The IL23R R381Q gene variant protects against immune-mediated diseases by impairing IL-23-induced Th17 effector response in humans

IL-23 and Th17 cells are key players in tissue immunosurveillance and are implicated in human immune-mediated diseases. Genome-wide association studies have shown that the IL23R R381Q gene variant protects against psoriasis, Crohn's disease and ankylosing spondylitis. We investigated the immunological consequences of the protective IL23R R381Q gene variant in healthy donors. The IL23R R381Q gene variant had no major effect on Th17 cell differentiation as the frequency of circulating Th17 cells was similar in carriers of the IL23R protective (A) and common (G) allele. Accordingly, Th17 cells generated from A and G donors produced similar amounts of Th17 cytokines. However, IL-23-mediated Th17 cell effector function was impaired, as Th17 cells from A allele carriers had significantly reduced IL-23-induced IL-17A production and STAT3 phosphorylation compared to G allele carriers. Our functional analysis of a human disease-associated gene variant demonstrates that IL23R R381Q exerts its protective effects through selective attenuation of IL-23-induced Th17 cell effector function without interfering with Th17 differentiation, and highlights its importance in the protection against IL-23-induced tissue pathologies.     

Cutting edge: a variant of the IL-23R gene associated with inflammatory bowel disease induces loss of microRNA regulation and enhanced protein production

IL-23R gene variants have been identified as risk factors for two major inflammatory bowel diseases (IBDs), Crohn's disease and ulcerative colitis, but how they contribute to disease is poorly understood. In this study, we show that the rs10889677 variant in the 3'-untranslated region of the IL-23R gene displays enhanced levels of both mRNA and protein production of IL-23R. This can be attributed to a loss of binding capacity for the microRNAs (miRNAs) Let-7e and Let-7f by the variant allele. Indeed, inhibition and overexpression of these miRNAs influenced the expression of the wild type but not the variant allele. Our data clearly demonstrate a role for miRNA-mediated dysregulation of IL-23R signaling, correlated with a single nucleotide polymorphism in the IL-23R strongly associated with IBD susceptibility. This implies that this mutation, in combination with other genetic risk factors, can lead to disease through sustained IL-23R signaling, contributing to the chronicity of IBD.     

IL-10R1 S138G loss-of-function polymorphism is associated with extrapulmonary tuberculosis risk development in Tunisia

There is considerable evidence that host genetic factors are important in determining susceptibility to mycobacterial infections. More recently, functional genetic mutations affecting IL-10 receptor 1 (IL-10R1) were described. In this study, we investigated the relationship of IL-10R1 S138G loss-of-function polymorphism (A536G: rs3135932) with susceptibility to active tuberculosis (TB) in Tunisian patients. A total of 168 patients with pulmonary TB, 55 with extrapulmonary TB, and 150 control subjects were studied. Genomic DNA samples were extracted from leukocytes and used to investigate S138G polymorphism in IL-10R1 gene by multiplex allele-specific polymerase chain reaction. Associations between G allele [odds ratio OR = 5.01; 95% confidence intervals CI = 2.58–9.77; P = 10⁻⁷], GG genotypes [OR=9.06; 95% CI (1.58–67.33); correcting P-values using the Bonferroni method for multiple tests Pc=0.015] and AG genotype [OR=3.75; 95% CI (1.62–8.7); Pc=0.0012] with the risk development of active extrapulmonary TB were found. In contrast, the AA genotype was found to be associated with resistance to extrapulmonary TB [OR=0.19; 95% CI (0.09–0.42); Pc=6.10⁻⁶]. No association was found between S138G SNP and pulmonary TB. In conclusion, our study suggested the possible role of IL-10R1 S138G loss-of-function polymorphism in extrapulmonary TB susceptibility-resistance in Tunisia.     

Functional characterization of Ape1 variants identified in the human population

Apurinic/apyrimidinic (AP) sites are common mutagenic and cytotoxic DNA lesions. Ape1 is the major human repair enzyme for abasic sites and incises the phosphodiester backbone 5′ to the lesion to initiate a cascade of events aimed at removing the AP moiety and maintaining genetic integrity. Through resequencing of genomic DNA from 128 unrelated individuals, and searching published reports and sequence databases, seven amino acid substitution variants were identified in the repair domain of human Ape1. Functional characterization revealed that three of the variants, L104R, E126D and R237A, exhibited ~40–60% reductions in specific incision activity. A fourth variant, D283G, is similar to the previously characterized mutant D283A found to exhibit ~10% repair capacity. The most common substitution (D148E; observed at an allele frequency of 0.38) had no impact on endonuclease and DNA binding activities, nor did a G306A substitution. A G241R variant showed slightly enhanced endonuclease activity relative to wild-type. In total, four of seven substitutions in the repair domain of Ape1 imparted reduced function. These reduced function variants may represent low penetrance human polymorphisms that associate with increased disease susceptibility.     

