A rare variant (c.863G>T) in exon 7 of SMN1 disrupts mRNA splicing and is responsible for spinal muscular atrophy

Abstract

Proximal spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by deletion or mutation of SMN1 (survival motor neuron 1). SMN exon 7 splicing is regulated by a number of exonic and intronic regulatory sequences and the trans-factors that bind them. Variants located in or near these regulated regions should be evaluated to determine their effect on splicing. We identified the rare variant c.863G4T (r.835_*3del, p.Gly279Glufs*5) in exon 7 of SMN1 in three patients affected with type I or type II SMA. Most of the SMN1 transcripts exhibited complete loss of exon 7 in vivo. The ex vivo splicing assay demonstrated that the variant disrupts inclusion of exon 7 (~85%) in the SMN1 mRNA; replacement with various bases yielded a variety of splicing effects in SMN1 and SMN2 pre-mRNA. The c.863G4T (r.835_*3del, p.Gly279Glufs*5) variant is located in a region that includes binding sites for multiple splicing factors including Tra2β1. Thus, the variant disrupts Tra2β1 binding, but does not affect binding of hnRNP A1. These findings demonstrate how rare variants influence pre-mRNA splicing of SMN and reveal the functional influence of c.863G4T (r.835_*3del, p.Gly279Glufs*5) variant in patients with SMA.

Spinal Muscular Atrophy (Werdnig-Hoffmann Atrophy/Disease): Two Case Presentations and Literature Review

“…The genetic method, which consists in determining the deletion of 7 and/or 8 exons in the SMN1 gene, is the "gold standard" for diagnosis 13,20 . The deletions in the specified areas of the gene, in a patient in a homozygous state, confirm the diagnosis of SMA.…”

Spinal Muscular Atrophy (Werdnig-Hoffmann Atrophy/Disease): Two Case Presentations and Literature Review

“…The genetic method, which consists in determining the deletion of 7 and/or 8 exons in the SMN1 gene, is the "gold standard" for diagnosis 13,20 . The deletions in the specified areas of the gene, in a patient in a homozygous state, confirm the diagnosis of SMA.…”

Mutation Spectrum of the Survival of Motor Neuron 1 and Functional Analysis of Variants in Chinese Spinal Muscular Atrophy

“…This variant influenced SMN1 pre-mRNA splicing by disrupting the exonic splicing enhancer that binds to Tra2b1. 20 The other variant, c.835-5 T>G, located in the intron 6 3 0 -splicing region, reached approximately 93.3% disruption of mRNA exon 7 inclusion. To determine the effects of the c.835-5 site on exon 7 splicing, we also constructed the other two base substitutions in the c.835-5 site (c.835-5 T>A and c.835-5 T>C).…”

Genetic diagnosis of Mendelian disorders via RNA sequencing

“…Some of them can be deeply located in intronic sequences 15 and are thus not covered by WES. Hence, direct probing of splice isoforms by RNA-seq is important, and has led to the discovery of multiple splicing defects based on single gene studies: skipping of multiple exons (exon 45–55) 16 and creation of a new exon by a deep intronic variant in DMD 17 , intron retention in LMNA caused by a 5′ splice site variant 18 , and skipping of exon 7 in SMN1 caused by a variant in a splicing factor binding site 19 . Altogether, RNA-seq promises to be an important complementary tool to facilitate molecular diagnosis of rare genetic disorders.…”

Spinal Muscular Atrophy (Werdnig-Hoffmann Atrophy/Disease): Two Case Presentations and Literature Review

“…The genetic method, which consists in determining the deletion of 7 and/or 8 exons in the SMN1 gene, is the "gold standard" for diagnosis 13,20 . The deletions in the specified areas of the gene, in a patient in a homozygous state, confirm the diagnosis of SMA.…”

Mutation Spectrum of the Survival of Motor Neuron 1 and Functional Analysis of Variants in Chinese Spinal Muscular Atrophy

“…This variant influenced SMN1 pre-mRNA splicing by disrupting the exonic splicing enhancer that binds to Tra2b1. 20 The other variant, c.835-5 T>G, located in the intron 6 3 0 -splicing region, reached approximately 93.3% disruption of mRNA exon 7 inclusion. To determine the effects of the c.835-5 site on exon 7 splicing, we also constructed the other two base substitutions in the c.835-5 site (c.835-5 T>A and c.835-5 T>C).…”

Genetic diagnosis of Mendelian disorders via RNA sequencing

“…Some of them can be deeply located in intronic sequences 15 and are thus not covered by WES. Hence, direct probing of splice isoforms by RNA-seq is important, and has led to the discovery of multiple splicing defects based on single gene studies: skipping of multiple exons (exon 45–55) 16 and creation of a new exon by a deep intronic variant in DMD 17 , intron retention in LMNA caused by a 5′ splice site variant 18 , and skipping of exon 7 in SMN1 caused by a variant in a splicing factor binding site 19 . Altogether, RNA-seq promises to be an important complementary tool to facilitate molecular diagnosis of rare genetic disorders.…”

Spinal Muscular Atrophy (Werdnig-Hoffmann Atrophy/Disease): Two Case Presentations and Literature Review

“…The genetic method, which consists in determining the deletion of 7 and/or 8 exons in the SMN1 gene, is the "gold standard" for diagnosis 13,20 . The deletions in the specified areas of the gene, in a patient in a homozygous state, confirm the diagnosis of SMA.…”

Mutation Spectrum of the Survival of Motor Neuron 1 and Functional Analysis of Variants in Chinese Spinal Muscular Atrophy

“…This variant influenced SMN1 pre-mRNA splicing by disrupting the exonic splicing enhancer that binds to Tra2b1. 20 The other variant, c.835-5 T>G, located in the intron 6 3 0 -splicing region, reached approximately 93.3% disruption of mRNA exon 7 inclusion. To determine the effects of the c.835-5 site on exon 7 splicing, we also constructed the other two base substitutions in the c.835-5 site (c.835-5 T>A and c.835-5 T>C).…”

Genetic diagnosis of Mendelian disorders via RNA sequencing

“…Some of them can be deeply located in intronic sequences 15 and are thus not covered by WES. Hence, direct probing of splice isoforms by RNA-seq is important, and has led to the discovery of multiple splicing defects based on single gene studies: skipping of multiple exons (exon 45–55) 16 and creation of a new exon by a deep intronic variant in DMD 17 , intron retention in LMNA caused by a 5′ splice site variant 18 , and skipping of exon 7 in SMN1 caused by a variant in a splicing factor binding site 19 . Altogether, RNA-seq promises to be an important complementary tool to facilitate molecular diagnosis of rare genetic disorders.…”