Fig. 4

Effect of adding adjusted PS4 on variant reclassification and reanalysis. A Pedigrees and molecular features of two probands diagnosed after reanalysis with the adjusted PS4 carrying the TMPRSS3: c.205+5G>C variant. Mini-gene splicing assay to investigate the influence of c.205+5G>C on TMPRSS3 splicing. Agarose gel electrophoresis of RT-PCR products (Lane 1: Marker; Lane 2: 263 bp; Lane 3: 374 bp (263 bp + 111 bp); Lane 4: empty vector (263 bp)) and sequencing results of the mini-gene product, confirming that the TMPRSS3 mutant mini-gene (c. 205+5G>C) caused a splicing abnormality, resulting in exon 3 skipping. B Pedigrees and molecular features of two probands diagnosed after reanalysis with the adjusted PS4 carrying the OTOA: p.Leu12Arg variant. C Overview of variants upgraded from VUS to P/LP. s: strong; m: moderate; p: supporting; a: PS4 based on proposed optimized thresholds determined in this study; b: evidence strength level was assigned according to the ACMG/AMP-HL guideline recommendations, with minor modification; F1: Family 1; F2: Family 2; F3: Family 3; F4: Family 4