Comparison of three molecular genetic methods for determining the human SMN2 gene copy number

Abstract

Spinal muscular atrophy (SMA) is an inheritable neuromuscular disease, which causes atrophy and progressive muscle weakness. The severity and the phenotype of the disease varies. SMA is caused by a lack of SMN protein, which is usually caused by the homozygous deletion of the SMN1 gene. The SMN2 gene also produces the SMN protein, but only 15% of the protein produced is functional. The copy number of the SMN2 gene affects the phenotype of SMA. SMA can be treated with nusinersen, but the cost of the treatment is extremely high. Due to the expensiveness of nusinersen, more accurate diagnostics are required. The main aim of this thesis was to compare three molecular genetic methods for determining the human SMN2 gene copy number, using pre-existing SMA, carrier and healthy samples. The three methods used were: Asuragen’s AmplideX PCR/CE SMN1/2 Kits utilising PCR and capillary electrophoresis, multiplex ligationdependent probe amplification (MLPA) and droplet digital PCR (ddPCR). The SMN1 copy number was also determined, as well as the usability and the accuracy of the methods. The SMN2 copy number results yielded with the three methods were varying. With the AmplideX kit and the ddPCR, the SMN2 copy number could be determined for all the samples and the results were nearly identical. The MLPA results showed the most variation and were in most cases low in quality. Also, MLPA seemed to be comparatively impractical. Overall, ddPCR seemed to fit the best to the original requirement of having more accurate diagnostics.