Discussion
Normal pre-mRNA splicing depends on accurate recognition of exons and
introns, so many variations that break the correct definition of introns
and exons will lead to defects in pre-mRNA splicing, causing or
modifying the severity of many genetic
diseases[4]. It is
currently clear that these variations affect splicing not only by
disrupting classic splice sites (DS or AS), but also by altering other
important RNA elements (splicing enhancers or silencers binding sites)
or by creating new splice
sites[4,5,22,23].
Traditionally, exonic variants outside the conserved splice site
dinucleotide have been classified as missense, nonsense or synonymous
variants when gene analysis is performed only at the DNA level.
Currently, it is widely believed that RNA analysis should be performed
to assess the pathogenicity of sequence variations, especially splicing
abnormalities caused by variants affecting the splicing regulatory
sequences. In many cases, however, RNA samples from the affected
individual are unavailable, and mRNAs are difficult to detect due to the
activation of the nonsense-mediated mRNA decay
pathway[24].
Fortunately, a functional splicing assay based on the minigene assay was
confirmed as an effective, reliable and relatively simple tool to
functionally assay potential
splicing[25], which
has been extensively validated in our previous
studies[20,21,26].
Single-base substitutions in the SLC4A1 , ATP6V1B1 ,ATP6V0A4 , FOXI1 , WDR72 and ATP6V1C2 gene,
many of which are predicted to lead to missense, nonsense or synonymous
variants, are often identified in patients with primary dRTA. Herein, we
assumed that the pathogenic effect of some of these variants is the
alteration of pre-mRNA splicing. Using bioinformatics tools, a total
of 15 single-base variants in SLC4A1 , ATP6V1B1 andATP6V0A4 gene were selected and tested with a minigene
assay. These variants were predicted to modify splicing regulatory
sequences (disruption of ESEs or creation of ESSs), generate new splice
sites or induce a significant reduction of splice site
strength. Minigene plasmids ligated to either WT or mutant genomic
sequences were constructed. The constructs were transfected into HEK293T
cells, and the mRNA was analyzed by RT-PCR and DNA
sequencing. Consequently, 8 of them were found to cause exon skipping.
Transcript analysis by quantitative PCR of four variants of
c.1765C>T in SLC4A1 , c.481G>T and
c.1102G>A in ATP6V1B1 , and c.322C>T inATP6V0A4 gene, located in the internal position of the exon, have
shown that they affect pre-mRNA splicing by causing a significant
imbalance in the proportion of ESEs/ESSs. Many splicing regulatory
sequences, including ESEs and ESS, can promote or inhibit the
recognition of surrounding splicing sites by recruiting different
protein factors, thus coordinating to regulate the correct splicing of
exons[27].
Therefore, an improper ESE/ ESS ratio will prevent correct preRNA
splicing. Variant c.1765C>T (p.Arg589Cys) in SLC4A1gene was reported to cause dRTA in the form of autosomal dominant
inheritance[28]. In
the previous study, the Arg509 was considered as a mutation hotspot,
which is located in the intracellular domain between the 6th and the 7th
transmembrane regions of the AE1 protein and at the apex of the
cytoplasm of the 6th transmembrane region, and two other forms of the
variant in the same site (p.Arg589His, p.Arg589Ser) have been confirmed
to be
pathogenic[29,30].
Variants at p. Arg589 site may lead to failure to completely transported
to the cell membrane of AE1 protein, or the defective loading of the
advanced structure after transferring to the cell membrane, resulting in
structural and functional
abnormalities[28-30].
In this study, online software HSF was used to predict that
c.1765C>T could cause a significant imbalance in the
proportion of ESEs/ESSs. Further in vitro minigene analysis revealed
that this variant produced part of transcripts lacking exon 14, which
did not change the open reading frame. We hypothesized that it not only
generated a transcript containing c.1765C>T but also
produced a transcript lacking exon 14, resulting in abnormal
morphogenesis of the encoded protein and leading to a phenotype of dRTA.
Nonsense variant, a single-nucleotide substitution, is generally
considered to generate a premature stop codon. However, many studies
have found that nonsense variants can occasionally alter exonic elements
and affect the splicing pathway, which can be demonstrated by the loss
of correlation between phenotype and
genotype[30,31].
The result of Zhu’s research has elucidated the detailed molecular
mechanisms of exon skipping induced by nonsense mutations in theDMDgene[30]. In this
context, variant c.484G>T in ATP6V1B1 was predicted
as nonsense mutation, p.Glu162*, and the minigene splicing assay for
c.484G>T showed that it prevents exon 6 inclusion with a
subsequent frameshift and premature termination at the 22th codon in
exon 7 in the mRNA probably by affecting a functional ESE site and/or
creating a functional ESS site. The bioinformatics analysis predicted
the loss of five ESE and the gain of two ESS motifs with HSF. Moreover,
missense variant c.1102G>A (p.Glu368Lys) in ATP6V1B1also generated a part of aberrant transcripts that lack exon 11,
resulting in a subsequent frameshift and premature termination at the
25th codon in exon 13. We hypothesize that this is probably due to the
disruption of seven functional ESE sites and the generation of a
functional ESS site, which binds hnRNPA1. Similar to
c.484G>T in ATP6V1B1, variant c.322C>T
in ATP6V0A4 was initially defined as nonsense variant (p.Gln108*)
leading to an unable mRNA due to the nonsense-mediated mRNA decay. Our
results showed that it influenced related ESEs and ESSs motifs and
disturbed the normal splicing in vitro causing exon 6 skipping.
