Variant Collection and In Silico Analyses
All point variants in SLC4A1 , ATP6V1B1 , ATP6V0A4 ,FOXI1 , WDR72 and ATP6V1C2 gene were collected from the Human Gene Mutation Database (April, 2019) and literatures[15,17,18], except c.409C>T (p.Pro137Ser) and c.904C>T (p.Arg302Trp) in ATP6V1B1 that was identified in our previous studies[19].
Bioinformatics analysis was carried out to predict the splicing effect of each of the variants. Online software Human Splice Finder (version 3.1, http://www. umd.be/HSF3) was performed to investigate the presence of potential splicing regulatory sequences (ESEs and ESSs), and identify the putative effect of variants on splicing regulatory motifs. Analysis of BDGP (http://www.fruitfly.org) was employed to determinate the potential effects of variation on classic 5’ donor or 3’ acceptor consensus sites and to predict the generation and/or activation of new sites. Default thresholds were performed for all analyses. Variants that satisfy the following criteria will be selected for further minigene splicing assay: elimination of enhancers or creation of silencers; and close to the 5’ or 3’ ends of exons.
Minigene Constructions
To investigate the effect on the splicing process of the candidate variants, in vitro analysis was performed using a minigene splicing assay based on the pSPL3 exon trapping vector shown at Figure 1 A. Minigene constructions were described as previously reported[20,21]. Briefly, for each variation, the fragments with the wild-type (WT) alleles involving interested exon, flanked by approximately 50-200 nucleotides of upstream intronic sequence and downstream intronic sequence, were cloned into the splicing vector pSPL3 using specific primers linking the XhoI and NheI restriction enzyme sites (XhoI: TGGAGC^TCGAG; NheI: AATTTG^CTAGC). Primers were designed for each target fragment using PP5 (SUP Table. 1). The variant sequence change was introduced in the WT plasmid through the GeneArt™ Site-Directed Mutagenesis PLUS System (Thermo Fisher Scientific, Massachusetts, USA) as instructed by the manufacturer. Mutagenesis primers were demonstrated at SUP Table. 2. All constructed vectors were further transformed into E. coli DH5α competent cells (Takara, Shiga, Japan), followed by screening by conventional sanger sequencing.