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.

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.

Study on the Structure and Properties of Silk Fibers Obtained from Factory All-Age Artificial Diets.

The traditional production mode of the sericulture industry is no longer suitable for the development requirements of modern agriculture; to facilitate the sustainable development of the sericulture industry, factory all-age artificial diet feeding came into being. Understanding the structural characteristics and properties of silk fibers obtained from factory all-age artificial diet feeding is an important prerequisite for application in the fields of textiles, clothing, biomedicine, and others. However, there have been no reports so far. In this paper, by feeding silkworms with factory all-age artificial diets (AD group) and mulberry leaves (ML group), silk fibers were obtained via two different feeding methods. The structure, mechanical properties, hygroscopic properties, and degradation properties were studied by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and thermogravimetric analysis (TGA). Structurally, no new functional groups appeared in the AD group. Compared with the ML group, the structure of the two groups was similar, and there was no significant difference in mechanical properties and moisture absorption. The structure of degummed silk fibers is dominated by crystalline regions, but α-chymotrypsin hydrolyzes the amorphous regions of silk proteins, so that after 28 d of degradation, the weight loss of both is very small. This provides further justification for the feasibility of factory all-age artificial diets for silkworms.

[Clinical characteristics and genetics functional analysis of two children with Spinal muscular atrophy].

OBJETIVE: To explore the characteristics of SMN1 gene variants and carry out functional verification for two children with Spinal muscular atrophy (SMA). METHODS: Two male children with complicated SMA diagnosed at the Children's Hospital Affiliated to Capital Institute of Pediatrics respectively in July 2021 and April 2022 due to delayed or retrograde motor development were selected as the study subjects. Clinical data of the children were collected. Primary culture of skin fibroblasts was carried out, and peripheral blood samples were collected from both children and their parents. Multiplex ligation-dependent probe amplification, combined long-range PCR and nested PCR, and Sanger sequencing were carried out to detect the copy number and variants of the SMN1 gene. Absolute quantitative real-time PCR, Western blotting and immunofluorescence were used to determine the transcriptional level of the SMN gene, expression of the SMN protein, and the number of functional SMN protein complexes (gems body), respectively. This study was approved by the Children's Hospital Affiliated to Capital Institute of Pediatrics (Ethics No. SHERLLM2021009). RESULTS: Child 1, a 1-year-old boy, was clinically diagnosed with type 1 SMA. Child 2, a 2-and-a-half-year-old boy, was clinically diagnosed with type 3 SMA. Both children were found to harbor a paternally derived SMN1 deletion and a maternally derived SMN1 gene variant, namely c.824G>T (p.Gly275Val) and c.884A>T (p.*295Leu). Compared with the normal controls and carriers, the levels of full-length SMN1 transcripts in their peripheral blood and skin fibroblast cell lines were significantly decreased (P < 0.05), and the levels of SMN protein normalized to that of ß-actin, and the numbers of gems bodies in the primary fibroblast cells were also significantly lower (P < 0.05). Based on the guidelines from the American College of Medical Genetics and Genomics, both variants were classified as likely pathogenic (PS3+PM3+PM5+PP3; PS3+PM3+PM4+PP3). Following the diagnosis, both children had received nusinersen treatment. Although their motor function was improved, child 1 still died at the age of 2 due to severe pulmonary infection. The walking ability of child 2 was significantly improved, and his prognosis appeared to be good. CONCLUSION: Two cases of clinically complicated SMA have been confirmed by genetic testing and experimental studies, which has provided a reference for their accurate treatment.

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.

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.

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.

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.

Effect of Buffer Conditions and Organic Cosolvents on the Rate of Strain-Promoted Azide-Alkyne Cycloaddition.

We investigate the effect of buffer identity, ionic strength, pH, and organic cosolvents on the rate of strain-promoted azide-alkyne cycloaddition with the widely used DIBAC cyclooctyne. The rate of reaction between DIBAC and a hydrophilic azide is highly tolerant to changes in buffer conditions but is impacted by organic cosolvents. Thus, bioconjugation reactions using DIBAC can be carried out in the buffer that is most compatible with the biomolecules being labeled, but the use of organic cosolvents should be carefully considered.

