Cell-mediated cytotoxicity within CSF and brain parenchyma in spinal muscular atrophy unaltered by nusinersen treatment.

5q-associated spinal muscular atrophy (SMA) is a motoneuron disease caused by mutations in the survival motor neuron 1 (SMN1) gene. Adaptive immunity may contribute to SMA as described in other motoneuron diseases, yet mechanisms remain elusive. Nusinersen, an antisense treatment, enhances SMN2 expression, benefiting SMA patients. Here we have longitudinally investigated SMA and nusinersen effects on local immune responses in the cerebrospinal fluid (CSF) - a surrogate of central nervous system parenchyma. Single-cell transcriptomics (SMA: N = 9 versus Control: N = 9) reveal NK cell and CD8+ T cell expansions in untreated SMA CSF, exhibiting activation and degranulation markers. Spatial transcriptomics coupled with multiplex immunohistochemistry elucidate cytotoxicity near chromatolytic motoneurons (N = 4). Post-nusinersen treatment, CSF shows unaltered protein/transcriptional profiles. These findings underscore cytotoxicity's role in SMA pathogenesis and propose it as a therapeutic target. Our study illuminates cell-mediated cytotoxicity as shared features across motoneuron diseases, suggesting broader implications.

Systemic LPS Administration Stimulates the Activation of Non-Neuronal Cells in an Experimental Model of Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by deficiency of the survival motor neuron (SMN) protein. Although SMA is a genetic disease, environmental factors contribute to disease progression. Common pathogen components such as lipopolysaccharides (LPS) are considered significant contributors to inflammation and have been associated with muscle atrophy, which is considered a hallmark of SMA. In this study, we used the SMNΔ7 experimental mouse model of SMA to scrutinize the effect of systemic LPS administration, a strong pro-inflammatory stimulus, on disease outcome. Systemic LPS administration promoted a reduction in SMN expression levels in CNS, peripheral lymphoid organs, and skeletal muscles. Moreover, peripheral tissues were more vulnerable to LPS-induced damage compared to CNS tissues. Furthermore, systemic LPS administration resulted in a profound increase in microglia and astrocytes with reactive phenotypes in the CNS of SMNΔ7 mice. In conclusion, we hereby show for the first time that systemic LPS administration, although it may not precipitate alterations in terms of deficits of motor functions in a mouse model of SMA, it may, however, lead to a reduction in the SMN protein expression levels in the skeletal muscles and the CNS, thus promoting synapse damage and glial cells' reactive phenotype.

Transcriptome- and proteome-wide effects of a circular RNA encompassing four early exons of the spinal muscular atrophy genes.

Spinal muscular atrophy (SMA) genes, SMN1 and SMN2 (hereinafter referred to as SMN1/2), produce multiple circular RNAs (circRNAs), including C2A-2B-3-4 that encompasses early exons 2A, 2B, 3 and 4. C2A-2B-3-4 is a universally and abundantly expressed circRNA of SMN1/2. Here we report the transcriptome- and proteome-wide effects of overexpression of C2A-2B-3-4 in inducible HEK293 cells. Our RNA-Seq analysis revealed altered expression of ~ 15% genes (4172 genes) by C2A-2B-3-4. About half of the affected genes by C2A-2B-3-4 remained unaffected by L2A-2B-3-4, a linear transcript encompassing exons 2A, 2B, 3 and 4 of SMN1/2. These findings underscore the unique role of the structural context of C2A-2B-3-4 in gene regulation. A surprisingly high number of upregulated genes by C2A-2B-3-4 were located on chromosomes 4 and 7, whereas many of the downregulated genes were located on chromosomes 10 and X. Supporting a cross-regulation of SMN1/2 transcripts, C2A-2B-3-4 and L2A-2B-3-4 upregulated and downregulated SMN1/2 mRNAs, respectively. Proteome analysis revealed 61 upregulated and 57 downregulated proteins by C2A-2B-3-4 with very limited overlap with those affected by L2A-2B-3-4. Independent validations confirmed the effect of C2A-2B-3-4 on expression of genes associated with chromatin remodeling, transcription, spliceosome function, ribosome biogenesis, lipid metabolism, cytoskeletal formation, cell proliferation and neuromuscular junction formation. Our findings reveal a broad role of C2A-2B-3-4, and expands our understanding of functions of SMN1/2 genes.