Subsequently, the ligation of exons 5 and 7 would result in a lack of 42
amino acids and the production of a truncated protein. As a consequence,
this mutated subunit A4 of apical H+-ATP protein would
destroy the first topological domain of cytoplasmic and may reduce or
abolish the transport activity of H+-ATPase.
Minigene assay allows us to categorize three exonic variants associated
with dRTA, c.368G>T and c.370C>T inATP6V1B1 , c.1571C>T and c.1572G>A inATP6V0A4 , as splicing mutation. These variants located at or near
to classical splice sites (DS or AS), may affect normal splicing either
by decreasing the recognition efficiency of the 5′ss DS or 3′ss AS or by
creating new splice sites. Sequence variations that occur in the
conserved GT and AG dinucleotides flanking exons will cause splicing
aberration, because the invariant GT and AG are required for the
spliceosome to recognize 5’s and 3’ss,
respectively[1,2,25].
Variants c.368G>T and c.370C>T
in ATP6V1B1 involving the first and +3 nucleotide of exon
5, respectively, were initially reported as missense mutations
p.Gly123Val and p.Arg124Trp, respectively. Analysis of variants
c.368G>T and c.370C>T in BDGP showed a
reduction in the score of the 3’ splice site. The minigene analysis of
these two mutations revealed that they certainly caused the skipping of
the exon 5.
Variants c.1571C>T and c.1572G>A inATP6V0A4 , located at the -2 and the last nucleotide of exon 15,
were identified as missense (p.Pro524Leu) and synonymous variant
(p.Pro524Pro), respectively. Such substitution usually reduces the
recognition of classic splicing sites. Our BDGP analysis further showed
that c.1571C>T resulted in the reduction of
recognition strength of donor
splicing site, while c.1572G>A did not. But HSF predicted
that c.1572G>A destroyed the WT donor sites. Consequently,
the minigene analysis revealed that these two variants certainly altered
normal splicing by increasing
approximately 3.74% and 84.48% exon 15 exclusion compared with WT,
respectively. The skipping of exon 15 results in a loss of 94
nucleotides with a subsequent frameshift and premature termination at
the 13th in exon 16. Significantly, synonymous substitutions are
generally considered to be benign from a protein perspective. But from
the mRNA’s point of view, these substitutions may lead to abnormal
splicing. Just as synonymous variant c.1572G>A of theATP6V0A4 in this study produced the aberrant transcript, the five
synonymous variants were reclassified as splicing mutations using a
minigene assay by Yohann Jourdy, which highlight that it is necessary to
investigate the splicing impact of all synonymous substitutions to
evaluate their clinical
significance[2].
Among 8 variants, the PCR product of c.368G>T inATP6V1B1 was a unique transcript lacking exon 5, however,
c.1765C>T in SLC4A1 , c.370C>T,
c.481G>T and c.1102G>A in ATP6V1B1 , and
c.322C>T, c.1571C>T and c.1572G>A
in ATP6V0A4 gene, still produced one transcript with the size of
the WT product except that one RT-PCR product was the transcript missing
corresponding exon, so they probably have a double damaging effect:
while the some part of the transcript is deleterious due to the skipping
of the exon, which would cause loss of part of some important domains or
premature termination of codons due to frameshift, the remaining mRNA is
damaging due to the mutated amino acid change, which could result in
non-functional protein, truncated polypeptide or nonsense mediated mRNA
decay.
In addition, variants of c.1437C>G and
c.1564G>A in SLC4A1 , c.481G>A inATP6V1B1 and c.52C>T, c.2190C>G inATP6V0A4 , were found to influence surrounding ESEs and ESSs
motifs by the assessment of HSF or to cause the change of recognition
strength of splicing site. However, results of our minigenes assay
demonstrated that these variants did not affect pre-mRNA splicing, which
was inconsistent with the predicted results of software, indicating
that the results of the online software had a certain high sensitivity
and low specificity. Also, we did not introduce these variants into cDNA
and detect the cell surface expression and ion transport activity after
AE1 or H+-ATPase mutations, which require further
study.
In conclusion, we have performed an extensive analysis of exonic
variants in SLC4A1 , ATP6V1B1 and ATP6V0A4associated with dRTA using bioinformatics tools and mini-genes. This
study allowed to reclassify 8 previously presumed missense, nonsense or
synonymous variants as splicing variations, which should be taken their
pathogenicity into account. These variants either disrupt ESEs and
produce ESSs, or interfere with the recognition of the splicing site of
3 ’ AS and a 5’ DS, resulting in exon skipping. This study
emphasized the importance of
assessing the effect of exon point mutations at the mRNA level in the
dRTA, especially under the condition of the failure to obtain the
patients’ RNA or kidney specimens, minigene assay may be a valuable
tool.