3.1.2 Variants of c.1437C>G (p.Cys479Trp) and
c.1564G>A (p.Glu522Lys) in exon 13 did not alter pre-mRNA
splicing
Variant c.1437C>G (p.Cys479Trp), located at nucleotide
position +6 from the 5’ end of exon 13, was predicted to lead to the
disruption of two ESEs and the generation of four new ESS sites using
bioinformatic tool HSF. Presumed missense variant
c.1564G>A
(p.Glu522Lys) is located 133 bp upstream of exon 13 (Table 1). The
results of HSF analysis indicated that this variant inactivated four
potential ESE sites and generated two potential ESSs.
However,
analysis of the minigenes containing variant c.1437C>G and
c.1564G>A in exon 13 of SLC4A1 resulted in RT-PCR
products that matched in size with those generated by the respective WT
constructs (Figure 2A). This was further confirmed by sequencing
analysis. Therefore, these variants did not affect pre-mRNA splicing.
3.2 Splicing outcome of sequences variations
of ATP6V1B1
3.2.1 Variants c.368G>T (p.Gly123Val) andc.370C>T
(p.Arg124Trp) led to exon 5 skipping compared with the WT plasmids.
Presumed missense variant
c.368G>T (p.Gly123Val)
is caused by the first nucleotide substitution in exon 5 ofATP6V1B1 . Bioinformatic analysis with BDGP demonstrated that this
variant drastically reduces the score of the WT 3’ splice site from 0.96
to 0.76 (Table 1). Variant c.370C>T
(p.Arg124Trp)
result from substitutions at the +3 nucleotide of exon 5 and was
demonstrated that this variant marginally reduces the score of the WT 3’
splice site from 0.96 to 0.94 with BDGP.
Additionally, analysis of HSF to
two variants predicted that they both generated three
ESSs. To determine the splicing
effect of variants c.368G>T and c.370C>T, a
minigene containing exon 5 and flanking intronic sequences was used. The
result of RT-PCR analysis showed that the WT lane demonstrated 2
different fragments of 263 bp and 368 bp, respectively (Figure 2B).
Direct sequencing showed that the larger fragment contained exon 5
flanked by two exons of the pSPL3 vector, while the smaller one included
only the 3’ and 5’ pSPL3 exons. In contrast, the mutant lane of
c.368G>T revealed one unique fragment of 263 bp
corresponding to skipping of exon 9 in the mRNA; whereas, the result of
variant c.370C>T revealed two different electrophoresis
bands whose sizes correspond to the WT products.
Analysis of cDNA prepared from
HEK293T showed that there was a significant increase of exon 5-skipping
transcript of c.370C>T compared with the WT plasmids
(28.19% versus 4.78%, P<0.05, Figure 2D). Deletion of exon 5
in the mutant transcript would result in the abnormal connection between
exons 3 and 4, leading to the loss of 26 amino acids at the protein
level without changing the open reading frame. Taken together, both of
variants c.368G>T and c.370C>T broken proper
recognition of the acceptor splicing site, resulting in exon 6 skipping
of ATP6V1B1 .
3.2.2 Nonsense variant c.484G>T
( p.Glu162*) led to the
skipping of exon 6 .
Variant c.484G>T of the internal position of exon 6 inATP6V1B1 alters a GAG codon for Glu to a premature TAG stop codon
((p.Glu162*), which is predicted to produce a truncated and
nonfunctional protein (Table 1). However, the in silico analysis
by HSF software indicated that c.484G>T not only disrupts
five ESEs (CCCGAGG A, CCGAGG , CCGAGG AG,
CGAGG A, GAGG AG), but also creates two ESSs
(GT AGAT, T AGATG). Besides, HSF also predicted that
this variant would cause the activation of a cryptic donor site
(CGAGT AGAT). To further clarify the impact of variant
c.484G>T on the splicing process, we inserted either the WT
or c.484G>T-mutated exon 6, along with the nearby intron
sequences, into the vector pSPL3, and analyzed the spicing pattern in
HEK293T. The WT minigene resulted
in two different transcripts
corresponding to a mature mRNA and a truncated mRNA (1.22%) by
sequencing analysis. The mutated minigene, also, produced two mature
transcripts: a larger one corresponded to the single band produced by
the WT, and a shorter one with the splicing of exon 6 (Figure 2B).
Because exon 6 has 140 nucleotides, loss of exon 6 would disrupt the
reading frame of the ATP6V1B1 transcript. Quantitative analysis
of the RT-PCR products revealed that the rate of transcript lacking exon
6 to the full transcripts was 60.35% (P<0.05, Figure 2D).
Taking these findings together, the splicing pattern of the mutant
minigene indicated that nonsense variant c.484G>T induces
exon 6 skipping.
