Introduction
Precursor messenger RNAs (Pre-mRNAs) composed of exons and introns are
transcribed from the protein-coding genes of most eukaryotic
cells[1]. Exons,
consisting of two terminal untranslated regions and protein-coding
regions, are interrupted by non-coding insertion segments called
introns. Mature translatable mRNAs are generated by pre-mRNAs splicing,
involving the removal of introns and the connection of exons, which is
an important process of gene expression. Splicing is catalyzed by the
spliceosome, a large ribonucleoprotein (RNP) complex constituted by 5
small nuclear ribonucleoproteins (snRNPs) U1, U2, U4, U5 and U6, and
many other associated non‐snRNP proteins, that can recognize a large
number of splicing signals in pre-mRNA and stimulate intron
removal[2,3].
The splicing signals include the
conserved splice sites (5′ splice donor site (5′ss), and 3′ splice
acceptor site (3′ss)), the branch site, polypyrimidine track,
exonic/intronic splicing enhancers (ESEs/ISEs) and silencers
(ESSs/ISSs), as well as other regulatory components or the RNA secondary
structure[4,5]. All
these factors cooperate with splicing factors and the spliceosome, to
accurately remove introns and join exons.
In
the past, only intronic mutations affecting donor or acceptor splice
sites (DS or AS) were considered to potentially alter transcriptional
processing. Currently, given the importance of core and auxiliary
splicing signals in the pre-mRNA splicing process, it has been suggested
that up to 50% of exonic variants including all types (missense,
nonsense, and small insertions or deletions) that
alter splicing regulatory elements,
may disrupt the splicing pathway and cause various diseases in
human[6,7].
Primary distal renal tubular acidosis (dRTA) is a rare tubular disease
characterized by impaired tubular secretion of hydrogen ions in the
distal nephron, resulting in normal serum anion gap metabolic acidosis
that often progresses to hypokalemia, nephrocalcinosis, and
nephrolithiasis[8].
The vast majority of primary dRTA patients are associated with variants
in SLC4A1 , ATP6V0A4 and ATP6V1B1 genes, transmitted
as either an autosomal dominant (AD) or recessive (AR) trait. TheSLC4A1 gene, located on chromosome 17q21, contains 20 exons and
encodes a membrane protein the chloride–bicarbonate exchanger composed
of 911 amino acids, also known as AE1 or band 3 protein, which is
primarily responsible for the reabsorption of
HCO3- coupled with the urinary
excretion of
chloride[9]; the
gene ATP6V1B1 is found on the 2p13 chromosome with a total of 14
exons, which codifies the B1 subunit of the V-ATPase in
the α-intercalated cells of the renal collecting duct and endolymphatic
sac
epithelia[10,11];
the ATP6V0A4 gene is located on chromosome 7q33-34 and includes
23 exons, of which 20 encode transmembrane A4 subunits, which may be
involved in H+translocation or transport and/or
assembly of the
H+-ATPase[12].
In addition, recent literatures reported that variants inFOXⅠ1 [13],WDR72 [14]andATP6V1C2gene[15] were found
in primary dRTA patients.
DNA sequence analysis has been now widely used in the clinical diagnosis
of related genetic diseases, resulting in the identification of
thousands of rare variations in affected and unaffected individuals. The
number of these variants is expected to increase rapidly with the
application of high-throughput sequencing technology based on
large-scale parallel sequencing. So far, a total
of 201 different dRTA-related gene
variants have been described in the Human Gene Mutation Database (HGMD,
Professional 2019.4), including 29 in SLC4A1 , 73 inATP6V1B1 , 94 in ATP6V0A4 , 2 in FOXⅠ1 and 3 inWDR72 . Among them, missense/nonsense variants account for about
50% (101/201) of all variants. However, most variants analysis was
performed to assess the effects on mRNA and protein
at
the genome level, but only in a few cases at both DNA and RNA
levels[16].
There are few studies on the effect of splicing in exonic variants of
pathogenic genes associated with primary dRTA. The purpose of this study
was to gain insight into the effects of the previously described
dRTA-related pathogenic variants including missense and nonsense
variants on pre-mRNA splicing using a minigene technology.