Unveiling the genetic tapestry: Rare disease genomics of spinal muscular atrophy and phenylketonuria proteins.

Rare diseases, defined by their low prevalence, present significant challenges, including delayed detection, expensive treatments, and limited research. This study delves into the genetic basis of two noteworthy rare diseases in Saudi Arabia: Phenylketonuria (PKU) and Spinal Muscular Atrophy (SMA). PKU, resulting from mutations in the phenylalanine hydroxylase (PAH) gene, exhibits geographical variability and impacts intellectual abilities. SMA, characterized by motor neuron loss, is linked to mutations in the survival of motor neuron 1 (SMN1) gene. Recognizing the importance of unveiling signature genomics in rare diseases, we conducted a quantitative study on PAH and SMN1 proteins of multiple organisms by employing various quantitative techniques to assess genetic variations. The derived signature-genomics contributes to a deeper understanding of these critical genes, paving the way for enhanced diagnostics for disorders associated with PAH and SMN1.

Results of a Pilot External Quality Assessment Scheme for Genetic Testing of Newborns with Spinal Muscular Atrophy.

BACKGROUND: This study aims to evaluate the ability of laboratories to perform spinal muscular atrophy (SMA) genetic testing in newborns based on dried blood spot (DBS) samples, and to provide reference data and advance preparation for establishing the pilot external quality assessment (EQA) scheme for SMA genetic testing of newborns in China. METHODS: The pilot EQA scheme contents and evaluation principles of this project were designed by National Center for Clinical Laboratories (NCCL), National Health Commission. Two surveys were carried out in 2022, and 5 batches of blood spots were submitted to the participating laboratory each time. All participating laboratories conducted testing upon receiving samples, and test results were submitted to NCCL within the specified date. RESULTS: The return rates were 75.0% (21/28) and 95.2% (20/21) in the first and second surveys, respectively. The total return rate of the two examinations was 83.7% (41/49). Nineteen laboratories (19/21, 90.5%) had a full score passing on the first survey, while in the second survey twenty laboratories (20/20, 100%) scored full. CONCLUSIONS: This pilot EQA survey provides a preliminary understanding of the capability of SMA genetic testing for newborns across laboratories in China. A few laboratories had technical or operational problems in testing. It is, therefore, of importance to strengthen laboratory management and to improve testing capacity for the establishment of a national EQA scheme for newborn SMA genetic testing.

Early spinal muscular atrophy treatment following newborn screening: A 20-month review of the first Italian regional experience.

OBJECTIVES: Mandatory newborn screening (NBS) for spinal muscular atrophy (SMA) was implemented for the first time in Italy at the end of 2021, allowing the identification and treatment of patients at an asymptomatic stage. METHODS: DNA samples extracted from dried blood spot (DBS) from newborns in Apulia region were analysed for SMA screening by using a real-time PCR-based assay. Infants harbouring homozygous deletion of SMN1 exon 7 confirmed by diagnostic molecular tests underwent clinical and neurophysiological assessment and received a timely treatment. RESULTS: Over the first 20 months since regional NBS introduction, four out of 42,492 (0.009%) screened children were found to carry a homozygous deletion in the exon 7 of SMN1 gene, with an annual incidence of 1:10,623. No false negatives were present. Median age at diagnosis was 7 days and median age at treatment was 20.5 days. Three of them had two copies of SMN2 and received gene therapy, while the one with three SMN2 copies was treated with nusinersen. All but one were asymptomatic at birth, showed no clinical signs of disease after a maximum follow-up of 16 months and reached motor milestones appropriate with their age. The minimum interval between diagnosis and the treatment initiation was 9 days. INTERPRETATION: The timely administration of disease-modifying therapies prevented presymptomatic subjects to develop disease symptoms. Mandatory NBS for SMA should be implemented on a national scale.