In addition, although variant c.481G>A (p.Glu161Lys) close
to c.484G>T was also predicted by the software HSF to
destroy five ESEs and produce two ESSs, it did not affect splicing
through the minigene analysis (P>0.05).
3.2.3 Variant
c.1102G>A
( p.Glu368Lys) resulted in
partial skipping of exon 11.
Variant c.1102G>A identified at nucleotide position 43 of
exon 11 in ATP6V1B1 were predicted by HSF to broke seven ESEs
(CACAG A, CACAG AG, ACAG AGGG, CAG AGG,
CAG AGGG, G AGGGA) and create one ESS site
(AA AGGG) corresponding to a new hnRNPA1 binding site (Table 1).
In order to verify whether this variant affected mRNA splicing, minigene
splicing experiments in vitro were also carried. As a result, two
products of 346 bp and 236 bp were both found from the RT-PCR products
of the WT minigene and mutant (Figure 2B). Direct sequencing of two
products showed that the larger amplicon was the transcript containing
exon 11, and the smaller was the transcript excluding exon 11.
Quantitative analysis of two products showed that c.1102G>A
altered weakly splicing resulting in a 9.51% of the aberrant transcript
that broke the open reading frame compared with WT of 2.60%
(P<0.05, Figure 2D). In addition, the skipping of exon 11 in
the mutant transcript would result in the loss of 83 nucleotides that
disrupt the reading frame.
3.3 Splicing outcome of sequences variations ofATP6V0A4.
3.3.1 Variant
c.322C>T (p.Gln108*) resulted in skipping of exon 6.
Variant c.322C>T, as a nonsense variant (p.Gln108*) located
at nucleotide position +31 from the 5’ end of exon 6, is predicted to
generate a truncated and nonfunctional protein (Table 1). The software
HSF demonstrated that c.322C>T not only destroyed six ESEs
(C AGGAA, TAC AGGA,
TAC AGGAA,
TTAC AG, GTTAC A, AGTTAC ), but also engenders
three ESSs (TAT AGG, TTAT AG, AGTTAT A).
Consequently, two fragments of 389 bp and 263 bp were detected from that
of the mutant vector by minigene assay, respectively (Figure 2C). Direct
sequencing results showed that the larger fragment containedATP6V0A4 exon 6 flanked by two exons of the pSPL3 vector, while
the smaller one included only the 3’ and 5’ pSPL3 exons (24.22%).
However, the WT vector also revealed a smaller fragment (1.78%) of 263
bp corresponding to incorrect skipping of exon 6 in ATP6V0A4expect for a mature transcript (Figure 2D). The skipping of exon 6 in
the mutant transcript would result in the loss of 42 amino acids
(residues 98-139) at the protein level without altering the open reading
frame. Therefore, variant c.322C>T (p.Gln108*) causes
partial exon 9 skipping because of ESEs destruction and ESSs generation.
3.3.2Missense
variant c.1571C>T (p.Pro524Leu) and synonymous variant
c.1572G>A (p.Pro524Pro) resulted in skipping of exon 15.
Missense variant c.1571C>T (p.Pro524Leu) caused by
substitutions at the -2 nucleotide of exon 15 downstream of the 3’
splice site, was demonstrated that this variant reduces the score of the
WT donor splice site of intron 15 from 0.8 to 0.68 with BDGP (Table 1).
Synonymous variant c.1572G>A (p.Pro524Pro) affected the G
the last nucleotide substitution in exon 15. Bioinformatic analysis with
BDGP demonstrated that the score of the donor site of intron 15 is 0.8,
whereas it could not be analyzed after variation. Additionally, analysis
of this variant with HSF predicted to break the WT donor sites
(CCG GTAATA), most probably affecting splicing. Taken together,
to examine the splicing effect of two variants, we also used a minigene
containing exon 15 and surrounding intronic sequences. RT-PCR analysis
results showed the splicing products produced by the mutant and WT
minigenes were different. The WT lane demonstrated one fragment of 357
bp that contains ATP6V0A4 exon 15, whereas both of mutant
c.1571C>T and c.1572G>A generated two
different fragments of 263 bp and 357 bp, respectively (Figure 2C).
Direct sequencing of all products showed that the larger amplicons were
the exons-included transcripts and the smaller amplicons are the
exons-excluded transcripts. Analysis of cDNA prepared from HEK293T
revealed that there was a significant increase of exon 15-skipping in
c.1571C>T and c.1572G>A with the control
plasmid (3.74% versus 0 and 84.48% versus 0, respectively, Figure 2D).
Besides, the skipping of exon 15 in the mutant transcript would result
in the alteration of the open reading frame due to the loss of 94
nucleotides.