Broadly, independent-tunable, dual-wavelength mid-infrared ultrafast optical parametric oscillator.

We demonstrate a two-crystal mid-infrared dual-wavelength optical parametric oscillator, synchronously pumped by a high power femtosecond Yb:fiber laser. The singly-resonant ring cavity, containing two periodically poled lithium niobate crystals, is capable of generating two synchronized idler wavelengths, independently tunable over 30 THz in the 2.9 - 4.2 µm wavelength region, due to the cascaded quadratic nonlinear effect. The independent tunability of the two idlers makes the optical parametric oscillator a promising source for ultrafast pulse generation towards the THz wavelength region, based on different frequency generation. In addition, the observed frequency doubled idler within the crystal indicates the possibility to realize a broadband optical self-phase locking between pump, signal, idler and higher order generated parametric lights.

[Screening for genetic mutations in hyperphenylalaninemia using Ion Torrent PGM sequencing].

OBJECTIVE: To establish a hyperphenylalaninemia related genes screening method using Ion Torrent Personal Genome Machine (PGM) for early detection and differential diagnosis of hyperphenylalaninemia (HPA). METHODS: Three children with known HPA mutations and a healthy control were used for setting up the method. Ten children with HPA with known mutations were recruited for validating the method. Ion Ampliseq PCR was used to amplify the 5' and 3' untranslated region, coding sequence, and flanking introns of PAH, GCH1, PTS, QDPR, and PCBD1 genes. After the enrichment with the Ion OneTouch system, the products were sequenced by PGM. Data from the PGM were processed with Torrent Suite v2.2 software package. All variations were confirmed by Sanger sequencing. RESULTS: For the 4 samples, the PGM output was 94.22 Mb, with approximately 99.5% of reads mapping to the target regions. Among these samples, we detected 74 variations (28 positions) including 6 known mutations. Compared with database and results of Sanger sequencing, 55 (18 positions) polymorphisms and 13 (4 positions) false positive calls were confirmed. For the 10 samples, all the known mutations were successfully identified. CONCLUSION: Ion Torrent PGM sequencing is suitable for screening genetic mutation underlying HPA from the perspective of metabolic pathways, which can meet the clinical demand for individualized diagnosis and treatment.

[Analysis of SMN1 gene partial deletion of spinal muscular atrophy based on MLPA].

OBJECTIVE: To explore the diversity of mutations in survival motor neuron gene 1 (SMN1) by analyzing seven cases of partial deletion of SMN1 gene. METHODS: Seven patients suspected spinal muscular atrophy (SMA) were recruited from 2011 to 2013. Multiplex ligation-dependent probe amplification (MLPA) for genetic testing of SMA was based on the commercially available SALSA MLPA kit P021-A2. Then the data were analyzed by the software Coffalyser.Negative control samples were chosen with two copies of SMN1 and SMN2. Positive control samples were chosen with zero copies of SMN1 and two copies of SMN2. According to the product description (www.mlpa.com): for exon 7 and 8 of SMN1 and SMN2: a ratio of <0.7 indicates 1 copy, a ratio of 0.7-1.3 2 copies, a ratio of 1.3-1.7 3 copies and a ratio of 1.7-2.3 4 copies. For exon 1, 4, 6, 8 of SMN gene (SMN1+SMN2): a ratio <0.4 indicates 1 copy, a ratio of 4.0-0.6 2 copies, a ratio of 0.7-0.9 3 copies and a ratio of 0.9-1.1 4 copies. All samples were analyzed in duplicate. RESULTS: Using MLPA for clinical diagnostics, two types of partial deletions of SMN1were identified in 7 patients.Since exon 8 is not translated and has no effect on the function of SMN protein, exons 1, 4, 6, 7 were targeted.One had an isolated deletion of exon 7 while the other ones were caused by the deletions of exon 1, 4 and 7. These mutations were not detected by conventional diagnostic methods. Both types of partial deletions of SMN1 gene contained a deletion of exon 7. CONCLUSIONS: Two types of partial deletions of SMN1 gene indicate that the structure of SMN gene is unstable leading to a variety of mutation forms. But the major cause of SMA lies in a deletion of exon 7 of SMN1 gene.