Optimized MLPA workflow for spinal muscular atrophy diagnosis: identification of a novel variant, NC_000005.10:g.(70919941_70927324)del in isolated exon 1 of SMN1 gene through long-range PCR.

BACKGROUND: Spinal muscular atrophy (SMA) is a rare autosomal recessive hereditary neuromuscular disease caused by survival motor neuron 1 (SMN1) gene deletion or mutation. Homozygous deletions of exon 7 in SMN1 result in 95% of SMA cases, while the remaining 5% are caused by other pathogenic variants of SMN1. METHODS: We analyzed two SMA-suspected cases that were collected, with no SMN1 gene deletion and point mutation in whole-exome sequencing. Exon 1 deletion of the SMN gene was detected using Multiplex ligation-dependent probe amplification (MLPA) P021. We used long-range polymerase chain reaction (PCR) to isolate the SMN1 template, optimized-MLPA P021 for copy number variation (CNV) analysis within SMN1 only, and validated the findings via third-generation sequencing. RESULTS: Two unrelated families shared a genotype with one copy of exon 7 and a novel variant, g.70919941_70927324del, in isolated exon 1 of the SMN1 gene. Case F1-II.1 demonstrated no exon 1 but retained other exons, whereas F2-II.1 had an exon 1 deletion in a single SMN1 gene. The read coverage in the third-generation sequencing results of both F1-II.1 and F2-II.1 revealed a deletion of approximately 7.3 kb in the 5' region of SMN1. The first nucleotide in the sequence data aligned to the 7385 bp of NG_008691.1. CONCLUSION: Remarkably, two proband families demonstrated identical SMN1 exon 1 breakpoint sites, hinting at a potential novel mutation hotspot in Chinese SMA, expanding the variation spectrum of the SMN1 gene and corroborating the specificity of isolated exon 1 deletion in SMA pathogenesis. The optimized-MLPA P021 determined a novel variant (g.70919941_70927324del) in isolated exon 1 of the SMN1 gene based on long-range PCR, enabling efficient and affordable detection of SMN gene variations in patients with SMA, providing new insight into SMA diagnosis to SMN1 deficiency and an optimized workflow for single exon CNV testing of the SMN gene.

Carrier Screening and Diagnosis for Spinal Muscular Atrophy Using Droplet Digital PCR Versus MLPA: Analytical Validation and Early Test Outcome.

Background: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular life-threatening disorder. Owing to high carrier frequency, population-wide SMA screening to quantify the copy number of SMN gene is recommended by American College of Medical Genetics and Genomics. An accurate, reliable, short runaround time and cost-effective method may be helpful in mass population screening for SMA. Methods: Multiplex ligation-dependent probe amplification (MLPA) is a gold standard to estimate the copy number variation (CNV) for SMN1 and SMN2 genes. In this study, we validated droplet digital polymerase chain reaction (ddPCR) for the determination of CNV for both SMN1 and SMN2 exon 7 for a diagnostic purpose. In total, 66 clinical samples were tested using ddPCR, and results were compared with the MLPA as a reference test. Results: For all samples, CNV for SMN1 and SMN2 exon 7 was consentaneous between ddPCR and MLPA test results (κ = 1.000, p < 0.0001). In addition, ddPCR also showed a significant acceptable degree of test repeatability, coefficient of variation < 4%. Conclusion: ddPCR is expected to be utilitarian for CNV detection for carrier screening and diagnosis of SMA. ddPCR test results for CNV detection for SMN1/SMN2 exon 7 are concordant with the gold standard. ddPCR is a more cost-effective and time-saving diagnostic test for SMA than MLPA. Furthermore, it can be used for population-wide carrier screening for SMA.

No significant sex differences in incidence or phenotype for the SMNΔ7 mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive disease that affects 1 out of every 6,000-10,000 individuals at birth, making it the leading genetic cause of infant mortality. In recent years, reports of sex differences in SMA patients have become noticeable. The SMNΔ7 mouse model is commonly used to investigate pathologies and treatments in SMA. However, studies on sex as a contributing biological variable are few and dated. Here, we rigorously investigated the effect of sex on a series of characteristics in SMA mice of the SMNΔ7 model. Incidence and lifespan of 23 mouse litters were tracked and phenotypic assessments were performed at 2-day intervals starting at postnatal day 6 for every pup until the death of the SMA pup(s) in each litter. Brain weights were also collected post-mortem. We found that male and female SMA incidence does not differ significantly, survival periods are the same across sexes, and there was no phenotypic difference between male and female SMA pups, other than for females exhibiting lesser body weights at early ages. Overall, this study ensures that sex is not a biological variable that contributes to the incidence ratio or disease severity in the SMNΔ7 mouse model.

