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.

Analytical validation of the amplification refractory mutation system polymerase chain reaction-capillary electrophoresis assay to diagnose spinal muscular atrophy.

OBJECTIVES: Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by homozygous deletion and compound heterozygous mutations in survival motor neuron 1 (SMN1), with severity tied to the copy number of survival motor neuron 2 (SMN2). This study aimed to develop a rapid and comprehensive method for the diagnosis of SMA. METHODS: A total of 292 children with clinically suspected SMA and 394 family members were detected by the amplification refractory mutation system polymerase chain reaction-capillary electrophoresis (ARMS-PCR-CE) method, which targeted 19 reported mutations, and the results were compared with those in multiplex ligation-dependent probe amplification (MLPA). Individuals with identified point mutations were further confirmed by SMN1 long-range PCR and Sanger sequencing. RESULTS: A total of 202 children with SMA, 272 carriers, and 212 normal individuals were identified in this study. No difference was found in the R-value distribution of exons 7 and 8 in SMN1 and SMN2 among these cohorts, with coefficients of variation consistently below 0.08. To detect exon 7 and 8 copy numbers in SMN1 and SMN2, the ARMS-PCR-CE results were concordant with those of MLPA. Approximately 4.95 % (10/202) of the study patients had compound heterozygous mutations. CONCLUSIONS: The ARMS-PCR-CE assay is a comprehensive, rapid, and accurate diagnostic method for SMA that simultaneously detects copy numbers of exons 7 and 8 in SMN1/SMN2, as well as 19 point mutations in SMN1 and 2 enhancers in SMN2. This approach can effectively reduce the time frame for diagnosis, facilitating early intervention and preventing birth defects.

ATP purinergic receptor signalling promotes Sca-1+ cell proliferation and migration for vascular remodelling.

AIMS: Vascular resident stem cells expressing stem cell antigen-1 (Sca-1+ cells) promote vascular regeneration and remodelling following injury through migration, proliferation and differentiation. The aim of this study was to examine the contributions of ATP signalling through purinergic receptor type 2 (P2R) isoforms in promoting Sca-1+ cell migration and proliferation after vascular injury and to elucidate the main downstream signalling pathways. METHODS AND RESULTS: ATP-evoked changes in isolated Sca-1+ cell migration were examined by transwell assays, proliferation by viable cell counting assays and intracellular Ca2+ signalling by fluorometry, while receptor subtype contributions and downstream signals were examined by pharmacological or genetic inhibition, immunofluorescence, Western blotting and quantitative RT-PCR. These mechanisms were further examined in mice harbouring TdTomato-labelled Sca-1+ cells with and without Sca-1+-targeted P2R knockout following femoral artery guidewire injury. Stimulation with ATP promoted cultured Sca-1+ cell migration, induced intracellular free calcium elevations primarily via P2Y2R stimulation and accelerated proliferation mainly via P2Y6R stimulation. Enhanced migration was inhibited by the ERK blocker PD98059 or P2Y2R-shRNA, while enhanced proliferation was inhibited by the P38 inhibitor SB203580. Femoral artery guidewire injury of the neointima increased the number of TdTomato-labelled Sca-1+ cells, neointimal area and the ratio of neointimal area to media area at 3 weeks post-injury, and all of these responses were reduced by P2Y2R knockdown. CONCLUSIONS: ATP induces Sca-1+ cell migration through the P2Y2R-Ca2+-ERK signalling pathway, and enhances proliferation through the P2Y6R-P38-MAPK signalling pathway. Both pathways are essential for vascular remodelling following injury. Video Abstract.

Natural killer cells in sepsis: Friends or foes?

