Variants located in intron 6 of SMN1 lead to misdiagnosis in genetic detection and screening for SMA.

Accurate genetic diagnosis is necessary for guiding the treatment of spinal muscular atrophy (SMA). An updated consensus for the diagnosis and management of SMA was published in 2018. However, clinicians should remain alert to some pitfalls of genetic testing that can occur when following a routine diagnosis. In this study, we report the diagnosis of three unrelated individuals who were initially misdiagnosed as carrying a homozygous deletion of SMN1 exon 7. MLPA (P060 and P021) and qPCR were used to detect the copy number of SMN. SMN1 variants were identified by SMN1 clone and next-generation sequencing (NGS). Transcription of SMN1 variants was detected using qRT-PCR and ex vivo splicing analysis. Among the three individuals, one was identified as a patient with SMA carrying a heterozygous deletion and a pathogenic variant (c.835-17_835-14delCTTT) of SMN1, one was a healthy carrier only carrying a heterozygous deletion of SMN1 exon 7, and the third was a patient with nemaline myopathy 2 carrying a heterozygous deletion of SMN1 exon 7. The misdiagnosis of these individuals was attributed to the presence of the c.835-17_835-14delCTTT or c.835-17C > G variants in SMN1 intron 6, which affect the amplification of SMN1 exon 7 during MLPA-P060 and qPCR testing. However, MLPA-P021 and NGS analyses were unaffected by these variants. These results support that additional detection methods should be employed in cases where the SMN1 copy number is ambiguous to minimize the misdiagnosis of SMA.

Public perception on active aging after COVID-19: an unsupervised machine learning analysis of 44,343 posts.

Introduction: To analyze public perceptions of active aging in China on mainstream social media platforms to determine whether the "14th Five Year Plan for the Development of the Aging Career and Older Adult Care System" issued by the CPC in 2022 has fully addressed public needs. Methods: The original tweets posted on Weibo between January 1, 2020, and June 30, 2022, containing the words "aging" or "old age" were extracted. A bidirectional encoder representation from transformers (BERT)-based model was used to generate themes related to this perception. A qualitative thematic analysis and an independent review of the theme labels were conducted by the researchers. Results: The findings indicate that public perceptions revolved around four themes: (1) health prevention and protection, (2) convenient living environments, (3) cognitive health and social integration, and (4) protecting the rights and interests of the older adult. Discussion: Our study found that although the Plan aligns with most of these themes, it lacks clear planning for financial security and marital life.

Molecular dynamics simulation of the 3-15alkyphenol compatibilizer in highly toughened and robust polyamide 10,12/MWCNT composites.

Highly toughened and stiff polyamide 10,12 (PA10,12) composites present a promising alternative to metal products for high-impact environments. However, it is challenging to toughen PA10,12 composites without compromising their robustness. Herein, we report a facile and scalable route to simultaneously develop reinforced and toughened PA10,12 composites via compounding PA10,12, carbon nanotubes (CNTs) and 3-15alkyphenol (PDP). The PDP acted as a compatibilizer to well-disperse MWCNTs since they tended to be adsorbed onto the CNT surface, which was revealed by molecular dynamics simulation. According to the simulation statistics, the vertical PDP conformations (to the CNT surface) were predominant in the ternary composites with ∼78.7% probability. Moreover, the hydrogen bonds (H-bonds) between the PDP and the PA matrix were confirmed using FTIR. A crystallization kinetics study also revealed that the crystallization temperature increased from 166.7 °C for the neat PA10,12 to 168.7 °C for the ternary PA/PDP/CNT composites containing 1.5 wt% CNTs, while the crystallization half-time increased from 0.58 s for the neat PA10,12 to 1.2 s for the ternary composites. It was also found that the notched impact strength of the ternary composites reached 75.2 kJ m-2, which was 970% higher than that of the neat PA10,12 without compromising their tensile strength of 50.5 MPa much. This work provides a new insight into PDP as a compatibilizer to develop simultaneously stiff and toughened nylon composites.

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.

Fast Automatic Registration of UAV Images via Bidirectional Matching.