Diagnosis of Challenging Spinal Muscular Atrophy Cases with Long-Read Sequencing.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder primarily caused by the deletion or mutation of the survival motor neuron 1 (SMN1) gene. This study assesses the diagnostic potential of long-read sequencing (LRS) in three patients with SMA. For Patient 1, who has a heterozygous SMN1 deletion, LRS unveiled a missense mutation in SMN1 exon 5. In Patient 2, an Alu/Alu-mediated rearrangement covering the SMN1 promoter and exon 1 was identified through a blend of multiplex ligation-dependent probe amplification, LRS, and PCR across the breakpoint. The third patient, born to a consanguineous family, bore four copies of hybrid SMN genes. LRS determined the genomic structures, indicating two distinct hybrids of SMN2 exon 7 and SMN1 exon 8. However, a discrepancy was found between the SMN1/SMN2 ratio interpretations by LRS (0:2) and multiplex ligation-dependent probe amplification (0:4), which suggested a limitation of LRS in SMA diagnosis. In conclusion, this newly adapted long PCR-based third-generation sequencing introduces an additional avenue for SMA diagnosis.

Rare homozygous disease-associated sequence variants in children with spinal muscular atrophy: a phenotypic description and review of the literature.

5q-associated spinal muscular atrophy (SMA) is the most common autosomal recessive neurological disease. Depletion in functional SMN protein leads to dysfunction and irreversible degeneration of the motor neurons. Over 95 % of individuals with SMA have homozygous exon 7 deletions in the SMN1 gene. Most of the remaining 4-5 % are compound heterozygous for deletion and a disease-associated sequence variant in the non-deleted allele. Individuals with SMA due to bi-allelic SMN1 sequence variants have rarely been reported. Data regarding their clinical phenotype, disease progression, outcome and treatment response are sparse. This study describes six individuals from three families, all with homozygous sequence variants in SMN1, and four of whom received treatment with disease-modifying therapies. We also describe the challenges faced during the diagnostic process and intrafamilial phenotypic variability observed between siblings.

Development of a low-cost and accurate carrier screening method for spinal muscular atrophy in developing countries.

Heterozygous carriers of the survival of motor neuron 1 (SMN1) gene deletion in parents account for approximately 95% of neonatal spinal muscular atrophy cases. Given the severity of the disease, professional organizations have recommended periconceptional spinal muscular atrophy carrier screening to all couples, regardless of race or ethnicity. However, the prevalence of screening activities in mainland China remains suboptimal, mainly attributed to the limitations of the existing carrier screening methods. Herein, we aimed to develop a low-cost, accessible, and accurate carrier screening method based on duplex droplet digital PCR (ddPCR), to cover a wider population in developing countries, including China. The receiver operating characteristic curve was used to determine the cut-off value of SMN1 copy numbers. Performance validation was conducted for linearity, precision, and accuracy. In total, 482 cases were considered to validate the concordance between the developed ddPCR assay and multiplex ligation-dependent probe amplification. Linear correlations were excellent between the expected concentration of the reference gene and the observed values (R2 > 0.99). Both the intra- and inter-assay precision of our ddPCR assays were less than 6.0%. The multiplex ligation-dependent probe amplification and ddPCR results were consistent in 480 of the 482 cases (99.6%). Two cases with multiplex ligation-dependent probe amplification, suggestive of two copies of SMN1 exon 7, were classified into three copies by ddPCR analysis. The overall correct classification of the samples included in our ddPCR assay was 100%. This study demonstrates that an appropriate cut-off value is an important prerequisite for establishing a semi-quantitative method to determine the SMN1 copy numbers. Compared to conventional methods, our ddPCR assay is low-cost, highly accurate, and has full potential for application in population spinal muscular atrophy carriers screening.

