Spinal muscular atrophy with respiratory distress type 1: Clinical phenotypes,molecular pathogenesis and therapeutic insights

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is a rare autosomal recessive neuromuscular disorder caused by mutations in the IGHMBP2 gene, which encodes immunoglobulin μ‐binding protein 2, leading to progressive spinal motor neuron degeneration. We review the data available in the literature about SMARD1. The vast majority of patients show an onset of typical symptoms in the first year of life. The main clinical features are distal muscular atrophy and diaphragmatic palsy, for which permanent supportive ventilation is required. No effective treatment is available yet, but novel therapeutic approaches, such as gene therapy, have shown encouraging results in preclinical settings and thus represent possible methods for treating SMARD1. Significant advancements in the understanding of both the SMARD1 clinical spectrum and its molecular mechanisms have allowed the rapid translation of preclinical therapeutic strategies to human patients to improve the poor prognosis of this devastating disease.     

脊髓性肌萎缩症患者尿液细胞模型的建立

脊髓性肌萎缩症(Spinal muscular atrophy, SMA)大多数在儿童或婴幼儿期发病,表现为进行性、对称性的肢体无力和肌肉萎缩,迄今尚无有效的治疗方法,是婴幼儿最常见的致死性遗传病之一。患者来源的细胞系是该病研究的重要工具,但依赖于肌肉或皮肤活检等创伤性手术的成纤维细胞培养较难被患者及家属接受。文章收集SMA患者及健康对照的新鲜尿液,进行离心、尿液沉渣培养,观察尿液细胞的生长状况,用酶联免疫吸附实验(Enzyme-linked immunosorbent assay,ELISA)分析患者尿液细胞中SMN(Survival of motor neuron)蛋白的表达量,应用免疫荧光染色观察SMN蛋白在细胞内的定位。共建立了11例SMA患者和14例健康对照的尿液细胞系,尿液细胞体外增殖旺盛,细胞形态及生长速度较稳定。患者来源的尿液细胞SMN1(Survival of motor neuron 1) 基因缺失突变、SMN蛋白表达量降低,荧光染色提示SMN蛋白在胞浆和胞核中均有定位。尿液细胞培养步骤简单、无创伤性、患儿及其家属的依从性好,是获取和保存病人来源标本的有效方法,在脊髓性肌萎缩症发病机制研究和临床应用方面具有较好的应用价值。     

Smn deficiency causes neuritogenesis and neurogenesis defects in the retinal neurons of a mouse model of spinal muscular atrophy

The eye is an excellent model for the study of neuronal development and pathogenesis of central nervous system disorders because of its relative ease of accessibility and the well‐characterized cellular makeup. We have used this model to study spinal muscular atrophy (SMA), an autosomal recessive neuromuscular disease caused by deletions or mutations in the survival of motor neuron 1 gene (SMN1). We have investigated the expression pattern of mouse Smn mRNA and protein in the neural retina and the optic nerve of wild type mice. Smn protein is present in retinal ganglion cells and amacrine cells within the neural retina as well as in glial cells in the optic nerve. Histopathological analysis in phenotype stage SMA mice revealed that Smn deficiency is associated with a reduction in ganglion cell axon and glial cell number in the optic nerve, as well as compromised cellular processes and altered organization of neurofilaments in the neural retina. Whole mount preparation and retinal neuron primary culture provided further evidence of abnormal synaptogenesis and neurofilament accumulation in the neurites of Smn‐deficient retinal neurons. A subset of amacrine cells is absent, in a cell‐autonomous fashion, in the retina of SMA mice. Finally, the retinas of SMA mice have altered electroretinograms. Altogether, our study has demonstrated defects in axodendritic outgrowth and cellular composition in Smn‐depleted retinal neurons, indicating a role for Smn in neuritogenesis and neurogenesis, and providing us with an insight into pathogenesis of SMA. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 71: 153‐169, 2011     

Recapitulation of spinal motor neuron-specific disease phenotypes in a human cell model of spinal muscular atrophy