Image registration plays a vital role in the mosaic process of multiple UAV (Unmanned Aerial Vehicle) images acquired from different spatial positions of the same scene. Aimed at the problem that many fast registration methods cannot provide both high speed and accuracy simultaneously for UAV visible light images, this work proposes a novel registration framework based on a popular baseline registration algorithm, ORB-the Oriented FAST (Features from Accelerated Segment Test) and Rotated BRIEF (Binary Robust Independent Elemental Features) algorithm. First, the ORB algorithm is utilized to extract image feature points fast. On this basis, two bidirectional matching strategies are presented to match obtained feature points. Then, the PROSRC (Progressive Sample Consensus) algorithm is applied to remove false matches. Finally, the experiments are carried out on UAV image pairs about different scenes including urban, road, building, farmland, and forest. Compared with the original version and other state-of-the-art registration methods, the bi-matching ORB algorithm exhibits higher accuracy and faster speed without any training or prior knowledge. Meanwhile, its complexity is quite low for on-board realization.

IKBKE regulates angiogenesis by modulating VEGF expression and secretion in glioblastoma.

PURPOSE: As a noncanonical inflammatory kinase, IKBKE is frequently overexpressed and activated and has been identified as an oncogenic protein in glioblastoma. However, the potential function and underlying mechanism of IKBKE contributing to tumor angiogenesis remain elusive. METHODS: First, we analyzed the correlation between IKBKE and VEGF expression in glioma samples by immunohistochemistry (IHC). Second, HUVEC-related assays and Western blot were used to detect the regulatory effect of IKBKE on angiogenesis by modulating VEGF expression. Third, IKBKE depletion could alleviate the influence of VEGF expression on IHC of intracranial glioma model. RESULTS: We demonstrate that depletion of IKBKE markedly inhibits tumor growth and angiogenesis in glioblastoma. Mechanistically, IKBKE induces VEGF expression and secretion by regulating AKT/FOXO3a in glioblastoma. CONCLUSIONS: This study reveals that IKBKE is a novel oncogenic molecule that induces angiogenesis through the promotion of VEGF expression and highlights the potential of targeting IKBKE for glioblastoma therapy.

Novel Alu-mediated deletions of the SMN1 gene were identified by ultra-long read sequencing technology in patients with spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by biallelic variants of the survival motor neuron 1 (SMN1) gene. In this study, our aim was to make a molecular diagnosis in two patients with SMA carrying only one SMN1 copy number. Using ultra-long read sequencing (Ultra-LRS), 1415 bp deletion and 3348 bp deletion of the SMN1 gene were identified in patient 1 and the father of patient 2, respectively. Ultra-LRS revealed two novel deletions, starting from the SMN1 promoter to intron 1. It also accurately provided the location of the deletion breakpoints in the SMN1 gene: chr5 g.70,924,798-70,926,212 for a 1415 bp deletion; chr5 g.70,922,695-70,926,042 for a 3348 bp deletion. By analyzing the breakpoint junctions, we identified that these genomic sequences were composed of Alu sequences, including AluJb, AluYm1, AluSq, and AluYm1, indicating that Alu-mediated rearrangements are a mechanism of SMN1 deletion events. In addition, full-length SMN1 transcripts and SMN protein in patient 1 were significantly decreased (p < 0.01), suggesting that a 1415 bp deletion that included the transcription and translation initiation sites of the SMN1 gene had severe consequences for SMN expression. Ultra-LRS can easily distinguish highly homozygous genes compared to other detection technologies, which is useful for detecting SMN1 intragenic mutations, to quickly discover structural rearrangements and to precisely present the breakpoint positions.

Rhenium Suppresses Iridium (IV) Oxide Crystallization and Enables Efficient, Stable Electrochemical Water Oxidation.

IrO2 as benchmark electrocatalyst for acidic oxygen evolution reaction (OER) suffers from its low activity and poor stability. Modulating the coordination environment of IrO2 by chemical doping is a methodology to suppress Ir dissolution and tailor adsorption behavior of active oxygen intermediates on interfacial Ir sites. Herein, the Re-doped IrO2 with low crystallinity is rationally designed as highly active and robust electrocatalysts for acidic OER. Theoretical calculations suggest that the similar ionic sizes of Ir and Re impart large spontaneous substitution energy and successfully incorporate Re into the IrO2 lattice. Re-doped IrO2 exhibits a much larger migration energy from IrO2 surface (0.96 eV) than other dopants (Ni, Cu, and Zn), indicating strong confinement of Re within the IrO2 lattice for suppressing Ir dissolution. The optimal catalysts (Re: 10 at%) exhibit a low overpotential of 255 mV at 10 mA cm-2 and a high stability of 170 h for acidic OER. The comprehensive mechanism investigations demonstrate that the unique structural arrangement of the Ir active sites with Re-dopant imparts high performance of catalysts by minimizing Ir dissolution, facilitating *OH adsorption and *OOH deprotonation, and lowering kinetic barrier during OER. This study provides a methodology for designing highly-performed catalysts for energy conversion.

