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.