Establishing human cell models of spinal muscular atrophy (SMA) to mimic motor neuron-specific phenotypes holds the key to understanding the pathogenesis of this devastating disease. Here, we developed a closely representative cell model of SMA by knocking down the disease-determining gene, survival motor neuron (SMN), in human embryonic stem cells (hESCs). Our study with this cell model demonstrated that knocking down of SMN does not interfere with neural induction or the initial specification of spinal motor neurons. Notably, the axonal outgrowth of spinal motor neurons was significantly impaired and these disease-mimicking neurons subsequently degenerated. Furthermore, these disease phenotypes were caused by SMN-full length (SMN-FL) but not SMN-Δ7 (lacking exon 7) knockdown, and were specific to spinal motor neurons. Restoring the expression of SMN-FL completely ameliorated all of the disease phenotypes, including specific axonal defects and motor neuron loss. Finally, knockdown of SMN-FL led to excessive mitochondrial oxidative stress in human motor neuron progenitors. The involvement of oxidative stress in the degeneration of spinal motor neurons in the SMA cell model was further confirmed by the administration of N-acetylcysteine, a potent antioxidant, which prevented disease-related apoptosis and subsequent motor neuron death. Thus, we report here the successful establishment of an hESC-based SMA model, which exhibits disease gene isoform specificity, cell type specificity, and phenotype reversibility. Our model provides a unique paradigm for studying how motor neurons specifically degenerate and highlights the potential importance of antioxidants for the treatment of SMA.     

In vitro and ex vivo evaluation of second-generation histone deacetylase inhibitors for the treatment of spinal muscular atrophy

Among a panel of histone deacetylase (HDAC) inhibitors investigated, suberoylanilide hydroxamic acid (SAHA) evolved as a potent and non-toxic candidate drug for the treatment of spinal muscular atrophy (SMA), an alpha-motoneurone disorder caused by insufficient survival motor neuron (SMN) protein levels. SAHA increased SMN levels at low micromolar concentrations in several neuroectodermal tissues, including rat hippocampal brain slices and motoneurone-rich cell fractions, and its therapeutic capacity was confirmed using a novel human brain slice culture assay. SAHA activated survival motor neuron gene 2 (SMN2), the target gene for SMA therapy, and inhibited HDACs at submicromolar doses, providing evidence that SAHA is more efficient than the HDAC inhibitor valproic acid, which is under clinical investigation for SMA treatment. In contrast to SAHA, the compounds m-Carboxycinnamic acid bis-Hydroxamide, suberoyl bishydroxamic acid and M344 displayed unfavourable toxicity profiles, whereas MS-275 failed to increase SMN levels. Clinical trials have revealed that SAHA, which is under investigation for cancer treatment, has a good oral bioavailability and is well tolerated, allowing in vivo concentrations shown to increase SMN levels to be achieved. Because SAHA crosses the blood-brain barrier, oral administration may allow deceleration of progressive alpha-motoneurone degeneration by epigenetic SMN2 gene activation.     

Salbutamol inhibits ubiquitin-mediated survival motor neuron protein degradation in spinal muscular atrophy cells

Spinal muscular atrophy (SMA) is a common autosomal recessive neuromuscular disorder that is currently incurable. SMA is caused by decreased levels of the survival motor neuron protein (SMN), as a result of loss or mutation of SMN1. Although the SMN1 homolog SMN2 also produces some SMN protein, it does not fully compensate for the loss or dysfunction of SMN1. Salbutamol, a β2-adrenergic receptor agonist and well-known bronchodilator used in asthma patients, has recently been shown to ameliorate symptoms in SMA patients. However, the precise mechanism of salbutamol action is unclear. We treated SMA fibroblast cells lacking SMN1 and HeLa cells with salbutamol and analyzed SMN2 mRNA and SMN protein levels in SMA fibroblasts, and changes in SMN protein ubiquitination in HeLa cells. Salbutamol increased SMN protein levels in a dose-dependent manner in SMA fibroblast cells lacking SMN1, though no significant changes in SMN2 mRNA levels were observed. Notably, the salbutamol-induced increase in SMN was blocked by a protein kinase A (PKA) inhibitor and deubiquitinase inhibitor, respectively. Co-immunoprecipitation assay using HeLa cells showed that ubiquitinated SMN levels decreased in the presence of salbutamol, suggesting that salbutamol inhibited ubiquitination. The results of this study suggest that salbutamol may increase SMN protein levels in SMA by inhibiting ubiquitin-mediated SMN degradation via activating β2-adrenergic receptor-PKA pathways.     