IrO2-Stablized La2IrO6 perovskite nanotubes via corner-shared interconnections as highly-efficient oxygen evolution electrocatalysts.

One-dimensional nanotube heterostructures with IrO2-stabilized La2IrO6 is obtained by an electrospinning approach. The La2IrO6/IrO2 catalyst exhibits superior catalytic activity and strong stability for the oxygen evolution reaction. The synergistic cooperation between the two types of Ir as the active sites in La2IrO6/IrO2 is demonstrated by in situ Raman spectrum and DFT calculation.

Antisense oligonucleotides targeting the SMN2 promoter region enhance SMN2 expression in spinal muscular atrophy cell lines and mouse model.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease caused by homozygous deletions or mutations in survival motor neuron gene 1 (SMN1). Currently, the primary therapeutic strategy for SMA is to increase the level of SMN via correcting SMN2 splicing (nusinersen and risdiplam). However, some patients with SMA do not respond to such treatments, thereby warranting a need to develop new therapeutic strategies. We have previously reported that SMN2 expression is epigenetically regulated by DNA methylation levels of the SMN2 promoter region. In the present study, we determined that methyl-CpG-binding protein 2 (MeCP2) may bind to this critical promoter region (nt-167 to 43). Antisense oligonucleotides (ASO-P1 and ASO-P2) were designed to target the key methylation sites in the SMN2 promoter region, which enhanced the overall transcription and functional protein expression levels in the SMA cell lines. These results were similar to those observed in nusinersen-treated SMA cells. Moreover, a combined treatment of ASO-P1 and ASO-NUS in SMA cell lines further increases fl-SMN2 transcript and SMN protein levels. The delivery of ASO-P1 to the central nervous system of severe SMA mice corrected the molecular, pathological, and functional phenotypes of this disease and increased survival rates. Our findings suggest that the key methylation regions in the SMN2 promoter region may be a novel therapeutic target for SMA.

NaBH4-reduction induced tunable oxygen vacancies in LaNiO2.7 to enhance the oxygen evolution reaction.

Tunable oxygen vacancies of LaNiO3 (LNO-Vo) are realized by theoretical prediction and the NaBH4-reduction approach. The LNO2.7 catalyst exhibits superior catalytic activity and long-term stability for water oxidation. Direct evidence of the active site center and the intermediates is observed from in situ Raman spectra and DFT calculations.

Factors associated with delayed diagnosis of spinal muscular atrophy in China and changes in diagnostic delay.

Spinal muscular atrophy (SMA) is a rare neuromuscular disease, which often occurs in childhood. Early SMA treatment may be highly beneficial to SMA patients, their families, and society. However, delayed diagnosis is common. To identify the factors that affect the SMA diagnostic time window, we analyzed disease characteristics, family factors, and medical factors of 205 SMA families. We compared the data with those of our previous cohort to explore the dynamic changes in the diagnostic time window. The median diagnostic time windows for SMA types I, II, and III were 3.38 [interquartile range (IQR): 2.01-4.98], 4.08 (IQR: 2.07-8.17), and 11.37 (IQR: 4.92-24.07) months, respectively. The diagnostic time window in patients who were clinically diagnosed with SMA at their first hospital visit was 49.42% shorter than that in other patients. Type I/II patients visited approximately 2.56 doctors before diagnosis, while type III patients visited approximately 3.94 doctors before diagnosis. The diagnostic time windows for types II and III were 54.67 and 62.10% shorter, respectively, than those in the previous cohort, which is mainly due to improvements in medical capacity. Therefore, with public awareness, increased medical personnel understanding, and increased neonatal screening, the SMA diagnostic time window is expected to further reduce.

