Spinal muscular atrophy (SMA) is one of the most common inherited forms of neurological disease leading to infant mortality. Patients exhibit selective loss of lower motor neurons resulting in muscle weakness, paralysis, and often death. Although patient fibroblasts have been used extensively to study SMA, motor neurons have a unique anatomy and physiology which may underlie their vulnerability to the disease process. Here we report the generation of induced pluripotent stem (iPS) cells from skin fibroblast samples taken from a child with SMA. These cells expanded robustly in culture, maintained the disease genotype, and generated motor neurons that showed selective deficits compared to those derived from the child's unaffected mother. This is the first study to show human iPS cells can be used to model the specific pathology seen in a genetically inherited disease. As such, it represents a promising resource to study disease mechanisms, screen novel drug compounds, and develop new therapies.Spinal muscular atrophy (SMA) is an autosomal recessive genetic disorder caused by mutations in the survival motor neuron 1 gene (SMN1) significantly reducing SMN protein expression 1, 2 and resulting in the selective degeneration of lower α-motor neurons 3 . Clinically, patients with SMA 1 typically show symptoms at 6 months of age and die by age 2 4 . The SMN2 gene is an almost identical copy of SMN1 except that SMN2 has a single nucleotide difference that results in only 10% of full-length protein being produced and high levels of a truncated, unstable protein lacking exon 7 (SMNΔ7) 5 . However, patients with multiple copies of SMN2 produce more full-length protein and have a less severe phenotype 6 . While current model systems using worms, flies, or mice have provided invaluable data concerning the genetic cause of SMA, mechanisms of motor neuron death, and potential drug therapies 7 , they have Correspondence: Reprints and permissions information is available at npg.nature.com/reprintsandpermissions Correspondence should be addressed to ADE (ebert@waisman.wisc.edu) or CNS (cnsvendsen@wisc.edu). Requests for material should be addressed to CNS. Contributions: ADE participated in all aspects and prepared the manuscript; JY generated and aided in characterization of iPS-SMA and iPS-WT clones; FR, VBM, and CLL performed SMN analysis and manuscript preparation; JAT participated in the generation of the iPS clones; CNS conceived the project and participated in planning, data analysis, and manuscript preparation. The authors declare no competing financial interest.Supplementary Information accompanies the paper on www.nature.com/nature. A schematic outlining the main results is included as Supplementary Figure 1.

SMN1 and SMN2 (survival motor neuron) encode identical proteins. A critical question is why only the homozygous loss of SMN1, and not SMN2, results in spinal muscular atrophy (SMA). Analysis of transcripts from SMN1/SMN2 hybrid genes and a new SMN1 mutation showed a direct relationship between presence of disease and exon 7 skipping. We have reported previously that the exon-skipped product SMNDelta7 is partially defective for self-association and SMN self-oligomerization correlated with clinical severity. To evaluate systematically which of the five nucleotides that differ between SMN1 and SMN2 effect alternative splicing of exon 7, a series of SMN minigenes was engineered and transfected into cultured cells, and their transcripts were characterized. Of these nucleotide differences, the exon 7 C-to-T transition at codon 280, a translationally silent variance, was necessary and sufficient to dictate exon 7 alternative splicing. Thus, the failure of SMN2 to fully compensate for SMN1 and protect from SMA is due to a nucleotide exchange (C/T) that attenuates activity of an exonic enhancer. These findings demonstrate the molecular genetic basis for the nature and pathogenesis of SMA and illustrate a novel disease mechanism. Because individuals with SMA retain the SMN2 allele, therapy targeted at preventing exon 7 skipping could modify clinical outcome.