Depletion of SMN protein in mesenchymal progenitors impairs the development of bone and neuromuscular junction in spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neuromuscular disorder characterized by the deficiency of the survival motor neuron (SMN) protein, which leads to motor neuron dysfunction and muscle atrophy. In addition to the requirement for SMN in motor neurons, recent studies suggest that SMN deficiency in peripheral tissues plays a key role in the pathogenesis of SMA. Using limb mesenchymal progenitor cell (MPC)-specific SMN-depleted mouse models, we reveal that SMN reduction in limb MPCs causes defects in the development of bone and neuromuscular junction (NMJ). Specifically, these mice exhibited impaired growth plate homeostasis and reduced insulin-like growth factor (IGF) signaling from chondrocytes, rather than from the liver. Furthermore, the reduction of SMN in fibro-adipogenic progenitors (FAPs) resulted in abnormal NMJ maturation, altered release of neurotransmitters, and NMJ morphological defects. Transplantation of healthy FAPs rescued the morphological deterioration. Our findings highlight the significance of mesenchymal SMN in neuromusculoskeletal pathogenesis of SMA and provide insights into potential therapeutic strategies targeting mesenchymal cells for the treatment of SMA.

Validation of SMA screening kits with SMN1 gene analysis in a Turkish cohort.

OBJECTIVE: It is crucial to start early treatment in Spinal Muscular Atrophy (SMA) with available drugs to stop the progression of the disease, therefore making SMA screening preferable. This study assessed Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) compared to Multiplex Ligation-dependent Probe Amplification (MLPA) for detecting Spinal Muscular Atrophy (SMA) through SMN1 gene copy number analysis in a Turkish cohort. METHODS: We analyzed 249 DNA samples, previously tested for SMN1 and SMN2 gene deletion via MLPA, using qRT-PCR kits from three different companies. Accuracy, sensitivity, and specificity of qRT-PCR in identifying deletions of SMN1 copy number variations. RESULTS: High accuracy (96.2-98.7%) achieved with qRT-PCR for detecting homozygous deletions, heterozygous deletions, and copy number variations in the SMN1 gene. Minor discrepancies between qRT-PCR and MLPA were observed, possibly due to single nucleotide polymorphisms affecting primer binding. CONCLUSIONS: The qRT-PCR method proved to be a rapid, cost-effective, and accurate technique, aligning well with the demands of routine SMA screening, suggesting its general suitability for application in SMA screening programs. This research highlights the importance of improving molecular methodologies and the value of collaborations between government and relevant sectors to overcome rare diseases, particularly through the enhancement of screening initiatives which is the first and most effective strategy to protect the public health.

[An evaluation of carrier detection for Spinal muscular atrophy using digital PCR assay].

OBJECTIVE: To assess the effectiveness and feasibility of carrier detection for Spinal muscular atrophy (SMA) by using digital PCR assay. METHODS: Peripheral blood samples were collected from 214 pregnant women who were routinely screened for SMA carriers, of which 204 were randomly selected samples and 10 were samples with known copy numbers of SMN1 exons 7 and 8. Samples with known copy numbers of SMN1 exons 7 and 8 were randomly mixed into the experiment to validate the performance of the digital PCR assay. RESULTS: The copy numbers of SMN1 exons 7 and 8 and SMN2 exons 7 and 8 in peripheral blood samples were detected by digital PCR assay. The results of SMN1 exons 7 and 8 were compared with those of the quantitative PCR method to assess the reliability and clinical performance of the digital PCR assay. Among the 204 random samples, digital PCR has detected five samples with simultaneous heterozygous deletion of SMN1 exons 7 and 8, three samples with heterozygous deletion of SMN1 exon 8 only, and 196 samples with no deletion of SMN1 exons 7 and 8. Ten samples with known SMN1 exons 7 and 8 copy numbers were detected with the expected values. The digital PCR test results were fully consistent with that of the quantitative PCR. CONCLUSION: The results of digital PCR for the detection of copy number variation of SMN1 exons 7 and 8 were consistent with qPCR. Digital PCR assay was able to clearly distinguish the copy number of the target genes, therefore can be used for SMA carrier screening. Moreover, it can also detect copy number of SMN2 exons 7 and 8, which can provide more information for genetic counseling.