Long non-coding RNA ROR recruits histone transmethylase MLL1 to up-regulate TIMP3 expression and promote breast cancer progression.

BACKGROUND: As a significant cause of cancer deaths worldwide, breast cancer continues to be a troublesome malignancy. Long non-coding RNAs (lncRNAs) have been implicated in the development of breast cancer. Abnormal methylation has been associated with unfavorable breast cancer prognosis. Herein, the current study aimed to elucidate the role of lncRNA ROR in breast cancer. METHODS: RT-qPCR was performed to determine whether lncRNA ROR was highly expressed in breast cancer tissues, while lncRNA ROR expression was detected in both the nuclear and cytoplasm of breast cancer cells. MCF-7 cells were subsequently introduced with oe-lncRNA ROR, sh-lncRNA ROR to explore the effects of lncRNA ROR on cell proliferation, invasion and apoptosis. RESULTS: RIP, RNA pull-down and ChIP assays provided evidence suggesting that lncRNA ROR recruited transmethylase MLL1 to promote H3K4 trimethylation that enhanced TIMP3 transcription. The rescue experiments demonstrated that lncRNA ROR knockdown could inhibit the progression of breast cancer via the downregulation of TIMP3. Finally, the in vivo experiment findings consistently highlighted the suppressive effects of lncRNA ROR silencing on tumor growth. CONCLUSION: Taken together, our study demonstrates that silencing of lncRNA ROR inhibits breast cancer progression via repression of transmethylase MLL1 and TIMP3, emphasizing the potential of lncRNA ROR as a novel target against breast cancer.

Dual Mechanism of a New SMN1 Variant (c.835G>C, p.Gly279Arg) by Interrupting Exon 7 Skipping and YG Oligomerization in Causation of Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by deletion or subtle variant of survival motor neuron 1 (SMN1) gene. By multiplex ligation-dependent probe amplification, genomic sequencing, and T-A cloning on cDNA level, we identified one novel SMN1 subtle variant c.835G>C (p.Gly279Arg) in a non-homozygous patient with type 1 SMA. Full-length SMN1 (fl-SMN1) transcripts in the peripheral bloods of the patient were significantly decreased compared with those in healthy individuals and the carries (p < 0.05). And two fragments of SMN1 transcripts including fl-SMN1 and △7-SMN1 were observed by RT-PCR, which indicated Exon 7 skipping of SMN1 gene. To further evaluate its splicing effects on Exon 7, we performed ex vivo splicing analysis, which showed that the mutant mini gene with c.835G>C reduced Exon 7 inclusion to 54%. In addition, self-oligomerization between mutant SMN protein with the c.835G>C (p.Gly279Arg) and wild SMN was decreased in self-interaction assays. Our study clearly demonstrates that the c.835G>C (p.Gly279Arg) variant can lead to a decrease in fl-SMN1 transcripts by interrupting correct splicing of SMN1. What is more, the variant also affects SMN self-oligomerization via amino acid substitution from Gly to Arg at amino acid position of 279. This work presents the first evidence that it does exit double-hit events for the novel variant, which is crucial to understanding a severe SMA phenotype (type 1).

An Overview on Noble Metal (Group VIII)-based Heterogeneous Electrocatalysts for Nitrogen Reduction Reaction.

The typically the Haber-Bosch process of nitrogen (N2 ) reduction to ammonia (NH3 ) production, expends a lot of energy, resulting in severe environmental issues. Electro-catalytic N2 reduction to NH3 formation by renewable resources is one of the effective ways to settle the issue. However, the electro-catalytic performances and selectivity of catalysts for electrochemical nitrogen reduction reaction (NRR) are very low. Therefore, it is of great significance to develop more efficient electro-catalysts to satisfy the needs of practical use. Among the reported catalysts, those based on Group VIII noble metals heterogeneous catalysts display excellent NRR activities and high selectivity because of their good conductivity, rich active surface area, unfilled d-orbitals, and the abilities with easy adsorption of reactants and stable reaction intermediates. Herein, we will introduce the progress of Group VIII precious metals heterogeneous catalysts applied in the electrocatalytic N2 reduction reaction. Then single precious metal electrocatalysts, precious metal alloy electrocatalysts, heterojunction structure electrocatalysts, and precious metal compounds based on the strategies of morphology engineering, crystal facet engineering, defect engineering, heteroatom doping, and synergetic interface engineering will be discussed. Finally, the challenges and prospects of the NH3 synthesis have been put forward. In the review, we will provide helpful direction to the development of effective electro-catalysts for catalytic N2 reduction reaction.