Spinal muscular atrophy (SMA) is a recessive disorder characterized by loss of motor neurons in the spinal cord. It is caused by mutations in the telomeric survival motor neuron 1 ( SMN1 ) gene. Alterations within an almost identical copy gene, the centromeric survival motor neuron 2 ( SMN2 ) gene produce no known phenotypic effect. The exons of the two genes differ by just two nucleotides, neither of which alters the encoded amino acids. At the genomic level, only five nucleotides that differentiate the two genes from one another have been reported. The entire genomic sequence of the two genes has not been determined. Thus, differences which might explain why SMN1 is the SMA gene are not readily apparent. In this study, we have completely sequenced and compared genomic clones containing the SMN genes. The two genes show striking similarity, with the homology being unprecedented between two different yet functional genes. The only critical difference in an approximately 32 kb region between the two SMN genes is the C->T base change 6 bp inside exon 7. This alteration but not other variations in the SMN genes affects the splicing pattern of the genes. The majority of the transcript from the SMN1 locus is full length, whereas the majority of the transcript produced by the SMN2 locus lacks exon 7. We suggest that the exon 7 nucleotide change affects the activity of an exon splice enhancer. In SMA patients, the loss of SMN1 but the presence of SMN2 results in low levels of full-length SMN transcript and therefore low SMN protein levels which causes SMA.

Spinal muscular atrophy (SMA) is a motor-neuron disorder resulting from anterior-horn-cell death. The autosomal recessive form has a carrier frequency of 1 in 50 and is the most common genetic cause of infant death. SMA is categorized as types I-III, ranging from severe to mild, based upon age of onset and clinical course. Two closely flanking copies of the survival motor neuron (SMN) gene are on chromosome 5q13 (ref. 1). The telomeric SMN (SMN1) copy is homozygously deleted or converted in >95% of SMA patients, while a small number of SMA disease alleles contain missense mutations within the carboxy terminus. We have identified a modular oligomerization domain within exon 6 of SMN1. All previously identified missense mutations map within or immediately adjacent to this domain. Comparison of wild-type to mutant SMN proteins of type I, II and III SMA patients showed a direct correlation between oligomerization and clinical type. Moreover, the most abundant centromeric SMN product, which encodes exons 1-6 but not 7, demonstrated reduced self-association. These findings identify decreased SMN self-association as a biochemical defect in SMA, and imply that disease severity is proportional to the intracellular concentration of oligomerization-competent SMN proteins.

The survival motor neuron genes, SMN1 and SMN2, encode identical proteins; however, only homo- zygous loss of SMN1 correlates with the development of spinal muscular atrophy (SMA). We have previously shown that a single non-polymorphic nucleotide difference in SMN exon 7 dramatically affects SMN mRNA processing. SMN1 primarily produces a full-length RNA whereas SMN2 expresses dramatically reduced full-length RNA and abundant levels of an aberrantly spliced transcript lacking exon 7. The importance of proper exon 7 processing has been underscored by the identification of several mutations within splice sites adjacent to exon 7. Here we show that an AG-rich exonic splice enhancer (ESE) in the center of SMN exon 7 is required for inclusion of exon 7. This region functioned as an ESE in a heterologous context, supporting efficient in vitro splicing of the Drosophila double-sex gene. Finally, the protein encoded by the exon-skipping event, Delta7, was less stable than full-length SMN, providing additional evidence of why SMN2 fails to compensate for the loss of SMN1 and leads to the development of SMA.

