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.