Transmission characteristics of SMN from 227 spinal muscular atrophy core families in China.

To define the relationship between the survival motor neuron 1 gene (SMN1) and SMN2, and explore the variability of these two genes within the generations, SMN1 and SMN2 copy numbers were determined for 227 SMA families. The association analysis indicated that there was a negative correlation between the copy number of SMN1 and SMN2 (Spearman = -0.472, P < 0.001) in 227 SMA children and 454 of their parents. The average SMN copies from father and mother in each SMA family were used to represent the copy number in the parent's generation. Subsequently, SMN transmission analysis showed that the similar distribution trend of SMN1 and SMN2 copy number was not only in the SMA children and their parents' generation but also in the non-SMA families. Moreover, when the SMN2 copy number was one in the parent's generation, 75% of their SMA children had type I and 25% of them had type II/III. However, when the SMN2 copies were three in the parent's generation, all of their SMA children were type II/III. Therefore, the diversity of SMN copies was mostly inherited and the SMN2 copy number in the parent's generation could predict the disease severity of SMA children to some extent.

Diagnosis of Spinal Muscular Atrophy: A Simple Method for Quantifying the Relative Amount of Survival Motor Neuron Gene 1/2 Using Sanger DNA Sequencing.

BACKGROUND: Spinal muscular atrophy (SMA) is caused by homozygous deletion or compound heterozygous mutation of survival motor neuron gene 1 (SMN1), which is the key to diagnose SMA. The study was to establish and evaluate a new diagnostic method for SMA. METHODS: A total of 1494 children suspected with SMA were enrolled in this study. Traditional strategy, including multiplexed ligation-dependent probe amplification (MLPA) and TA cloning, was used in 1364 suspected SMA children from 2003 to 2014, and the 130 suspected SMA children were tested by a new strategy from 2015 to 2016, who were also verified by MLPA combined with TA cloning. The SMN1 and SMN2 were simultaneously amplified by polymerase chain reaction using the same primers. Mutation Surveyor software was used to detect and quantify the SMN1 variants by calculating allelic proportions in Sanger sequencing. Finally, turnaround time and cost of these two strategies were compared. RESULTS: Among 1364 suspected SMA children, 576 children had SMN1 homozygous deletion and 27 children had SMN1 compound heterozygous mutation. Among the 130 cases, 59 had SMN1 homozygous deletion and 8 had heterozygous deletion: the SMN1-specific peak proportion on exon 7 was 34.6 ± 1.0% and 25.5 ± 0.5%, representing SMN1:SMN2 to be 1:2 and 1:3, respectively. Moreover, five variations, including p.Ser8Lysfs *23 (in two cases), p.Leu228*, p.Pro218Hisfs *26, p.Ser143Phefs*5, and p.Tyr276His, were detected in 6/8 cases with heterozygous deletion, the mutant allele proportion was 31.9%, 23.9%, 37.6%, 32.8%, 24.5%, and 23.6%, which was similar to that of the SMN1-specific site on exon 7, suggesting that those subtle mutations were located in SMN1. All these results were consistent with MLPA and TA cloning. The turnaround times of two strategies were 7.5 h and 266.5 h, respectively. Cost of a new strategy was only 28.5% of the traditional strategy. CONCLUSION: Sanger sequencing combined with Mutation Surveyor analysis has potential application in SMA diagnosis.

The SMN1 common variant c.22 dupA in Chinese patients causes spinal muscular atrophy by nonsense-mediated mRNA decay in humans.

Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder that is mostly caused by homozygous deletion of the SMN1 gene. Approximately 5%-10% of SMA patients are believed to have SMN1 variants. c.22 dupA (p.Ser8lysfs*23) has been identified as the most frequent variant in the Chinese SMA population and to be associated with a severe phenotype. However, the exact molecular mechanism of the variant on the pathogenesis of SMA is unclear. We observed that SMN1 mRNA and the SMN protein in the peripheral blood cells of a patient with c.22 dupA were lower than those of controls. The aim of this study is to investigate whether nonsense-mediated mRNA decay (NMD) plays a role in the mechanism of the c.22 dupA variant of the SMN1 gene as it causes SMA. Two lymphoblasts cell lines from two patients (patient 1 and 2) with the c.22 dupA, and one dermal fibroblasts cell line from patient 2 were included in our study. Two-stage validation of the NMD mechanism was supplied. We first measured the changes in the transcript levels of the SMN1 gene by real-time quantitative PCR after immortalized B-lymphoblasts and dermal fibroblasts cells of the SMA patients were treated with inhibitors of the NMD pathway, including puromycin and cyclohemide. Next, lentivirus-mediated knockdown of the key NMD factor-Up-frameshift protein 1 (UPF1)-was performed in the fibroblasts cell line to further clarify whether the variant led to NMD, as UPF1 recognizes abnormally terminated transcripts as NMD substrates during translation. SC35 1.7-kb transcripts, a physiological NMD substrate was determined to be a NMD positive gene in our experiments. The two inhibitors resulted in a dramatic escalation of the levels of the full-length SMN1 (fl-SMN1) transcripts. Additionally, the SC35 1.7-kb mRNA levels were also increased, suggesting that NMD pathway is suppressed by the two inhibitors. For the 3 cell lines, the fold increase of the SMN1 transcript levels of cycloheximide ranged from 2.5±0.4 to 8.3±0.1, 1.9±0.2 to 5.0±0.7 and 2.2±0.1 to 4.9±0.2 for two lymphoblastoid cell lines and one fibroblasts cell line, respectively. For these cell lines, the fold increases of the SMN1 transcript levels of puromycin were as follows: 5.5±0.2 to 19.5±4.0, 3.1±0.3 to 9.9±1.8 and 1.5±0.2 to 6.5±0.5. Meanwhile, the SC35 1.7-kb transcript levels were markedly increased in all 3 cell lines. In addition, lentivirus-mediated UPF1 knockdown lead to a reduction of the UPF1 protein level to 22.5% compared to the negative control lentivirus. Additionally, knockdown of the UPF1 gene also promoted mRNA expression of the SC35 1.7kb and fl-SMN1 genes. The increases of the SMN1 and SC35 1.7-kb mRNA levels reached about 4- and 6.5-fold in fibroblasts derived from the patient 2, respectively. Altogether, our study provides the first evidence that the c.22 dupA variant in the SMN1 gene triggers NMD. SMA pathogenesis in the patient is associated with mRNA degradation of SMN1, but not the truncated SMN protein.

p.Val19Glyfs*21 and p.Leu228* variants in the survival of motor neuron 1 trigger nonsense-mediated mRNA decay causing the SMN1 PTC+ transcripts degradation.

Spinal Muscular Atrophy (SMA) results from loss-of-function mutations in the survival of motor neuron 1 (SMN1) gene. Our previous research showed that 40% of variants were nonsense or frameshift variants and SMN1 mRNA levels in the patients carrying these variants were significantly decreased. Here we selected one rare variant (p.Val19Glyfs*21) and one common variant (p.Leu228*) to explore the degradation mechanism of mutant transcripts. The levels of full-length (FL)-SMN1 transcripts and SMN protein in the cell lines from the patients with these variants were both significantly reduced (p<0.01). Treatment with two translation inhibitors (puromycin and Cycloheximide (CHX)) markedly increased the levels of FL-SMN1 transcripts with premature translation termination codons (PTCs) (p<0.01) and showed time-dependent (10h>5.5h) but not dose-dependent effects. Moreover, the knockdown of UPF1, a key factor in nonsense-mediated mRNA decay (NMD) by lentivirus, led to a 3.1-fold increase (p<0.01) in FL-SMN1 transcript levels in patient fibroblasts. Our research provides evidence that these two PTC-generating variants (p.Val19Glyfs*21 and p.Leu228*) can trigger NMD, causing rapid degradation of SMN1 transcripts thereby resulting in SMN protein deficiency. These two variants are highly pathogenic and are associated with more severe SMA phenotypes. Varying NMD efficiency after treatment with puromycin and CHX in different cell types was also observed.