A rareSMN2variant in a previously unrecognized composite splicing regulatory element induces exon 7 inclusion and reduces the clinical severity of spinal muscular atrophy

Abstract

ABSTRACT:Spinal muscular atrophy (SMA) is a common neuromuscular disorder caused by homozygous inactivation of the SMN1 (Survival Motor Neuron 1) gene. The disease severity is mainly influenced by the copy number of SMN2, a nearly identical gene from which only low amounts of full-length mRNA are produced. This correlation is not absolute, suggesting the existence of yet unknown factors modulating disease progression. We identified and characterized the rare variant c.859G>C (p.Gly287Arg) in exon 7 in both SMN2 copies of a male patient affected with type III SMA, a milder form of the disease rarely associated with only two SMN2 copies. We demonstrated in vivo, in this patient and in a second unrelated patient, and ex vivo, using SMN splicing assays, that the variant induces inclusion of exon 7 into SMN2 mRNA. Moreover, we show that the c.859G>C variation is located in a composite splicing regulatory element in the centre of exon 7. The variation does not affect binding of HTra2β nor creates a novel SF2/ASF enhancer, but disrupts an hnRNP A1 binding site. The natural occurrence of enhanced inclusion of SMN2 exon 7 in milder SMA cases supports the current therapeutic strategies based on splicing modulation in SMA patients.

Clinical and molecular cross-sectional study of a cohort of adult type III spinal muscular atrophy patients: clues from a biomarker study

“…This hypothesis is supported by the previous report of the same mutation in another patient, 35 affected by SMA type III as well; however, in that patient, SMN2 gene copy number was not assessed. Also, the frequency of the G287R variant in our cohort was much higher than previously reported (about 9% vs 1% 38 ). This variant has a positive effect in the inclusion of exon 7 in SMN2 transcripts.…”

“…This variant has a positive effect in the inclusion of exon 7 in SMN2 transcripts. 32,38 All our patients bearing the G287R variant were type IIIb subjects, thus raising the prevalence of the mutation in this group of patients up to 16% (4/25 individuals). Because of the positive effect of this variant in exon 7 inclusion in SMN2 transcripts, this finding is not unexpected.…”

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

“…19,25,34 A study of the c.859G4C variant (at position +25 of exon 7) showed that the central portion of exon 7 (positions +16-+39) also bind the splicing-inhibitor hnRNP A1 and the splicing activator SF2/ASF. 35 Our RNA pull-down assays showed that SF2/ASF does not bind exon 7 at positions +19-+41, consistent with predictions generated in ESEfinder 2.0. The findings of Vezain et al 35 led us to suggest that the important nucleotides for SF2/ASF binding may lie within positions +16-+19 of exon 7, not +19-+41.…”

Germline copy number variation of genes involved in chromatin remodelling in families suggestive of Li-Fraumeni syndrome with brain tumours

“…To assess the impact of the CNV on gene expression, we developed, according to the same strategy, a QMPSF assay performed on RT, as previously described. 18 Total RNA was extracted from peripheral blood samples, using the PAXgene Blood RNA kit (Qiagen). Reverse transcription-QMPSF (RT-QMPSF) was performed on 100 ng of RNA, using SF3A1 and TOP1 amplicons as controls.…”