Spinal muscular atrophy (SMA), a common motor neuron disease in humans, results from loss of functional survival motor neuron (SMN1) alleles. A nearly identical copy of the gene, SMN2, fails to provide protection from SMA because of a single translationally silent nucleotide difference in exon 7. This likely disrupts an exonic splicing enhancer and causes exon 7 skipping, leading to abundant production of a shorter isoform, SMN2⌬7. The truncated transcript encodes a less stable protein with reduced self-oligomerization activity that fails to compensate for the loss of SMN1. This report describes the identification of an in vivo regulator of SMN mRNA processing. Htra2-␤1, an SR-like splicing factor and ortholog of Drosophila melanogaster transformer-2, promoted the inclusion of SMN exon 7, which would stimulate full-length SMN2 expression. Htra2-␤1 specifically functioned through and bound an AG-rich exonic splicing enhancer in SMN exon 7. This effect is not speciesspecific as expression of Htra2-␤1 in human or mouse cells carrying an SMN2 minigene dramatically increased production of full-length SMN2. This demonstrates that SMN2 mRNA processing can be modulated in vivo. Because all SMA patients retain at least one SMN2 copy, these results show that an in vivo modulation of SMN RNA processing could serve as a therapeutic strategy to prevent SMA. P roximal spinal muscular atrophy (SMA) is a neurodegenerative disorder with progressive paralysis caused by the loss of ␣-motor neurons in the spinal cord. With an incidence of 1 in 10,000 live births and a carrier frequency of 1 in 50, SMA is the second most common autosomal recessive disorder and the most frequent genetic cause of infantile death (1). SMA patients are subdivided into types I-III according to age of onset and achieved motor abilities (2). All three forms of proximal SMA are caused by mutations within the telomeric copy of the survival motor neuron gene, SMN1 (3). Some 96.4% of 5q-linked SMA patients show homozygous absence of SMN1 caused by deletions or gene conversions, whereas 3.6% display rare subtle mutations (3, 4). Homozygous absence of SMN2 is found in 5% of control individuals; however, loss of SMN2 has no phenotypic effect (3). SMN1 produces exclusively full-length (FL) SMN mRNA. In contrast, SMN2 expresses dramatically reduced FL and abundant levels of transcript lacking exon 7, SMN2⌬7. SMN2 is retained by all patients and a correlation between the SMN2 protein level and the disease state is established (5, 6). This spliced isoform encodes a truncated, less stable protein with reduced self-oligomerization activity (3,7,8). We have shown that inclusion of exon 7 in SMN1-derived transcripts and exclusion of this exon in SMN2-derived transcripts is determined by a single nucleotide difference at position ϩ6 in SMN exon 7 (C in SMN1; T in SMN2). This nucleotide difference is nonpolymorphic in the SMN2 gene and likely disrupts an exonic splicing enhancer (ESE) (9, 10).The removal of introns and joining of exons is performed by the spliceosome, a ma...

Spinal muscular atrophy (SMA) is an autosomal recessive disorder that is the leading genetic cause of infantile death. SMA is characterized by loss of motor neurons in the ventral horn of the spinal cord, leading to weakness and muscle atrophy. SMA occurs as a result of homozygous deletion or mutations in Survival Motor Neuron-1 (SMN1). Loss of SMN1 leads to a dramatic reduction in SMN protein, which is essential for motor neuron survival. SMA disease severity ranges from extremely severe to a relatively mild adult onset form of proximal muscle atrophy. Severe SMA patients typically die mostly within months or a few years as a consequence of respiratory insufficiency and bulbar paralysis. SMA is widely known as a motor neuron disease; however, there are numerous clinical reports indicating the involvement of additional peripheral organs contributing to the complete picture of the disease in severe cases. In this review, we have compiled clinical and experimental reports that demonstrate the association between the loss of SMN and peripheral organ deficiency and malfunction. Whether defective peripheral organs are a consequence of neuronal damage/ muscle atrophy or a direct result of SMN loss will be discussed.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been rapidly evolving in the form of new variants. At least eleven known variants have been reported. The objective of this study was to delineate the differences in the mutational profile of Delta and Delta Plus variants. High-quality sequences (n = 1756) of Delta (B.1.617.2) and Delta Plus (AY.1 or B.1.617.2.1) variants were used to determine the prevalence of mutations (≤20 %) in the entire SARS-CoV-2 genome, their co-existence, and change in prevalence over a period of time. Structural analysis was conducted to get insights into the impact of mutations on antibody binding. A Sankey diagram was generated using phylogenetic analysis coupled with sequence-acquisition dates to infer the migration of the Delta Plus variant and its presence in the United States. The Delta Plus variant had a significant number of high-prevalence mutations (≤20 %) than in the Delta variant. Signature mutations in Spike (G142D, A222V, and T95I) existed at a more significant percentage in the Delta Plus variant than the Delta variant. Three mutations in Spike (K417N, V70F, and W258L) were exclusively present in the Delta Plus variant. A new mutation was identified in ORF1a (A1146T), which was only present in the Delta Plus variant with ∼58 % prevalence. Furthermore, five key mutations (T95I, A222V, G142D, R158G, and K417N) were significantly more prevalent in the Delta Plus than in the Delta variant. Structural analyses revealed that mutations alter the sidechain conformation to weaken the interactions with antibodies. Delta Plus, which first emerged in India, reached the United States through England and Japan, followed by its spread to more than 20 the United States. Based on the results presented here, it is clear that the Delta and Delta Plus variants have unique mutation profiles, and the Delta Plus variant is not just a simple addition of K417N to the Delta variant. Highly correlated mutations may have emerged to keep the structural integrity of the virus.