A super minigene with a short promoter and truncated introns recapitulates essential features of transcription and splicing regulation of the SMN1 and SMN2 genes.

Here we report a Survival Motor Neuron 2 (SMN2) super minigene, SMN2Sup, encompassing its own promoter, all exons, their flanking intronic sequences and the entire 3'-untranslated region. We confirm that the pre-mRNA generated from SMN2Sup undergoes splicing to produce a translation-competent mRNA. We demonstrate that mRNA generated from SMN2Sup produces more SMN than an identical mRNA generated from a cDNA clone. We uncover that overexpression of SMN triggers skipping of exon 3 of SMN1/SMN2. We define the minimal promoter and regulatory elements associated with the initiation and elongation of transcription of SMN2. The shortened introns within SMN2Sup preserved the ability of camptothecin, a transcription elongation inhibitor, to induce skipping of exons 3 and 7 of SMN2. We show that intron 1-retained transcripts undergo nonsense-mediated decay. We demonstrate that splicing factor SRSF3 and DNA/RNA helicase DHX9 regulate splicing of multiple exons in the context of both SMN2Sup and endogenous SMN1/SMN2. Prevention of SMN2 exon 7 skipping has implications for the treatment of spinal muscular atrophy (SMA). We validate the utility of the super minigene in monitoring SMN levels upon splicing correction. Finally, we demonstrate how the super minigene could be employed to capture the cell type-specific effects of a pathogenic SMN1 mutation.

A transcriptomics-based drug repositioning approach to identify drugs with similar activities for the treatment of muscle pathologies in spinal muscular atrophy (SMA) models.

Spinal muscular atrophy (SMA) is a genetic neuromuscular disorder caused by the reduction of survival of motor neuron (SMN) protein levels. Although three SMN-augmentation therapies are clinically approved that significantly slow down disease progression, they are unfortunately not cures. Thus, complementary SMN-independent therapies that can target key SMA pathologies and that can support the clinically approved SMN-dependent drugs are the forefront of therapeutic development. We have previously demonstrated that prednisolone, a synthetic glucocorticoid (GC) improved muscle health and survival in severe Smn-/-;SMN2 and intermediate Smn2B/- SMA mice. However, long-term administration of prednisolone can promote myopathy. We thus wanted to identify genes and pathways targeted by prednisolone in skeletal muscle to discover clinically approved drugs that are predicted to emulate prednisolone's activities. Using an RNA-sequencing, bioinformatics, and drug repositioning pipeline on skeletal muscle from symptomatic prednisolone-treated and untreated Smn-/-; SMN2 SMA and Smn+/-; SMN2 healthy mice, we identified molecular targets linked to prednisolone's ameliorative effects and a list of 580 drug candidates with similar predicted activities. Two of these candidates, metformin and oxandrolone, were further investigated in SMA cellular and animal models, which highlighted that these compounds do not have the same ameliorative effects on SMA phenotypes as prednisolone; however, a number of other important drug targets remain. Overall, our work further supports the usefulness of prednisolone's potential as a second-generation therapy for SMA, identifies a list of potential SMA drug treatments and highlights improvements for future transcriptomic-based drug repositioning studies in SMA.

Preconception or prenatal acceptance of SMN1 gene carrier screening and carrier rate of spinal muscular atrophy: a retrospective study in 18,818 reproductive age women in Wuhan area of China.

