A-44G transition in SMN2 intron 6 protects patients with spinal muscular atrophy

Abstract

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by reduced expression of survival of motor neuron (SMN), a protein expressed in humans by two paralogous genes, SMN1 and SMN2. These genes are nearly identical, except for 10 single-nucleotide differences and a 5-nucleotide insertion in SMN2. SMA is subdivided into four main types, with type I being the most severe. SMN2 copy number is a key positive modifier of the disease, but it is not always inversely correlated with clinical severity. We previously reported the c.859G > C variant in SMN2 exon 7 as a positive modifier in several patients. We have now identified A-44G as an additional positive disease modifier, present in a group of patients carrying 3 SMN2 copies but displaying milder clinical phenotypes than other patients with the same SMN2 copy number. One of the three SMN2 copies appears to have been converted from SMN1, but except for the C6T transition, no other changes were detected. Analyzed with minigenes, SMN1C6T displayed a ∼20% increase in exon 7 inclusion, compared to SMN2. Through systematic mutagenesis, we found that the improvement in exon 7 splicing is mainly attributable to the A-44G transition in intron 6. Using RNA-affinity chromatography and mass spectrometry, we further uncovered binding of the RNA-binding protein HuR to the -44 region, where it acts as a splicing repressor. The A-44G change markedly decreases the binding affinity of HuR, resulting in a moderate increase in exon 7 inclusion.

Mechanism of Splicing Regulation of Spinal Muscular Atrophy Genes

“…1C). A C-to-T substitution at the sixth position (C6U) of exon 7, a G-to-A substitution at the -44th position (G-44A) of intron 6, and an A-to-G substitution at the 100th position (A100G) of intron 7 are associated with skipping of SMN2 exon 7 [37–40]. Recently discovered exon 6B is generated by exonization of an Alu element within intron 6 [33].…”

“…Interestingly, an A-to-G substitution at the -44th position (A-44G) of intron 6 has been found to promote SMN2 exon 7 inclusion (Fig. 3; [40]). The A-44G substitution is naturally present in human population and SMA patients carrying A-44G substitution show mild phenotype [40].…”

“…3; [40]). The A-44G substitution is naturally present in human population and SMA patients carrying A-44G substitution show mild phenotype [40]. …”

High-throughput screening reveals novel mutations in spinal muscular atrophy patients

“…Due to the complexity of 5q chromosome structure, the mechanism of SMA has not been fully elucidated. For example, the rare variations in SMN2 have been described by several studies [18][19][20], and some scholars believe that the variations in SMN2 locus, such as the deletion of adjacent NAIP1 gene, will affect or even change the severity of SMA [21,22]. With the development of bioinformatics, more and more mutations have been discovered in SMN1, and some of them possess signi cant clinical implications.…”

RNA-Targeted Therapies and High-Throughput Screening Methods

“…HuR inhibition can sensitise tumour cells to cancer therapies [119,120]. Interestingly, HuR represses SMN2 splicing by binding to the -44 region in intron 6, implying its potential to become an SMA target [121]. HuR contains three RRMs.…”