Fast clinical molecular diagnosis of hyperphenylalaninemia using next-generation sequencing-based on a custom AmpliSeq™ panel and Ion Torrent PGM sequencing.

Hyperphenylalaninemia (HPA) can be classified into phenylketonuria (PKU) and tetrahydrobiopterin deficiency (BH4D), according to the defect of enzyme activity, both of which vary substantially in severity, treatment, and prognosis of the disease. To set up a fast and comprehensive assay in order to achieve early etiological diagnosis and differential diagnosis for children with HPA, we designed a custom AmpliSeq™ panel for the sequencing of coding DNA sequence (CDS), flanking introns, 5' untranslated region (UTR) and 3' UTR from five HPA-causing genes (PAH, PTS, QDPR, GCH1, and PCBD1) using the Ion Torrent Personal Genome Machine (PGM) Sequencer. A standard group of 15 samples with previously known DNA sequences and a test group of 37 HPA patients with unknown mutations were used for assay validation and application, respectively. All variations were confirmed by Sanger sequencing. In the standard group, all the known mutations were detected and were consistent with the results of previous Sanger sequencing. In the test group, we identified mutations in 71 of 74 alleles, with a mutation detection rate of 95.9%. We also found a frame shift deletion p.Ile25Metfs*13 in PAH that was previously unreported. In addition, 1 of 37 in the test group was inconsistent with either the molecular diagnosis or clinical diagnosis by traditional differential methods. In conclusion, our comprehensive assay based on a custom AmpliSeq™ panel and Ion Torrent PGM sequencing has wider coverage, higher throughput, is much faster, and more efficient when compared with the traditional molecular detection method for HPA patients, which could meet the medical need for individualized diagnosis and treatment.

Two-crystal mid-infrared optical parametric oscillator for absorption and dispersion dual-comb spectroscopy.

We present a femtosecond optical parametric oscillator (OPO) containing two magnesium-doped periodically poled lithium niobate crystals in a singly resonant ring cavity, pumped by two mode-locked Yb-fiber lasers. As such, the OPO generates two idler combs (up to 220 mW), covering a wavelength range from 2.7 to 4.2 µm, from which a mid-infrared dual-comb Fourier transform spectrometer is constructed. By detecting the heterodyning signal between the two idler beams a full broadband spectrum of a molecular gas can be observed over 250 cm(-1) within 70 µs with a spectral resolution of 15 GHz. The absorption and dispersion spectra of acetylene and methane have been measured around 3000 cm(-1), indicating that this OPO represents an ideal broadband mid-infrared source for fast chemical sensing.

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.

Association among biomarkers, phenotypes, and motor milestones in Chinese patients with 5q spinal muscular atrophy types 1-3.

Background: Biomarkers can be used to assess the severity of spinal muscular atrophy (5q SMA; SMA). Despite their potential, the relationship between biomarkers and clinical outcomes in SMA remains underexplored. This study aimed to assess the association among biomarkers, phenotypes, and motor milestones in Chinese patients diagnosed with SMA. Methods: We collected retrospective clinical and follow-up data of disease-modifying therapy (DMT)-naïve patients with SMA at our center from 2019 to 2021. Four biomarkers were included: survival motor neuron 2 (SMN2) copies, neuronal apoptosis inhibitory protein (NAIP) copies, full-length SMN2 (fl-SMN2), and F-actin bundling protein plastin 3 (PLS3) transcript levels. Data were analyzed and stratified according to SMA subtype. Results: Of the 123 patients, 30 were diagnosed with Type 1 (24.3%), 56 with Type 2 (45.5%), and 37 with Type 3 (30.1%). The mortality rate for Type 1 was 50%, with median survival times of 2 and 8 months for types 1a and 1b, respectively. All four biomarkers were correlated with disease severity. Notably, fl-SMN2 transcript levels increased with SMN2 copies and were higher in Type 2b than those in Type 2a (p = 0.028). Motor milestone deterioration was correlated with SMN2 copies, NAIP copies, and fl-SMN2 levels, while PLS3 levels were correlated with standing and walking function. Discussion: Our findings suggest that SMN2 copies contribute to survival and that fl-SMN2 may serve as a valuable biomarker for phenotypic variability in SMA Type 2 subtypes. These insights can guide future research and clinical management of SMA.