OBJECTIVE: Spinal muscular atrophy (SMA) is an autosomal recessive disorder mainly affecting the neuromuscular system, which seriously threatens the life and health of patients. But few studies have reported the acceptance rate of SMA gene screening and SMA carrier rate in China. The present study aimed to clarify the two issues in China through a retrospective analysis of 18,818 reproductive age women in Wuhan area of China. METHODS: The copy number (CN) of exons 7 and 8 in survival motor neuron 1 (SMN1) gene was detected by real-time quantitative PCR, and the results were verified by multiplex ligation-dependent probe amplification. RESULTS: Carrier screening was offered to 44,953 women of childbearing age in our medical center from March, 2018, to February, 2022, of whom 18,818 were enrolled in the program. A total of 336 women were identified as carriers (1.73%; 326/18,808; without fertility history of the children with SMA). Among 18,818 reproductive age women, 286 spouses (85.12%; 286/336) were successfully recalled for screening. The results showed 17 couples at high risk of having children with SMA, of whom prenatal diagnosis was implemented in 11, and 6 fetuses were identified with SMA. All the 5 pregnant women bearing the 6 SMA fetuses chose to terminate the pregnancy by artificial abortion. CONCLUSION: Reproductive age women and their spouses in Wuhan area showed a positive attitude toward general screening for SMA carriers. Given the high early mortality of children with SMA, screening for SMA carriers in women of reproductive age is necessary and feasible.

An evaluation of carrier detection for Spinal muscular atrophy using digital PCR assay / 中华医学遗传学杂志

OBJECTIVE@#To assess the effectiveness and feasibility of carrier detection for Spinal muscular atrophy (SMA) by using digital PCR assay.@*METHODS@#Peripheral blood samples were collected from 214 pregnant women who were routinely screened for SMA carriers, of which 204 were randomly selected samples and 10 were samples with known copy numbers of SMN1 exons 7 and 8. Samples with known copy numbers of SMN1 exons 7 and 8 were randomly mixed into the experiment to validate the performance of the digital PCR assay.@*RESULTS@#The copy numbers of SMN1 exons 7 and 8 and SMN2 exons 7 and 8 in peripheral blood samples were detected by digital PCR assay. The results of SMN1 exons 7 and 8 were compared with those of the quantitative PCR method to assess the reliability and clinical performance of the digital PCR assay. Among the 204 random samples, digital PCR has detected five samples with simultaneous heterozygous deletion of SMN1 exons 7 and 8, three samples with heterozygous deletion of SMN1 exon 8 only, and 196 samples with no deletion of SMN1 exons 7 and 8. Ten samples with known SMN1 exons 7 and 8 copy numbers were detected with the expected values. The digital PCR test results were fully consistent with that of the quantitative PCR.@*CONCLUSION@#The results of digital PCR for the detection of copy number variation of SMN1 exons 7 and 8 were consistent with qPCR. Digital PCR assay was able to clearly distinguish the copy number of the target genes, therefore can be used for SMA carrier screening. Moreover, it can also detect copy number of SMN2 exons 7 and 8, which can provide more information for genetic counseling.

A high-fidelity long-read sequencing-based approach enables accurate and effective genetic diagnosis of spinal muscular atrophy.

BACKGROUND: We aimed to develop a high-fidelity long-read sequencing (LRS)-based approach to detect SMN gene variants in one step. It is challenging for conventional step-wise methods to simultaneously detect all kinds of variations between homologous SMN1 and SMN2. METHODS: In this study, LRS was developed to analyze copy numbers (CNs), full sequences, and structure of SMN1 and SMN2. The results were compared with those from the step-wise methods in 202 samples from 67 families. RESULTS: LRS achieved 100% (202/202) and 99.5% (201/202) accuracy for SMN1 and SMN2 CNs, respectively. It corrected SMN1 CNs from MLPA, which was caused by SNVs/indels that located in probe-binding region. LRS identified 23 SNVs/indels distributing throughout SMN1, including c.22dup and c.884A > T in trans-configuration, and a de novo variant c.41_42delinsC for the first time. LRS also identified a SMN2 variant c.346A > G. Moreover, it successfully determined Alu-mediated 8978-bp deletion encompassing exon 2a-5 and 1415-bp deletion disrupting exon 1, and the exact breakpoints of large deletions. Through haplotype-based pedigree trio analysis, LRS identified SMN1 2 + 0 carriers, and determined the distribution of SMN1 and SMN2 on two chromosomes. CONCLUSIONS: LRS represents a more comprehensive and accurate diagnosis approach that is beneficial to early treatment and effective management of SMA.