Reverse Transcription-PCR-based Sanger Sequencing-confirmed Exon-skipping Effect of a Novel GEN1 Intronic Variant (c.1408+4A>G).

Abstract

and corresponds to PM2, according to the 2015 American College of Medical Genetics guidelines [9]. Splice-site prediction algorithms, including Netgene2, AdaBoost (score: 0.9), and Random Forest (score: 0.7), predicted a splice-site change. The novel variant was tentatively classified as a variant of uncertain significance (VUS) because its splicing effect and clinical significance were not confirmed. RT-PCR and Sanger sequencing were performed to determine whether the splice donor-site change in the consensus sequence induced aberrant RNA splicing. Total RNA extracted from blood leukocytes of a control subject and the patient using the High Pure RNA Isolation Kit (Roche, India-napolis, IN, USA) was reverse-transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher