Copy Number Variations in the Survival Motor Neuron Genes: Implications for Spinal Muscular Atrophy and Other Neurodegenerative Diseases

Proximal spinal muscular atrophy (SMA), a leading genetic cause of infant death worldwide, is an early-onset, autosomal recessive neurodegenerative disease characterized by the loss of spinal α-motor neurons. This loss of α-motor neurons is associated with muscle weakness and atrophy. SMA can be classified into five clinical grades based on age of onset and severity of the disease. Regardless of clinical grade, proximal SMA results from the loss or mutation of SMN1 (survival motor neuron 1) on chromosome 5q13. In humans a large tandem chromosomal duplication has lead to a second copy of the SMN gene locus known as SMN2. SMN2 is distinguishable from SMN1 by a single nucleotide difference that disrupts an exonic splice enhancer in exon 7. As a result, most of SMN2 mRNAs lack exon 7 (SMNΔ7) and produce a protein that is both unstable and less than fully functional. Although only 10–20% of the SMN2 gene product is fully functional, increased genomic copies of SMN2 inversely correlates with disease severity among individuals with SMA. Because SMN2 copy number influences disease severity in SMA, there is prognostic value in accurate measurement of SMN2 copy number from patients being evaluated for SMA. This prognostic value is especially important given that SMN2 copy number is now being used as an inclusion criterion for SMA clinical trials. In addition to SMA, copy number variations (CNVs) in the SMN genes can affect the clinical severity of other neurological disorders including amyotrophic lateral sclerosis (ALS) and progressive muscular atrophy (PMA). This review will discuss how SMN1 and SMN2 CNVs are detected and why accurate measurement of SMN1 and SMN2 copy numbers is relevant for SMA and other neurodegenerative diseases.

SMA is an early-onset neurodegenerative disease characterized by the loss of α-motor neurons in the anterior horn of the spinal cord, i.e., lower motor neurons (LMNs; Crawford and Pardo, 1996;Kolb and Kissel, 2015). This loss of α-motor neurons is associated with muscle weakness and atrophy. In SMA, those muscles that are proximally innervated are preferentially affected over distal muscles. SMA can be classified into five clinical grades based on age of onset and severity of the disease (Munsat and Davies, 1992;Russman, 2007; Table 1). Type 0 SMA infants present with very severe hypotonia and require respiratory support from birth. These SMA infants cannot survive beyond 6 months. Type I SMA [listed in the Online Inheritance in Man (OMIM) database under accession number 253300; http://www.omim.org/entry/ 253300] patients have an age of onset before 6 months. They show hypotonia and weakness in limbs; they are unable to sit independently. Type I SMA infants show a bell-shaped chest due to weakness in the intercostals muscles but sparing of the diaphragm; this bell-shaped chest results in abnormal breathing patterns. These patients typically live <2 years. Type II SMA (http://www.omim.org/entry/253500) patients have an age of onset before 18 months. They are poor crawlers and weak sitters; most of these patients can rarely stand and only with support. Their legs are generally weaker than their arms. Due in part to better supportive care, these patients generally have a life expectancy into early adulthood. Type III SMA (http://www. omim.org/entry/253400) patients have an age of onset >18 months. These patients are able to walk with difficulty (waddling gait) and the legs are weaker than the arms. Type III patients usually have a normal lifespan. Adult-onset (type IV) SMA (http://www.omim.org/entry/271150) patients have an age of onset of 18-21 years. Type IV SMA exhibits as a slowly progressive limb weakness. The disease is fairly benign in these patients.
In more than 95% of cases, proximal SMA results from the loss of SMN1 but retention of SMN2, regardless of clinical grade (Lefebvre et al., 1995). Large-scale deletions in chromosome 5q13 that include SMN1, NAIP, SERF1A, and GTF2H2A are observed in patients with type I SMA (Wirth et al., 1995;Burlet et al., 1996;Rodrigues et al., 1996;Velasco et al., 1996;Bürglen et al., 1997;Carter et al., 1997). Smaller deletions only involving SMN1 have also been observed in type I SMA patients demonstrating that SMN1 is the most likely causative gene for SMA. In addition, the identification of intragenic SMA mutations in SMN1 (Lefebvre et al., 1995;see Burghes and Beattie, 2009 for a comprehensive listing of SMA-associated point mutations in SMN1) provides additional evidence to support SMN1 as the gene responsible for SMA. To date, no intragenic mutations in the other genes within this segmental duplication have been associated with SMA.
As mentioned earlier, the SMN gene is duplicated in humans to give rise to SMN1 and SMN2. This duplication of SMN is unique to humans (Rochette et al., 2001). What is the difference between SMN1 and SMN2? The major difference between these two SMN genes is a C-to-T transition in exon 7(SMN2 c.850C>T; Lorson et al., 1999;Monani et al., 1999). This nucleotide change is translationally silent. This position on exon 7 is in the middle of an exonic splicing enhancer (ESS) sequence that regulated the inclusion of exon 7 in SMN transcripts (Figure 2). For SMN1, the C at this position promotes inclusion of exon 7 in SMN1-derived mRNAs which leads to the production of full-length SMN protein. Full-length SMN protein is able to form functional complexes. For SMN2, the T at this position disrupts this ESS which results in the exclusion of exon 7 (SMN 7) from the majority of SMN2derived mRNAs. As a result, a truncated SMN 7 protein is produced by the majority (∼90%) of SMN2-derived mRNAs; this SMN 7 protein is unstable and is unable to associate with itself (Lorson and Androphy, 2000;Burnett et al., 2009;Cho and Dreyfuss, 2010). The SMN 7 protein is still partially functional given that transgenic overexpression of SMN 7 in severe SMA mice partially ameliorates their phenotype since these mice die at 14-15 days as opposed to 5-8 days (Le et al., 2005). About  10% of the mRNAs from SMN2 contain exon 7 and these fulllength mRNAs can produce some full-length, functional SMN protein.

