Intragenic and Structural Variation in the Locus and Clinical Variability in Spinal Muscular Atrophy

Clinical severity and treatment response vary significantly between patients with spinal muscular atrophy. The approval of therapies and the emergence of neonatal screening programmes urgently require a more detailed understanding of the genetic variants that underlie this clinical heterogeneity. We systematically investigated genetic variation other than copy number in the locus. Data were collected through our single-centre, population-based study on spinal muscular atrophy in the Netherlands, including 286 children and adults with spinal muscular atrophy Types 1-4, including 56 patients from 25 families with multiple siblings with spinal muscular atrophy. We combined multiplex ligation-dependent probe amplification, Sanger sequencing, multiplexed targeted resequencing and digital droplet polymerase chain reaction to determine sequence and expression variation in the locus. , and gene copy number were determined by multiplex ligation-dependent probe amplification. gene variant analysis was performed using Sanger sequencing and RNA expression analysis of by droplet digital polymerase chain reaction. We identified - hybrid genes in 10% of spinal muscular atrophy patients, including partial gene deletions, duplications or conversions within and genes. This indicates that copies can vary structurally between patients, implicating an important novel level of genetic variability in spinal muscular atrophy. Sequence analysis revealed six exonic and four intronic variants, which were associated with disease severity in individual cases. There are no indications that gene copy number or sequence variants add value in addition to copies in predicting the clinical phenotype in individual patients with spinal muscular atrophy. Importantly, 95% of spinal muscular atrophy siblings in our study had equal copy numbers and structural changes (e.g. hybrid genes), but 60% presented with a different spinal muscular atrophy type, indicating the likely presence of further inter- and intragenic variabilities inside as well as outside the locus. gene copies can be structurally different, resulting in inter- and intra-individual differences in the composition of and gene copies. This adds another layer of complexity to the genetics that underlie spinal muscular atrophy and should be considered in current genetic diagnosis and counselling practices.