Spinal muscular atrophy type I associated with a novel SMN1 splicing variant that disrupts the expression of the functional transcript

Abstract

Introduction: Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by pathogenic variants in the SMN1 gene. The majority of SMA patients harbor a homozygous deletion of SMN1 exon 7 (95%). Heterozygosity for a conventional variant and a deletion is rare (5%) and not easily detected, due to the highly homologous SMN2 gene interference. SMN2 mainly produces a truncated non-functional protein (SMN-d7) instead of the full-length functional (SMN-FL). We hereby report a novel SMN1 splicing variant in an infant with severe SMA.

Methods: MLPA was used for SMN1/2 exon dosage determination. Sanger sequencing approaches and long-range PCR were employed to search for an SMN1 variant. Conventional and improved Real-time PCR assays were developed for the qualitative and quantitative SMN1/2 RNA analysis.

Results: The novel SMN1 splice-site variant c.835-8_835-5delinsG, was identified in compound heterozygosity with SMN1 exons 7/8 deletion. RNA studies revealed complete absence of SMN1 exon 7, thus confirming a disruptive effect of the variant on SMN1 splicing. No expression of the functional SMN1-FL transcript, remarkable expression of the SMN1-d7 and increased levels of the SMN2-FL/SMN2-d7 transcripts were observed.

Discussion: We verified the occurrence of a non-deletion SMN1 variant and supported its pathogenicity, thus expanding the SMN1 variants spectrum. We discuss the updated SMA genetic findings in the Cypriot population, highlighting an increased percentage of intragenic variants compared to other populations.

Keywords: Cypriot population; novel variant; spinal muscular atrophy; splicing dysregulation; transcript analysis.

Figure 1

Identification of the SMN1 novel splice site variant. (A) Sanger sequencing electropherograms were obtained using a non-SMN1 specific PCR product and an SMN1 specific LR-PCR product in the proband. The SMN1 specific product analysis is also shown for the mother who carries the variant, the father and non-disease control representing the wild-type sequence. (B) Conservation of the identified variant. The DNA sequence of the SMN1 gene region encompassing the identified variant (c.835-8_835-5delinsG) is highly conserved in various mammals. Nucleotides in black belong to intron 6 and nucleotides in red to exon 7.

Figure 2

SMN1/SMN2 transcript analysis by conventional PCR, DdeI digestion and Sanger sequencing. (A) Agarose gel electrophoresis of the undigested and DdeI digested PCR products that resulted from amplifying the exons 6–8 cDNA region. Digested proband = 1, Undigested proband = 2, Digested non-disease control = 3, Undigested non-disease control = 4, Digested SMA affected control = 5, Undigested SMA affected control = 6, Size standard ladder (φX174 DNA-HaeIII Digest) = 7. It is shown that a smaller band (641 bp) than the expected SMN1/2-FL size (695 bp) is predominant in the proband. It corresponds to the SMN1-d7 form as it remains undigested. In the non-disease control, the predominant is the larger size band (695 bp), which corresponds to the SMN1-FL form as it remains undigested. In the SMA affected control having zero SMN1 copies, the detected bands correspond to the SMN2-FL and SMN2-d7 forms, which are fully digested (420/275 bp and 366/275 bp, respectively). (B) Sanger sequencing electropherograms were obtained by analyzing the remaining undigested products after performing the DdeI digestion in the proband and the non-disease control. The absence of exon 7 is clearly shown in the proband.

Figure 3

Relative mRNA expression levels of SMN transcripts in the proband (patient), the non-disease control having two copies of both SMN1 and SMN2 (control 2/2) and an SMA affected control having zero copies of SMN1, two copies of SMN2 (patient 0/2). qPCR revealed that the functional SMN1-FL transcript in the proband has almost zero expression (0.003), similar to the SMA affected control (0.0002). The SMN1-d7 transcript was found to be expressed only in the proband, and therefore a fold-change comparison with the non-disease control was not possible. Increased levels of the SMN2-FL and the SMN2-d7 transcripts were observed for the proband (1.3 and 2.5-fold respectively) and the SMA affected control (2.5 and 2.6-fold respectively), compared to the non-disease control. Determination of the total SMN-d7 levels also agrees with the findings observed by the SMN1-d7 and SMN2-d7 distinct analyses. Increased total d7 expression was observed in the proband (3.5-fold, attributed to both the SMN1-d7 and SMN2-d7 increase) and the SMA affected control (2.6-fold, attributed to the SMN2-d7 increase) compared to the non-disease control. Values were obtained after normalization with the L19 and B2M housekeeping genes. Data are represented as the mean of three independent triplicate experiments ± SE. The non-disease control has been used as the reference sample for the interpretation of the FL relative transcripts level. For the d7 relative transcripts level interpretation, the proband has been used as the reference sample, due to the absence of the SMN1-d7 in the non-disease control.