3-Pentadecylphenol (PDP) as a Novel Compatibilizer for Simultaneous Toughened and Reinforced PA10,12 Composites.

The utilization of polyamide 10,12 (PA10,12) composites in various industries has been limited constrained by their inherent low toughness, making it a challenge to achieve a balance between toughness and structural integrity through conventional elastomer addition strategies. Herein, we introduce a straightforward method for the concurrent toughening and reinforcement of PA10,12 composites. This is accomplished by blending polyolefin elastomer (POE) and 3-pentadecylphenol (PDP) with the PA10,12 matrix. The incorporation of 5 wt% PDP effectively blurred the PA10,12/POE interface due to PDP's role as a compatibilizer. This phenomenon is attributed to the formation of intermolecular hydrogen bonds, as evidenced by Fourier Transform Infrared Spectroscopy (FTIR) analysis. Further investigation, using differential scanning calorimetry (DSC), elucidated the crystallization thermodynamics and kinetics of the resulting binary PA10,12/POE and ternary PA10,12/POE/PDP composites. Notably, the crystallization temperature (Tc) was observed to decrease from 163.1 °C in the binary composite to 161.5 °C upon the addition of PDP. Increasing the PDP content to 10% led to a further reduction in Tc to 159.5 °C due to PDP's capacity to slow down crystallization. Consequently, the ternary composite of PA10,12/POE/PDP (92/3/5 wt%) demonstrated a synergistic improvement in mechanical properties, with an elongation at break of 579% and a notch impact strength of 61.54 kJ/m2. This represents an approximately eightfold increase over the impact strength of unmodified PA10,12. Therefore, our work provides the potential of PDP as a compatibilizer to develop nylon composites with enhanced stiffness and toughness.

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.

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.

[Detection of homozygous deletions in spinal muscular atrophy with genomic DNA sequencing].

OBJECTIVE: To detect homozygous deletions of survival motor neuron (SMN) gene with genomic DNA sequencing, and to assess the value of genetic testing for the diagnosis of spinal muscular atrophy (SMA). METHODS: Polymerase chain reaction (PCR) was used for amplifying SMN gene in 100 SMA patients and 110 controls. Four different bases (g.31957, g.32006, g.32154 and g.32269) between SMN1 and SMN2 within the amplified segments were identified with genomic DNA sequencing. Homozygous deletion of SMN1 or SMN2 was determined by the presence or absence of base peaks at such four sites. Multiplex ligation-dependent probe amplification (MLPA) was carried out to confirm the results of genomic DNA sequencing. RESULTS: In the 100 SMA samples, only SMN2 specific base peaks were detected at the four sites, for which the copy numbers of SMN1 and SMN2 was 0:2 or 0:3, suggesting homozygous deletion of SMN1 gene. By contrast, only SMN1 specific base peaks were detected in 5 samples, for which the ratio of SMN1:SMN2 was 2:0, indicating homozygous deletion of SMN2. At four different sites, SMN1/SMN2 heterozygous peaks were detected in the remaining 105 samples, for which SMN1:SMN2was 2:2, suggesting non-deletion of SMN1 or SMN2. The results of sequencing were consistent with those of MLPA. CONCLUSION: Genomic DNA sequencing is a rapid, accurate and economic method for the diagnosis of homozygous deletion of SMA.