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subtle variants in SMN1: (a) long-range PCR of the entire SMN1 genomic fragment of approximately 28 kb (exon 1 to exon 8) using primers that specifically amplify SMN1, followed by reamplification and sequencing of each exon (96), or (b) amplification and cloning of SMN cDNA products and PCR-based identification of those carrying the SMN1 gene. The laborious second version allows the identification of exonic and intronic variants that may cause splicing defects (106, 165). The first method, although fast, fails to detect deep intronic variants that affect splicing. The Mutation Spectrum of SMN1 Regardless of the disease severity, all 5q SMA patients have biallelic SMN1 mutations (100). In approximately 96% of SMA patients, the genetic cause is an SMN1 deletion or gene conversion of SMN1 into SMN2, which results in a homozygous loss of SMN1 exon 7 or exons 7 and 8 (48). While the majority of SMA type I patients have a true SMN1 deletion, in SMA types II and III, gene conversion of SMN1 leads to increased SMN2 copy number. Incomplete gene conversion results in hybrid SMN1/SMN2 genes, with exon 7 of SMN2 origin and exon 8 of SMN1 origin (23, 65, 172, 180). A compiled study on SMN1 deletion screening in SMA patients revealed 96% homozygous deletions in SMA type I, 94% in type II, and 86% in type III (180). Consequently, patients with the milder SMA forms carry a subtle mutation more often than patients with the severe form (6, 85, 106). In approximately 4% of SMA patients, subtle mutations can be found in combination with SMN1 loss on the second chromosome 5 (6, 106). Very rarely, and only in consanguineous families, two subtle SMN1 variants have been reported (23). Currently, 108 different pathogenic SMN1 variants have been described across SMN1 (32). The most frequently found subtle mutations are p.Tyr272Cys in the German population and p.Thr274Ile in the Polish population (85, 180); the frameshift mutation p.Arg133fs∗148, caused by a 4-bp deletion (c.399_402delAGAG), in the Spanish population; and the frameshift mutation p.Gly261fs∗269, caused by an 11-bp duplication (c.770_780dup11), in the Spanish, French, and US populations (6, 23, 31, 133). De Novo Mutations The SMA region on 5q13 is highly unstable due to a repeat unit of approximately 500 kb, which is duplicated and inverted and includes several genes other than SMN1 and SMN2 (100, 146). Physical and genetic maps of the region and, recently, long-read next-generation sequencing have shown that this region is extremely polymorphic. The various units vary not only in number (from zero to four per chromosome) but also in their orientation (39, 120, 153). The region is prone to unequal recombination and gene conversion, leading to frequent de novo mutations. Thus, 2% of SMA cases result from de novo mutations, which are more often due to unequal recombination than to gene conversion events (48, 185). Phenotype–Genotype Correlation The severity of the SMA phenotype is influenced mainly by the SMN2 copy number, with more copies resulting in a milder phenotype. Although this correlation is strong, it is not absolute. Thus, 73% of SMA type I patients carry two SMN2 copies, 78% of SMA type II patients carry 240 Wirth et al. Annu. Rev. Genom. Hum. Genet. 2020.21:231-261. Downloaded from www.annualreviews.org Access provided by 2600:1700:3c30:a3d0:89dc:b3d1:ac73:5085 on 01/23/22. For personal use only. three copies, 50% of SMA type IIIa patients carry three copies, 61% of SMA type IIIb patients carry four copies, and 75% of SMA type IV patients have four copies. The predictive value of three SMN2 copies is less than that of two or four copies; three SMN2 copies can be found in 20% of type I, 78% of type II, and 51% of type III patients (24, 48, 181) (Figure 1). Some of the missense mutations, such as p.Tyr272Cys, are associated with a severe phenotype, while p.Thr274Ile is associated with a milder phenotype (85, 180). However, the severity of the phenotype is additionally dependent on the SMN2 copy number accompanying the subtle mutation (180). Variants within SMN2 can also influence severity; for example, the missense variant c.859G>C (p.Gly287Arg) increases full-length SMN2
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