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.

Keywords: SMN2 copies; next-generation sequencing; paralogous variants; phenotype-genotype correlations; spinal muscular atrophy.

Figure 1

Structure of the SMN2–SMN1 hybrid detected in patients SMA4 and SMA39. PSVs were genotyped in all patients. In SMA4 and SMA39, six SMN1 PSVs located in intron 6 (Chr5: 69370451‐69370895) were detected, which indicates the presence of hybrid genes. The AB ratio indicates that in SMA04, two of the three copies are SMN2–SMN1 hybrids (SMN1 PSVs in 66%) while in SMA39, only one of the three copies is an SMN2–SMN1 hybrid (SMN1 PSVs in 33%). PSV, paralogous sequence variant

Figure 2

Utility of AB ratios to calculate the number of the copies in which variants are present. Patient SMA52 has two SMN2 copies (determined by MLPA) and the variant NM_017411.4:c.859G>C (p.(Gly287Arg)) was detected with a frequency of 56%, in agreement with the AB ratio expected for the variant in one over two alleles. Patient SMA21 has three SMN2 copies (determined by MLPA), and the variant c.835‐44A>G (NC_000005.9: g.69372304A>G) was detected with a frequency of 36%, in agreement with the AB ratio expected for the variant in one over three alleles. MLPA, multiplex ligation‐dependent probe amplification

Figure 3

Detection of the pathogenic variant NM_017411.4:c.815A>G in samples SMA55 and SMA55F. Patient SMA55 has one SMN1 copy (with the variant c.815A>G) and one SMN2 copy, while his father (SMA55F) has two SMN1 (one copy with the variant c.815A>G) and two SMN2 copies (determined by MLPA and Sanger). The pathogenic variant c.815A>G was detected in SMA55 and SMA55F through NGS with a frequency of 52% and 25%, respectively. The SMN1‐specific PCR performed confirms that the pathogenic variant c.815A>G is present in SMN1 since we observed the variant in hemizygous status in SMA55 and in heterozygous status in SMA55F. MLPA, multiplex ligation‐dependent probe amplification; NGS, next‐generation sequencing; PCR, polymerase chain reaction

Figure 4

Description of the utility of AB ratios to determine the presence of two partial SMN genes (SMN1/2Δ7/8). The patient (SMA55) has one SMN1 (with a pathogenic variant in exon 6*), one SMN2, and two partial SMNs from promoter to exon 6 (determined by MLPA). In the 5′ region (promoter‐ex6), the patient has a total of four SMN copies; consequently, we detected different SNPs with an allelic frequency of 25%, 50%, 75%, and 100%. By contrast, in the 3′ region (in6‐ex8), the patient has two SMN copies, and only variants with an allelic frequency of 50% are detected. Note that PSVs are located in the 3′ region; therefore, it is not possible to determine whether these partial genes are derived from SMN1 or SMN2. MLPA, multiplex ligation‐dependent probe amplification; PSV, paralogous sequence variant; SNP, single‐nucleotide polymorphisms