BIOLOGY OF SMN
SMN is a ubiquitously expressed protein whose expression is reduced in SMA. There is a strong inverse correlation between SMN protein levels and disease severity in SMA fibroblasts and lymphoblastoid cells as well (Coovert et al., 1997;Lefebvre et al., 1997;Kolb et al., 2006). Changes in SMN mRNA and protein levels observed in SMA patient-derived PBMCs mirror those observed in SMA cell lines Simard et al., 2007;Vezain et al., 2007;Tiziano et al., 2010;Crawford et al., 2012). SMN protein is present within the nuclei in discreet foci known as gems (Liu and Dreyfuss, 1996). In SMA fibroblasts, the number of SMN-positive subnuclear gems is higher in cells derived from mild SMA individuals than in those from children with more severe forms of SMA (Coovert et al., 1997). SMN is required for the assembly of the small nuclear ribonucleoprotein (snRNP) complexes that mediate splicing (Pellizzoni, 2007;Burghes and Beattie, 2009). snRNP assembly is defective in SMN-deficient SMA cells (Wan et al., 2005). Since snRNP assembly is required for all cell types, why are motor neurons primarily affected in SMA? snRNP assembly is defective in tissues from mouse models for SMA and that the extent of reduced snRNP assembly correlates with phenotypic severity of these SMA mice (Gabanella et al., 2005;Zhang et al., 2008). snRNP assembly is more markedly reduced in SMA mouse neural tissues than in other tissues like the kidney (Gabanella et al., 2005) suggesting that motor neurons are more sensitive to deficits in snRNP assembly. SMN may also have a function that is unique to motor neurons. Axonal defects in Smn-knocked down zebrafish embryos (McWhorter et al., 2003) are corrected by overexpression of mutant SMNs which are incapable of snRNP assembly (Carrel et al., 2006).
Gene conversion is one mechanism to account for increased SMN2 copy number in the absence of SMN1 in SMA (Burghes, 1997). In this scenario, the SMN1 gene actually contains part of SMN2, in particular within exon 7 (Wirth et al., 1995;Devriendt et al., 1996;Hahnen et al., 1996;van der Steege et al., 1996;Campbell et al., 1997;DiDonato et al., 1997b). It is hypothesized that type I SMA patients have deletions of SMN1 on both chromosomes. Type II SMA patients have an SMN1 deletion on one chromosome and a SMN1-to-SMN2 conversion on the other chromosome (three copies of SMN2). Type III SMA patients have SMN1-to-SMN2 converted genes on both chromosomes (four copies).
While the inverse relationship between SMN2 copy number and disease severity generally holds true in SMA, there are some exceptions. For example, there are cases of type II and III SMA patients who harbor only two copies of SMN2 instead of the predicted three or four copies Bernal et al., 2010;Vezain et al., 2010). Sequencing of SMN2 in these cases revealed the presence of a rare single nucleotide variant (SMN2 c.859G>C) in exon 7 Bernal et al., 2010;Vezain et al., 2010). This variant regulates the splicing of SMN2 pre-mRNAs so that a greater proportion of SMN2 transcripts contain exon 7. This variant may either create an additional SF2/ASF binding to promote exon 7 inclusion  or disrupt a hnRNPA1-dependent splicing silencer element (Vezain et al., 2010).
Intrafamilial variability in clinical presentation has been reported in SMA families with more than one affected sibling (Burghes et al., 1994;Cobben et al., 1995;Hahnen et al., 1995;McAndrew et al., 1997;Cuscó et al., 2006). Even though the siblings are haploidentical with respect to SMN2 copy number, they have differing clinical presentations. This would suggest that there are SMN2-independent modifiers of disease severity for SMA. Plastin-3 (PLS3) mRNA levels were higher in females with milder SMA than in discordant siblings with a more severe SMA clinical presentation (Oprea et al., 2008;Stratigopoulos et al., 2010;Bernal et al., 2011;Yanyan et al., 2014). In some families, however, female siblings with a more severe SMA phenotype show high PLS3 mRNA levels . It is possible that the modifier property of PLS3 is age-and sex-dependent as well as incompletely penetrant; alternatively, PLS3 may not actually be a major modifier of SMA phenotype. There may be other non-SMN2 molecular modifiers of disease severity in SMA. It is important to identify and characterize these novel modifiers for the development of novel SMA biomarkers and targets for the development of therapeutic strategies for SMA as well as for the planning of current and future clinical trials in SMA (Wirth et al., 2013).
To overcome some of the limitations associated with the PCRbased assays described above, digital PCR (dPCR) distributed across a large number of partitions by limited dilution so that some partitions will lack the template DNA (Sykes et al., 1992;Vogelstein and Kinzler, 1999). The absolute abundance of the target gene can be measured by counting the number of positive partitions and the number of negative partitions. dPCR can reliably and accurately measure SMN1 and SMN2 copy numbers over a wide range, i.e., between 0 and 6 copies (Zhong et al., 2011;Stabley et al., 2015).

