Mild SMN missense alleles are only functional in the presence of SMN2 in mammals

Abstract

Spinal muscular atrophy (SMA) is caused by reduced levels of full-length SMN (FL-SMN). In SMA patients with one or two copies of the Survival Motor Neuron 2 (SMN2) gene there are a number of SMN missense mutations that result in milder-than-predicted SMA phenotypes. These mild SMN missense mutation alleles are often assumed to have partial function. However, it is important to consider the contribution of FL-SMN as these missense alleles never occur in the absence of SMN2. We propose that these patients contain a partially functional oligomeric SMN complex consisting of FL-SMN from SMN2 and mutant SMN protein produced from the missense allele. Here we show that mild SMN missense mutations SMND44V, SMNT74I or SMNQ282A alone do not rescue mice lacking wild-type FL-SMN. Thus, missense mutations are not functional in the absence of FL-SMN. In contrast, when the same mild SMN missense mutations are expressed in a mouse containing two SMN2 copies, functional SMN complexes are formed with the small amount of wild-type FL-SMN produced by SMN2 and the SMA phenotype is completely rescued. This contrasts with SMN missense alleles when studied in C. elegans, Drosophila and zebrafish. Here we demonstrate that the heteromeric SMN complex formed with FL-SMN is functional and sufficient to rescue small nuclear ribonucleoprotein assembly, motor neuron function and rescue the SMA mice. We conclude that mild SMN missense alleles are not partially functional but rather they are completely non-functional in the absence of wild-type SMN in mammals.

Comprehensive Modeling of Spinal Muscular Atrophy in Drosophila melanogaster

“…Given the conservation of SMN protein structure from fly to human, we expect that this finding holds true in human patients as well. Our results are in direct contrast to of those of a recent report using the SMNΔ7 mouse model, claiming that two mild patient-derived missense mutations (D44V and T741I) are non-functional in the absence of full-length SMN (Iyer et al, 2018). This assertion is based on a single finding: that mice expressing only SMN missense mutations do not survive to birth.…”

“…Missense alleles that fully lack function would be expected to arrest at the same stage as 580 the null allele. Unfortunately, neither this stage nor any other embryonic stage was assessed in 581 the context of the D44V or T274I mutations to determine if SMN missense mutations partially 582 rescue embryonic development (Iyer et al, 2018). Notably, all of the human SMN missense 583 alleles analyzed in the mouse to date have been expressed from randomly-integrated, variable 584 copy number, cDNA-based transgenes (Gavrilina et al, 2008;Workman et al, 2009;Iyer et al, 585 2018).…”

“…Unfortunately, neither this stage nor any other embryonic stage was assessed in the context of the D44V or T274I mutations to determine if SMN missense mutations partially rescue embryonic development (Iyer et al, 2018). Notably, all of the human SMN missense alleles analyzed in the mouse to date have been expressed from randomly-integrated, variable copy number, cDNA-based transgenes Workman et al, 2009;Iyer et al, 2018). Moreover, none of these studies uses a wild-type cDNA control, so it is difficult to compare the transgenic lines without the ability to control for expression levels, position effects, or secondary mutations caused by integration.…”

“…To date, work in the fly has 59 focused primarily on assessing the effects of strong Smn depletion on organismal development 60 (Rajendra et al, 2007;Shpargel et al, 2009) or larval synapses and musculature (Chan et al, 61 2003;Chang et al, 2008), reviewed in (Grice et al, 2013;Aquilina and Cauchi, 2018). Despite 62 this body of work, the validity and translational value of the fly as a model for SMA continues to 63 be called into question (Bowerman et al, 2017;Iyer et al, 2018). This appears to be due, at 64 least in part, to the lack of a systematic and comprehensive analysis of SMA-related phenotypes 65 at the organismal level.…”

Comprehensive Modeling of Spinal Muscular Atrophy in Drosophila melanogaster

“…To date, work in the fly has focused primarily on assessing the effects of strong Smn depletion on organismal development (Rajendra et al, 2007; Shpargel et al, 2009) or larval synapses and musculature (Chan et al, 2003; Chang et al, 2008), reviewed in (Grice et al, 2013; Aquilina and Cauchi, 2018). Despite this body of work, the validity and translational value of the fly as a model for SMA continues to be called into question (Bowerman et al, 2017; Iyer et al, 2018). This appears to be due, at least in part, to the lack of a systematic and comprehensive analysis of SMA-related phenotypes at the organismal level.…”