from DBS. Also, according to the results of this study, the test detects all possible molecular forms of homozygous SMN1 gene loss.