In silico screening and analysis of single-nucleotide polymorphic variants of the ABCC2 gene affecting Dubin–Johnson syndrome

Dubin–Johnson syndrome (DJS) is a congenital, rare, benign, autosomal recessive disorder, which was first described in 1954 [1]. This syndrome is distinguished by fluctuating conjugated hyperbilirubinemia and accumulation of dark-colored pigments, which causes a condition called “black liver.” The main cause of DJS is impairment in the transport of non-bile acid organic anions from hepatocytes into the canaliculi [2]. Based on histochemical staining and physicochemical properties of the pigments, these pigments are similar to melanin [3], [4]. Patients with this syndrome might suffer from abdominal discomfort and other nonspecific symptoms [1]. DJS occurs in all races and nationalities and is distributed equally in both males and females [5]. Although it is a rare disorder, a higher incidence is observed in Sephardic Jews (approximately 1:3,000) [6]. DJS is caused by the mutations in the adenosine triphosphate (ATP)-binding cassette subfamily C member (ABCC2) gene [7].

ABCC2 is a canalicular multispecific organic anion transporter (cMOAT) that encodes the multidrug resistance associated protein 2 (MRP2) protein and functions as a transporter protein. Among nine MRPs, only MRP2 is exclusively localized to the apical membrane of the polarized cells. The ABCC2 gene is clustered on the chromosome band 10q24.2, has approximately 65 kbp, and contains 32 exons with a high section of class O introns [6], [8]. The MRP2 protein consists of 1,545 amino acids and has a molecular mass of 190 kDa [9]. It consists of 17 transmembrane helices, which form three transmembrane domains (TMDs), also known as membrane-spanning domains, named as MSD0 (N-terminal), MSD1 (middle), and MSD2 (C-terminal). The intracellular region of this protein contains two conserved nucleotide-binding domains (NBDs) of which NBD1 is linked with TMD1 and NBD2 is linked with TMD2 (Fig. 1). MSD1 and MSD2 are involved in the substrate recognition and specificity, while MSD0 and the adjacent linker region are vital for the apical targeting of proteins [10]. NBDs are involved in ATP-binding and hydrolysis, which are crucial for the efflux of substrates.

ABCC2 is expressed at pharmacological barriers, for example, enterocytes, canalicular membrane of hepatocytes, intestinal epithelial cells, and bronchial epithelial cells [11], [12]. It plays a vital role in the pharmacokinetics of various compounds. This cMOAT mediates energy-dependent efflux of different xenobiotic organic ions, particularly glutathione conjugates, sulfate, and glucuronate, from hepatocytes to bile. Moreover, ABCC2 is involved in the biliary excretion of both exogenous and endogenous waste products. ABCC2 substrates include immunosuppression drugs, antitumor drugs, carcinogens, toxins, and antibiotics, and ABCC2 ensures the efflux of the aforementioned substrates, resulting in their detoxification [13], [14]. Apart from this, ABCC2 is a vital determinant of bile and bile flow. Mutations in the ABCC2 gene lead to a deficiency in the expression of canalicular MRP2 and impair the transportation of conjugated bilirubin to the bile duct system. Conjugated bilirubin deposits in hepatocytes, increasing their levels in the blood [13], [15]. Numerous sequence variants have been acknowledged throughout the ABCC2 gene; however, information concerning their potential effects on the transporter activity is scarce.

Single-nucleotide polymorphisms (SNPs) in membrane transporters and dysfunctional mutations are one of the laying reasons for these disorders. In the human genome field, SNPs are considered accountable for approximately 90% sequence variations [16]. The identification of common genetic variants for investigating their neutral and deleterious effects on protein function connected with various disorders and diseases is essential. Among various SNPs, missense SNPs cause amino acid substitutions. The effect of these substitutions on the structure and function of the MRP2 protein is essential for understanding the multifaceted mechanisms of DJS, which are caused by the substitution of variant amino acids [17]. SNPs are recognized as biomarkers for predicting disease susceptibility. An analysis of SNPs in the ABCC2 gene associated with DJS has observed a notable increase in these SNPs in the past years [18], [19]. Due to the expression of ABCC2 in clearance organs (i.e., the liver and kidney) and its broad specificity of substrates, polymorphisms of MRP2 can seriously affect the transport activity. Studies have identified that SNPs in the TMDs and NBDs of ABCC2 are harmful. SNPs result in rapid mRNA degradation and impaired protein maturation, leading to the production of non-functional MRP2 [20], [21]. According to previous reports, the deleterious effects of SNPs in the ABCC2 gene have not been studied using in silico methods.

Therefore, this study was designed to detect the deleterious SNPs in the ABCC2 gene using various computational tools, such as SIFT, PROVEAN, PolyPhen-2, PANTHER, SNPs&GO, PhD-SNP, SNAP2, and Mutpred-2. The deleterious SNPs retrieved from these tools were further examined for the physicochemical properties (mainly structure, domain, charge, size, and hydrophobicity) of amino acid residues of ABCC2 using mutation3D, ELASPIC, and HOPE. Most damaging non-synonymous SNPs (nsSNPs) were additionally analyzed using stability tools, such as I-mutant 3.0 and MuPro. Finally, MirSNP and PolymiRTS 3.0 were used to investigate the effects of these SNPs in the untranslated region (UTR). This study will provide a constructive method for rapid and cost-efficient screening of pathological SNPs.

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