A positive modifier of spinal muscular atrophy in the SMN2 gene

Abstract

Spinal muscular atrophy (SMA) is a common autosomal-recessive motor neuron disease caused by the homozygous loss of the SMN1 gene. A nearly identical gene, SMN2, has been shown to decrease the severity of SMA in a dose-dependent manner. However SMN2 is not the sole phenotypic modifier, because there are discrepant SMA cases in which the SMN2 copy number does not explain the clinical phenotype. This report describes three unrelated SMA patients who possessed SMN2 copy numbers that did not correlate with the observed mild clinical phenotypes. A single base substitution in SMN2, c.859G>C,, was identified in exon 7 in the patients' DNA. We now show that the change creates a new exonic splicing enhancer element and increases the amount of full-length transcripts, thus resulting in the less severe phenotypes. This demonstrates that the c.859G>C substitution is a positive modifier of the SMA phenotype and that not all SMN2 genes are equivalent. We have shown not only that the SMA phenotype is modified by the number of SMN2 genes but that SMN2 sequence variations can also affect the disease severity.

Figure 1

SMN2 Sequence Analysis of SMA Cases The inclusion of exon 7 in SMN1 transcripts, as well as the exclusion of this exon in SMN2 transcripts, is caused by the C-to-T change difference shown at nucleotide position 6 in SMN2 exon 7. The substitution of guanine by cytosine at nucleotide 859 (c.859G>C) is shown at nucleotide position 25 in SMN2 exon 7. (A) Wild-type SMN2 sequence chromatogram. (B) Case 1, showing a homozygous substitution of guanine by cytosine at nucleotide 859 (c.859G>C) at nucleotide position 25. (C) Case 2, showing a heterozygous substitution of guanine by cytosine at nucleotide 859 (c.859G>C) at nucleotide position 25. The patient has one wild-type SMN2 copy. (D) Case 3, also showing the substitution of guanine by cytosine at nucleotide 859 (c.859G>C) at nucleotide position 25. The patient has two wild-type SMN2 copies.

Figure 2

c.859G>C Creates a Putative SF2/ASF Motif (A) ESEfinder3.0, which includes four motif matrices of SR proteins, SF2/ASF, SC35, SRp40, and SRp55, predicts a strong SF2/ASF motif CGAAGGT with a score of 3.50 that was created by c.859G>C. Note that the wild-type sequence GGAAGGT score is 0.60 and the standard threshold is usually set at 1.88. (B) c.859G>C in the context of the SMN2 minigene showed a significant increase of exon 7 inclusion during pre-mRNA splicing in comparison to the wild-type SMN2 minigene.