Sepsis is one of the major causes of death in the hospital worldwide. The pathology of sepsis is tightly associated with dysregulation of innate immune responses. The contribution of macrophages, neutrophils, and dendritic cells to sepsis is well documented, whereas the role of natural killer (NK) cells, which are critical innate lymphoid lineage cells, remains unclear. In some studies, the activation of NK cells has been reported as a risk factor leading to severe organ damage or death. In sharp contrast, some other studies revealed that triggering NK cell activity contributes to alleviating sepsis. In all, although there are several reports on NK cells in sepsis, whether they exert detrimental or protective effects remains unclear. Here, we will review the available experimental and clinical studies about the opposing roles of NK cells in sepsis, and we will discuss the prospects for NK cell-based immunotherapeutic strategies for sepsis.

The Alterations of Gut Microbiome and Lipid Metabolism in Patients with Spinal Muscular Atrophy.

INTRODUCTION: Spinal muscular atrophy (SMA) can cause multiple system dysfunction, especially lipid metabolic disorders, for which management strategies are currently lacking. Microbes are related to metabolism and the pathogenesis of neurological diseases. This study aimed to preliminarily explore the alterations in the gut microbiota in SMA and the potential relationship between altered microbiota and lipid metabolic disorders. METHODS: Fifteen patients with SMA and 17 gender- and age-matched healthy controls were enrolled in the study. Feces and fasting plasma samples were collected. 16S ribosomal RNA sequencing and nontargeted metabolomics analysis were performed to explore the correlation between microbiota and differential lipid metabolites. RESULTS: No significant difference was found in microbial diversity (α- and ß-diversity) between the SMA and control groups, with both groups having a relatively similar community structure. However, compared to the control group, the SMA group showed an increased relative abundance of the genera Ruminiclostridium, Gordonibacter, Enorma, Lawsonella, Frisingicoccus, and Anaerofilum and a decreased abundance of the genera Catabacter, Howardella, Marine_Methylotrophic_Group_3, and Lachnospiraceae_AC2044_group. The concurrent metabolomic analysis showed that the SMA group had 56 different kinds of lipid metabolite levels than did the control group. Additionally, the Spearman correlation suggested a correlation between the altered differential lipid metabolites and the above-mentioned altered microbiota. CONCLUSIONS: The gut microbiome and lipid metabolites differed between the patients with SMA and the control subjects. The altered microbiota may be related with the lipid metabolic disorders in SMA. However, further study is necessary to clarify the mechanism of lipid metabolic disorders and develop management strategies to improve the related complications in SMA.

Decrease of MYPT1 Is Critical for Impairment of NO-mediated Vasodilation in Mesenteric Artery of the Older Spontaneously Hypertensive Rats.

Nitric oxide (NO)-mediated vasodilatation is a fundamental response of vasculature, however, regulation of NO signaling pathway on resistance vessels in the older adult with hypertension is still unclear. The 16-week-spontaneously hypertensive rats (SHR), the 18-month-SHR (OldSHR), and the age-matched Wistar-Kyoto rats were used to study the changes of mesenteric resistance artery dilatation caused by sodium nitroprusside (SNP). After pre-vasoconstriction by norepinephrine (NE), the response of endothelium-denuded mesenteric artery ring to SNP was observed, and the changes in vascular response after pharmacological interventions of key nodes in the NO/sGC/cGMP/PKG1α signaling pathway were observed as well. RNA sequencing and functional enrichment analyses were used to provide information for conducting validation experiments. Vasodilation of NO in OldSHR was decreased, which significantly correlated with the reduction of PKG-mediated effect. Functional enrichment analysis of RNA sequencing showed that genes encoding important proteins such as sGC and MYPT1 (protein phosphatase 1 regulatory subunit 12A) were downregulated in OldSHR. Molecular biology validation results showed that mRNA expression of both α and ß subunits of sGC were reduced, while mRNA and protein expression of PKG1α were reduced in OldSHR. More importantly, the expression of MYPT1 and pS668-MYPT1 was significantly reduced in OldSHR, even under the treatment of SNP. The experiment also revealed an enhanced cAMP system in vasodilation in hypertension, while this function was completely lost in the OldSHR. Therefore, an NO-mediated decrease in vascular smooth muscle relaxation was found in the OldSHR. The dysfunction in cGMP-PKG signaling, in particular, decreased pS668-MYPT1 was mechanistically involved.

