Clinical and Gene-Specific Sequencing to Enhance the Clinical Sensitivity of Spinal Muscular Atrophy Diagnostic Testing.

PURPOSE

Therapeutic advances in the treatment of spinal muscular atrophy (SMA) prompt the need for robust and efficient molecular diagnosis of this disease. Approximately five percent of SMA cases are attributable to one copy of with a hypomorphic or inactivating variant in with a deleted or converted allele. These intragenic variants are challenging to definitively localize to due to its sequence homology with the gene. To enhance the clinical sensitivity of SMA diagnostic testing, we present an optimized gene-specific sequencing assay to localize variants to either or .

METHODS

and genes are independently amplified by long-range allele-specific PCR. Long-range products are used in subsequent nested PCR reactions to amplify the coding exons of and . The resulting products are sequenced using standard Sanger-based methodologies and analyzed for disease-associated alterations.

RESULTS

83 probands suspicious for a clinical diagnosis of SMA with a nondiagnostic dosage result were sequenced for intragenic variants in the gene. Gene-specific sequencing revealed likely disease-associated variants in in 42 cases (50.6%). Of the 42 variants, 27 are unique including 16 loss-of-function variants, 9 missense variants, 1 in-frame deletion variant, and 1 splice site variant.

CONCLUSIONS

Herein, we describe an optimized assay for clinical sequencing of the full coding region of and . This assay uses standard techniques and equipment readily available to most molecular diagnostic laboratories.