SMN1 AND SMN2 CNVs IN ALS
Amyotrophic lateral sclerosis (ALS) is a mostly adult-onset motor neuron disease characterized by a progressive loss of motor function leading to paralysis and respiratory failure (Boylan, 2015;Statland et al., 2015). Unlike SMA, ALS is caused by degeneration of LMNs as well as upper motor neurons (UMNs). ALS is usually fatal within 3-5 years after disease onset but there is considerable variability with respect to duration as well as phenotypic presentation (Swinnen and Robberecht, 2014). Most cases of ALS are sporadic in nature since there is no apparent family history. Approximately 10% of ALS is considered familial since either a causative gene has been identified or there is strong family history. With the recent advents of whole exome and whole genome sequencing, the genetic bases of almost 70% of familial ALS and 10% of sporadic ALS have been identified (Renton et al., 2014).
There are many case studies reporting the co-occurrence of SMA and ALS within a family (Appelbaum et al., 1992;Camu and Billiard, 1993;Orrell et al., 1997;Corcia et al., 2002a) which suggests that SMN1 deficiency may lead to ALS in addition to SMA. SMN1 deletions, however, have not been observed in either familial or sporadic ALS patients (Orrell et al., 1997;Corcia et al., 2002a). Furthermore, no intragenic point mutations in SMN1 have been reported in the ALS population (Blauw et al., 2012). The intrafamilial coexistence of SMA and ALS, therefore, occurs by chance.
Even though loss of SMN1 is not associated with ALS, CNVs in the SMN genes may modulate the clinical severity of ALS in addition to SMA. Multiple studies suggest that deletion of SMN2 leads to increased risk of the sporadic forms of amyotrophic lateral sclerosis (ALS) (Veldink et al., 2001(Veldink et al., , 2005Kim et al., 2010;Corcia et al., 2012;Lee et al., 2012). Additionally, atypical SMN1 copy number-in other words, any number other than two-can affect the risk of ALS (Corcia et al., 2002b(Corcia et al., , 2006Blauw et al., 2012;Wang et al., 2014b). Other studies, however, have shown no association between deletion of either SMN1 or SMN2 in ALS (Jackson et al., 1996;Moulard et al., 1998;Parboosingh et al., 1999;Crawford and Skolasky, 2002;Gamez et al., 2002). The discrepant results from these studies may be due, in part, to different assays used to assess SMN1 and SMN2 CNVs as some reports using quantitative PCR while others used MLPA or RFLP.