Corrigendum: The alteration of left ventricular strain in later-onset spinal muscular atrophy children.

[This corrects the article DOI: 10.3389/fncel.2022.953620.].

The alteration of left ventricular strain in later-onset spinal muscular atrophy children.

Background: Patients with spinal muscular atrophy (SMA) may suffer from multisystem injury, including an impaired cardiovascular system. However, M-mode echocardiography, the current dominant echocardiographic modality, is limited in the detection of myocardial injury. We considered the use of left ventricular strain imaging in detecting myocardial injury and explored the serum lipid profile related to cardiovascular disease in later-onset SMA children. Methods: A case-control study involving 80 patients with later-onset SMA and 80 age-, gender-, and body surface area-matched control children was conducted in a single tertiary pediatric hospital in China. Data on the left ventricular strain measured using two-dimensional speckle tracking echocardiography, left ventricular function parameters assessed by M-mode echocardiography, and serum lipid profile of these two groups were retrospectively collected for differential analysis. Results: The mean age of the 80 SMA patients were (6.87 ± 2.87) years, of which 46 were type 2 and 34 were type 3 patients. The global longitudinal strain (GLS) of the SMA group (-18.7 ± 2.9%, p < 0.001) was lower than that of the control group; the time to peak longitudinal strain (TTPLS) of the SMA group (22.9 ± 13.6 ms, p < 0.001) was higher than that of the control group, while left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS), measured by the Teichholz method of M-mode echocardiography, showed no significant differences between the two groups. In addition, independent indicators for cardiovascular risk, including total cholesterol (TC)/HDL, low-density lipoprotein (LDL)/HDL, and Apo B/Apo A1 levels, were higher in SMA children than in the control group. Conclusion: Compared with healthy controls, later-onset SMA children presented with reduced GLS and prolonged TTPLS while the LVEF and LVFS values were within normal range. In particular, whether a reduced GLS or prolonged TTPLS in later-onset SMA compared to the control group can predict the risk of future cardiomyopathy remains to be investigated.

Thromboxane-induced contractile response of mesenteric arterioles is diminished in the older rats and the older hypertensive rats.

Nearly all physiological processes are controlled at some level by G-protein-coupled receptor (GPCR) signaling activity. The thromboxane A2 (TXA2) receptor (TP) is a member of the GPCR family. The ultimate effect of TP receptor activation depends on the availability of specific G proteins, which in turn depend on the cell type, tissue, and disease state. However, the roles of the TXA2-TP signaling pathway executed under disease states are poorly defined. In this study, 16-week-spontaneously hypertensive rats (SHR), the 18-month-SHR (OldSHR), and the age-matched Wistar-Kyoto (WKY) rats were used to study the vasoconstriction of mesenteric resistance artery induced by TP-specific agonist, U-46619. Vasoconstriction induced by U-46619 was significantly attenuated in OldWKY and OldSHR rats, and mesenteric arteries with impaired response to U-46619 responded strongly to the adrenergic receptor agonist, phenylephrine. Similar vascular responses to U-46619 were obtained in endothelium-denuded mesenteric arteries. Accordingly, the expression of TP membrane proteins in mesenteric vessels was decreased, and the endogenous TP competitor, 8, 9-EET, in serum was increased, which was partly responsible for the decreased vascular reactivity of U-46619. Decreased TP membrane expression was associated with TP endocytosis, which involved actin cytoskeletal remodeling, including increased ratio of F-actin/G-actin in OldWKY and OldSHR rats. Hence, we studied the effects of TXA2 and its receptors on blood vessels and found that the TXA2-TP prostaglandin signaling pathway was impaired in older adults, which would facilitate the creation of "precision therapeutics" that possess selective efficacy in diseases.