Beyond copy number: A new, rapid, and versatile method for sequencing the entire SMN2 gene in SMA patients

Abstract

Spinal muscular atrophy (SMA) is caused by bi-allelic loss or pathogenic variants in the SMN1 gene. SMN2, the highly homologous copy of SMN1, is considered the major phenotypic modifier of the disease. Determination of SMN2 copy number is essential to establish robust genotype-phenotype correlations and predict disease evolution, to stratify patients for clinical trials, as well as to define those eligible for treatment. Discordant genotype-phenotype correlations are not uncommon in SMA, some of which are due to intragenic SMN2 variants that may influence the amount of complete SMN transcripts and, therefore, of full-length SMN protein. Detection of these variants is crucial to predict SMA phenotypes in the present scenario of therapeutic advances and with the perspective of SMA neonatal screening and early diagnosis to start treatments.Here, we present a novel, affordable, and versatile method for complete sequencing of the SMN2 gene based on long-range polymerase chain reaction and next-generation sequencing. The method was validated by analyzing samples from 53 SMA patients who lack SMN1, allowing to characterize paralogous, rare variants, and single-nucleotide polymorphisms of SMN2 as well as SMN2-SMN1 hybrid genes. The method identifies partial deletions and can be adapted to determine rare pathogenic variants in patients with at least one SMN1 copy.

Genomic Variability in the Survival Motor Neuron Genes (SMN1 and SMN2): Implications for Spinal Muscular Atrophy Phenotype and Therapeutics Development

“…Most gene conversion events occur at the canonical c.840C>T nucleotide difference at exon 7 [ 59 , 60 ]. There are, however, at least 15 other paralogous structural variants (PSVs) between SMN1 and SMN2 ( Figure 3 ; [ 60 , 108 , 120 , 135 , 136 ]). Gene conversion events at exon 8 ( SMN2c.1155G>A ) as well as those within intron 6 ( SMN2c.835-44G>A ) and intron 7 ( SMN2c.888+100A>G and SMN2c.888+215A>G ) have been observed in SMA, as well as in control populations [ 127 , 129 , 133 ].…”

The Importance of Digging into the Genetics of SMN Genes in the Therapeutic Scenario of Spinal Muscular Atrophy

“…More studies are needed to determine which structure is correct or if both orientations are present in the population. Given that SMN2 was originated from SMN1, their sequences only differ in 16 paralogous sequence variants (PSVs), which represent a total of 20 different nucleotides between both genes (15 SNVs and 1 indel) [10] as represented in Figure 1. The PSV c.840C>T, located in exon 7, causes exon skipping in the majority of SMN2 pre-mRNA transcripts resulting in a truncated, nonfunctional and rapidly degraded protein that is not able to oligomerize (SMN-∆7) [11] and can only produce the complete functional protein in 10-15% of cases [12][13][14].…”

“…In addition, it has been postulated that this allele has originated from a common ancestor by haplotype analysis [39]. The second variant classified as a positive modifier is the variant c.835-44A>G, located in intron 6, and it is one of the 16 PSVs described between SMN1 and SMN2 [10,40]. Wu et al (2017) demonstrated that this transition decreases the affinity of the RNA-binding protein HuR, which acts as a splicing repressor, increasing in~20% the SMN2 exon 7 inclusion [38].…”

“…Aside from the previously described SMN2 modifiers (c.859G>C and c.835-44A>G), other variants in this gene have been proposed to modify the SMA phenotype, although functional studies to demonstrate an effect in the SMN protein have not been performed or more cases have not yet been reported [9,38,54]. For instance, variants c.835-1897C>T and c.835-549A>G in intron 6 of SMN2 and variant c.*3+100G>A in intron 7, later classified as a PSV [10], have been associated with a better-than-expected phenotype (Figure 1) [9,38]. Furthermore, a recent study suggested that variants c.81+45C>T in intron 1 of SMN2 and c.838_840del in exon 7 were related to a more severe SMA phenotype.…”

“…In addition, variants in the SMN1 promoter, which have been associated with non-functional SMN1 alleles, have also been reported, even though they are infrequent findings [45]. It is important to bear in mind that variants located in deep intronic regions would not be detected through MLPA or exome sequencing, highlighting the importance of recently developed strategies for the entire sequencing of SMN1 or SMN2 genes [10,18]. Another cause of discordance could be a clinical misclassification of the patient or an inaccurate SMN2_CN determination.…”