Muscle biopsy to end a diagnostic odyssey in a genetically complex case of spinal muscular atrophy type 3: the importance of "old school" in the next-generation sequencing era.

Spinal muscular atrophy (SMA) is an autosomal recessively inherited disease leading to severe and progressive muscle weakness due to loss of motor neurons in the spinal cord and the brainstem. Motor neuron degeneration is caused by the absence of a functional SMN1 gene copy, a gene located on 5q13.2, encoding the SMN ("survival of motor neuron") protein. Initially classified into five groups with varying severity (SMA type 0-4), SMA is now regarded as a progressive neurodegenerative disease with a continuum of clinical severity, which genetic and epigenetic modifiers can modulate. The highly homologous gene SMN2, which differs only at a single nucleotide position within the coding sequence (c.840C > T), can modify SMA disease severity due to its ability to drive low SMN protein expression levels. SMN2 copy number and at least two SMN2 variants are known as protective factors and are currently recommended for reporting in genetic testing. As therapies either replacing functional SMN1 or targeting RNA expression to increase SMN2 splicing became available since 2016, rapid genetic confirmation of SMA together with SMN2 copy number status became even more important. Here, we report on a pediatric case of SMA type 3 that could be solved only by functional transcript-level investigation using RNA sequencing following muscle biopsy after several iterations of inconclusive genetic testing. Due to an unusual genotype and multiple SMN2 copies concealing a subtle nonsense variant, DNA-based (next-generation) sequencing failed to provide a clear result. This case report is a cautionary tale hoping to trigger careful re-evaluation in similar unsolved cases.