Deep Molecular Characterization of Milder Spinal Muscular Atrophy Patients Carrying the c.859G>C Variant in SMN2

Abstract

Spinal muscular atrophy (SMA) is a severe neuromuscular disorder caused by biallelic loss or pathogenic variants in the SMN1 gene. Copy number and modifier intragenic variants in SMN2, an almost identical paralog gene of SMN1, are known to influence the amount of complete SMN proteins. Therefore, SMN2 is considered the main phenotypic modifier of SMA, although genotype−phenotype correlation is not absolute. We present eleven unrelated SMA patients with milder phenotypes carrying the c.859G>C-positive modifier variant in SMN2. All were studied by a specific NGS method to allow a deep characterization of the entire SMN region. Analysis of two homozygous cases for the variant allowed us to identify a specific haplotype, Smn2-859C.1, in association with c.859G>C. Two other cases with the c.859G>C variant in their two SMN2 copies showed a second haplotype, Smn2-859C.2, in cis with Smn2-859C.1, assembling a more complex allele. We also identified a previously unreported variant in intron 2a exclusively linked to the Smn2-859C.1 haplotype (c.154-1141G>A), further suggesting that this region has been ancestrally conserved. The deep molecular characterization of SMN2 in our cohort highlights the importance of testing c.859G>C, as well as accurately assessing the SMN2 region in SMA patients to gain insight into the complex genotype−phenotype correlations and improve prognostic outcomes.

Keywords: SMN2 copies; next-generation sequencing; phenotype–genotype correlations; positive modifiers; spinal muscular atrophy.

Figure 1

Pedigree representation of cases with two SMN2^859C copies. According to segregation studies, a cis or trans configuration was defined in each patient. (A) In patient 1, the father’s sample was not available, and SMN2 configuration was inferred based on the results from the mother and patient. Given that the father did not present symptoms, we can assume that he carries at least one SMN1 gene. In addition, being a consanguineous family, we assumed that Smn2-859.C1 was transmitted by both parents (untested inferred alleles are represented by a dashed line). (B) In patient 2, the Smn2-859.C1 haplotype was inherited from both parents, in agreement with the consanguinity in the family. (C) Patient 3 had two copies of SMN2 with Smn2-859.C1 in cis, inherited from his mother. (D) Patient 4 also had two copies of SMN2 in cis, one with Smn2-859.C1 and the other with Smn2-859.C2 haplotype, forming a complex allele inherited from the father. (E) Patient 5 inherited the complex allele from his mother and the other allele with a partial non-functional SMN1/2Δ7-8 gene from his father.

Figure 2

Expected SMA phenotype in cases with two SMN2 copies according to the presence of c.859G>C. An additive effect on SMA phenotype is observed depending on whether the c.859G>C variant is found in one or both SMN2 copies. SMN2 gene is represented as a rectangle, and the presence of the c.859G>C variant in exon 7 is indicated by an asterisk. Not all SMN2 genotypes represented in this figure were detected in this study (see Figure 1 for more details).

Figure 3

Proposed mechanism of origin and structure of Smn2-859C.1 and Smn2-859C.2 haplotypes. (A) Hypothetic origin of the Smn2-859C.2 haplotype through homologous recombination involving a double cross-over event between allele A (with two Smn2-859C.1 haplotypes represented in green) and allele B (containing the 5′ region of the Smn2-859C.2 haplotype, in blue, and the 3’ end with an unknown sequence, in grey). (B) SMN2 structure details (representing exons and introns from top to bottom) and location of variants of the Smn2-859C.1 and Smn2-859C.2 haplotypes, as well as the unknown original haplotype that presumably originated the Smn2-859C.2 haplotype. The c.154-1141G>A (69360651-G-A, hg19/GRCh37) variant is indicated in green, whereas the c.859G>C modifier is shown in red (further explanation in the text and Table 2).