SMN1 AND SMN2 CNVs IN PMA
Progressive muscular atrophy (PMA) is an adult-onset motor neuron disease characterized by loss of LMNs (Rowland, 2010;Liewluck and Saperstein, 2015). It is a rare and sporadic disorder that is clinically distinct from ALS even though subclinical involvement of UMNs has been observed in many PMA patients. Those PMA patients exhibiting a more severe clinical presentation tend to harbor higher SMN1 copy numbers (Kuzma-Kozakiewicz et al., 2013). No relationship between SMN2 copy number and disease severity was noted in these PMA patients. Moulard et al. (1998) noted that the frequency of SMN2 deletion was higher in a small group of patients with sporadic LMN disease.

CONCLUSIONS
CNVs in the SMN genes can modulate disease severity in SMA as well as in other motor neurons. It is well-established that SMN2 copy number is inversely correlated with disease severity in SMA. Because of this relationship, SMN2 is a primary target for the development of therapeutics for SMA (Arnold and Burghes, 2013;Cherry et al., 2014). Numerous approaches including promoter activation, increased inclusion of exon 7 and protein stabilization are currently being developed to increase SMN2 expression. With respect to other motor neuron diseases, it is presently unclear whether increasing SMN1 or SMN2 expression would be beneficial or detrimental. On one hand, increasing SMN expression provides neuroprotective benefit to cell culture and transgenic mouse models for ALS; however, some genetic studies suggest that duplication of SMN1 increases the risk of sporadic ALS. Future studies will assess the relationship between SMN1 and SMN2 CNVs and disease risk and progression in ALS and PMA as well as in other disorders affecting motor neurons.
SMN2 copy number is becoming an inclusion criterion for many clinical trials for SMA. Additionally, SMN2 copy number can be used to help guide the type of care SMA patients will receive. It is, therefore, essential to be able to accurately and reliably measure SMN2 CNVs in SMA patient samples, especially for those individuals harboring more than three copies of SMN2. Newly developed technologies like dPCR offer a means to accurately determine SMN2 copy number over a wider range.

AUTHOR CONTRIBUTIONS
The author confirms being the sole contributor of this work and approved it for publication.