Fig. 2

Minigene assays and identification of the variant’s impact on alternative splicing. a Sanger sequencing confirmed that wild-type and mutant fragments were successfully introduced into the minigene construct. Splicing variation c.339+2T>C in NPRL2 is indicated by the red box. b RT-PCR was performed to verify alternative splicing in the wild-type and mutant groups. Abnormal splicing bands in mutant groups, named “b” and “d”, were uncovered in both HeLa and 293 T cells. At the same time, normal bands, named “a” and “c”, were indicated in the wild-type group. c Alternative splicing was affected by the c.339+2T>C variation in NPRL2. PCR product sequencing revealed 18 bp intron 2 retention and exon 3 skipping. The alternative schematic is shown in (d). The red “*” symbolizes the variation site. Intron 2 retention is indicated in the mutant group by a red line. e Species conservation analysis of exon 3 in NPRL2.