A spinal muscular atrophy modifier implicates the SMN protein in SNARE complex assembly at neuromuscular synapses

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Molecular and phenotypic reassessment of an infrequently used mouse model for spinal muscular atrophy

Proximal spinal muscular atrophy (SMA) results from loss of the survival motor neuron 1 (SMN1) gene, with retention of its nearly identical homolog, SMN2. There is a direct correlation between disease severity and SMN2 copy number. Mice do not have a Smn2 gene, and thus cannot naturally replicate the disorder. However, two murine models of SMA have been generated using SMN2-BAC transgenic mice bred onto a mutant Smn background. In these instances mice die shortly after birth, have variable phenotypes within the same litter, or completely correct the SMA phenotype. Both models have been imported to The Jackson Laboratory for distribution to the research community. To ensure that similar results are obtained after importation to The Jackson Laboratory to what was originally reported in the literature, we have begun a molecular and phenotypic evaluation of these mouse models. Here we report our findings for the SMA mouse model that has been deposited by the Li group from Taiwan. These mice, JAX stock number TJL-005058, are homozygous for the SMN2 transgene, Tg(SMN2)2Hung, and a targeted Smn allele that lacks exon 7, Smn1^tm1Hung. Our findings are consistent with those reported originally for this line and clarify some of the original data. In addition, we have cloned and mapped the integration site for Tg(SMN2)2Hung to Chromosome 4, and provide a simple genotyping assay that is specific to the junction fragment. Finally, based upon the survival data from our genetic crosses, we suggest that this underused SMA model may be a useful compliment or alternative to the more commonly used “delta7” SMA mouse. We provide breeding schemes in which two genotypes of mice can be generated so that 50% of the litter will be SMA-like pups while 50% will be controls.     

脊髓性肌萎缩症SMN1基因2+0基因型携带者的家系研究

脊髓性肌萎缩症(spinal muscular atrophy, SMA)是一种儿童时期较为常见的神经肌肉病,属于常染色体隐性遗传。绝大多数SMA由运动神经元存活基因1 (survival motor neuron 1, SMN1)的纯合缺失突变所致。而SMN1的2+0基因型个体作为一种特殊的SMA携带者,给携带者筛查以及家系的遗传咨询带来了巨大的挑战。已有研究表明,g.27134T>G和g.27706₂7707delAT多态位点变异对于Ashkenazi犹太人群中的2+0基因型个体具有提示作用。为进一步探究这两个多态位点是否在中国人群也具有特异性,本研究纳入了44例家系成员和204例已知SMN1基因拷贝数的对照样本。44例家系成员来自于9个无关的SMN1基因纯合缺失的SMA家系,先证者双亲之一疑似为2+0基因型携带者。利用多重连接探针扩增(multiplex ligation-dependent probe amplification,MLPA)和短串联重复(short tandem repeat, STR)连锁分析进行基因型的鉴定以及多态位点的筛查,最终...     

Spinal muscular atrophy pathogenic mutations impair the axonogenic properties of axonal-survival of motor neuron

The axonal survival of motor neuron (a-SMN) protein is a truncated isoform of SMN1, the spinal muscular atrophy (SMA) disease gene. a-SMN is selectively localized in axons and endowed with remarkable axonogenic properties. At present, the role of a-SMN in SMA is unknown. As a first step to verify a link between a-SMN and SMA, we investigated by means of over-expression experiments in neuroblastoma-spinal cord hybrid cell line (NSC34) whether SMA pathogenic mutations located in the N-terminal part of the protein affected a-SMN function. We demonstrated here that either SMN1 missense mutations or small intragenic re-arrangements located in the Tudor domain consistently altered the a-SMN capability of inducing axonal elongation in vitro. Mutated human a-SMN proteins determined in almost all NSC34 motor neurons the growth of short axons with prominent morphologic abnormalities. Our data indicate that the Tudor domain is critical in dictating a-SMN function possibly because it is an association domain for proteins involved in axon growth. They also indicate that Tudor domain mutations are functionally relevant not only for FL-SMN but also for a-SMN, raising the possibility that also a-SMN loss of function may contribute to the pathogenic steps leading to SMA.     

Mildly affected patients with spinal muscular atrophy are partially protected by an increased SMN2 copy number

Spinal muscular atrophy (SMA) is a recessive neuromuscular disorder caused by loss of the SMN1 gene. The clinical distinction between SMA type I to IV reflects different age of onset and disease severity. SMN2, a nearly identical copy gene of SMN1, produces only 10% of full-length SMN RNA/protein and is an excellent target for a potential therapy. Several clinical trials with drugs that increase the SMN2 expression such as valproic acid and phenylbutyrate are in progress. Solid natural history data for SMA are crucial to enable a correlation between genotype and phenotype as well as the outcome of therapy. We provide genotypic and phenotypic data from 115 SMA patients with type IIIa (age of onset <3 years), type IIIb (age of onset >3 years) and rare type IV (onset >30 years). While 62% of type IIIa patients carry two or three SMN2 copies, 65% of type IIIb patients carry four or five SMN2 copies. Three type IV SMA patients had four and one had six SMN2 copies. Our data support the disease-modifying role of SMN2 leading to later onset and a better prognosis. A statistically significant correlation for ≥4 SMN2 copies with SMA type IIIb or a milder phenotype suggests that SMN2 copy number can be used as a clinical prognostic indicator in SMA patients. The additional case of a foetus with homozygous SMN1 deletion and postnatal measurement of five SMN2 copies illustrates the role of genotypic information in making informed decisions on the management and therapy of such patients.Database: SMN1—OMIM: 600354; GeneBank: U18423, SMN2—OMIM: 601627: GeneBank: NM_022875     

Counteracting chromatin effects of a splicing-correcting antisense oligonucleotide improves its therapeutic efficacy in spinal muscular atrophy

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Drugs,genes and screens: The ethics of preventing and treating spinal muscular atrophy

Spinal muscular atrophy (SMA) is the most common genetic disease that causes infant mortality. Its treatment and prevention represent the paradigmatic example of the ethical dilemmas of 21st-century medicine. New therapies (nusinersen and AVXS-101) hold the promise of being able to treat, but not cure, the condition. Alternatively, genomic analysis could identify carriers, and carriers could be offered in vitro fertilization and preimplantation genetic diagnosis. In the future, gene editing could prevent the condition at the embryonic stage. How should these different options be evaluated and compared within a health system? In this paper, we discuss the ethical considerations that bear on the question of how to prioritize the different treatments and preventive options for SMA, at a policy level. We argue that despite the tremendous value of what we call ‘ex-post’ approaches to treating SMA (such as using pharmacological agents or gene therapy), there is a moral imperative to pursue ‘ex-ante’ interventions (such as carrier screening in combination with prenatal testing and preimplantation genetic diagnosis, or gene editing) to reduce the incidence of SMA. There are moral reasons relating to autonomy, beneficence and justice to prioritize ex-ante methods over ex-post methods.     

Molecular,genetic and stem cell‐mediated therapeutic strategies for spinal muscular atrophy (SMA)

Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease. It is the first genetic cause of infant mortality. It is caused by mutations in the survival motor neuron 1 (SMN1) gene, leading to the reduction of SMN protein. The most striking component is the loss of alpha motor neurons in the ventral horn of the spinal cord, resulting in progressive paralysis and eventually premature death. There is no current treatment other than supportive care, although the past decade has seen a striking advancement in understanding of both SMA genetics and molecular mechanisms. A variety of disease modifying interventions are rapidly bridging the translational gap from the laboratory to clinical trials. In this review, we would like to outline the most interesting therapeutic strategies that are currently developing, which are represented by molecular, gene and stem cell‐mediated approaches for the treatment of SMA.     

Genetic carrier screening for spinal muscular atrophy and spinal muscular atrophy with respiratory distress 1 in an isolated population in Israel

Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by progressive muscle weakness. It is caused by a mutation in the survival motor neuron gene 1 (SMN1) gene. SMA with respiratory distress 1 (SMARD1), an uncommon variant of infantile SMA also inherited in an autosomal recessive manner, is caused by mutations in the immunoglobulin mu-binding protein 2 (IGHMBP2) gene. We carried out genetic carrier screening among the residents of an isolated Israeli Arab village with a high frequency of SMA in order to identify carriers of SMA type I and SMARD1. During 2006, 168 women were tested for SMA, of whom 13.1% were found to be carriers. Of 111 women tested for SMARD1, 9.9% were found to be carriers. Prenatal diagnosis was performed in one couple where both spouses were carriers of SMARD1; the fetus was found to be affected, and the pregnancy was terminated. To the best of our knowledge, this is the first example of the establishment of a large-scale carrier-screening program for SMA and SMARD1 in an isolated population. SMA has a carrier frequency of 1:33-1:60 in most populations and should be considered for inclusion in a population-based genetic-screening program.