Effect of the CALHM1 G330D and R154H Human Variants on the Control of Cytosolic Ca2+ and Aβ Levels

CALHM1 is a plasma membrane voltage-gated Ca²⁺-permeable ion channel that controls amyloid-β (Aβ) metabolism and is potentially involved in the onset of Alzheimer''s disease (AD). Recently, Rubio-Moscardo et al. (PLoS One (2013) 8: e74203) reported the identification of two CALHM1 variants, G330D and R154H, in early-onset AD (EOAD) patients. The authors provided evidence that these two human variants were rare and resulted in a complete loss of CALHM1 function. Recent publicly available large-scale exome sequencing data confirmed that R154H is a rare CALHM1 variant (minor allele frequency (MAF)  = 0.015%), but that G330D is not (MAF  = 3.5% in an African American cohort). Here, we show that both CALHM1 variants exhibited gating and permeation properties indistinguishable from wild-type CALHM1 when expressed in Xenopus oocytes. While there was also no effect of the G330D mutation on Ca²⁺ uptake by CALHM1 in transfected mammalian cells, the R154H mutation was associated with defects in the control by CALHM1 of both Ca²⁺ uptake and Aβ levels in this cell system. Together, our data show that the frequent CALHM1 G330D variant has no obvious functional consequences and is therefore unlikely to contribute to EOAD. Our data also demonstrate that the rare R154H variant interferes with CALHM1 control of cytosolic Ca²⁺ and Aβ accumulation. While these results strengthen the notion that CALHM1 influences Aβ metabolism, further investigation will be required to determine whether CALHM1 R154H, or other natural variants in CALHM1, is/are associated with EOAD.     

Germline EPHB2 receptor variants in familial colorectal cancer

Familial clustering of colorectal cancer occurs in 15-20% of cases, however recognized cancer syndromes explain only a small fraction of this disease. Thus, the genetic basis for the majority of hereditary colorectal cancer remains unknown. EPHB2 has recently been implicated as a candidate tumor suppressor gene in colorectal cancer. The aim of this study was to evaluate the contribution of EPHB2 to hereditary colorectal cancer. We screened for germline EPHB2 sequence variants in 116 population-based familial colorectal cancer cases by DNA sequencing. We then estimated the population frequencies and characterized the biological activities of the EPHB2 variants identified. Three novel nonsynonymous missense alterations were detected. Two of these variants (A438T and G787R) result in significant residue changes, while the third leads to a conservative substitution in the carboxy-terminal SAM domain (V945I). The former two variants were found once in the 116 cases, while the V945I variant was present in 2 cases. Genotyping of additional patients with colorectal cancer and control subjects revealed that A438T and G787R represent rare EPHB2 alleles. In vitro functional studies show that the G787R substitution, located in the kinase domain, causes impaired receptor kinase activity and is therefore pathogenic, whereas the A438T variant retains its receptor function and likely represents a neutral polymorphism. Tumor tissue from the G787R variant case manifested loss of heterozygosity, with loss of the wild-type allele, supporting a tumor suppressor role for EPHB2 in rare colorectal cancer cases. Rare germline EPHB2 variants may contribute to a small fraction of hereditary colorectal cancer.     

Functional variants in the lipoprotein lipase gene and risk cardiovascular disease.

The current report is a quantitative review of the relationship between lipoprotein lipase gene variants and cardiovascular disease based on published population-based studies. Sixteen studies, representing 17,630 individuals, report allelic distribution for lipoprotein lipase gene variants among patients and control individuals. Patient outcomes included clinical cardiovascular disease events, documented coronary disease based on angiography, or intimal media thickening by B-mode ultrasonography. Mantel-Haenszel stratified analysis was used to compute a summary odds ratio and 95% confidence intervals for the association between rare allele in the lipoprotein lipase gene and disease status. Because of potential differing effects associated with different lipoprotein lipase variants, each lipoprotein lipase mutant allele was considered separately. The lipoprotein lipase D9N/-93G to T allele has a summary odds ratio of 2.03 (95% confidence interval 1.30-3.18), indicating a twofold increase in risk of coronary disease for carriers with this allelic variant. The summary odds ratio for the relationship of the rare lipoprotein lipase G188E variant with cardiovascular disease is 5.25 (95% confidence interval 1.54-24.29). The lipoprotein lipase N291S allele is associated with a marginal increase in cardiovascular disease (summary odds ratio 1.25, 95% confidence interval 0.99-1.60, P = 0.07). However, there is stronger evidence for a positive association in certain populations. The summary odds ratio for lipoprotein lipase S447X allele is 0.81 (95% confidence interval 0.65-1.0), which indicates a cardioprotective effect of this lipoprotein lipase gene variant. Thus, lipoprotein lipase gene variants are associated with differential susceptibility to cardiovascular disease.     

Identification of a rare p.G320R alpha-1-antitrypsin variant in emphysema and lung cancer patients

The alpha-1-antitrypsin (A1AT) gene is highly polymorphic, with more than 100 genetic variants identified of which some can affect A1AT protein concentration and/or function and lead to pulmonary and/or liver disease. This study reports on the characterization of a p.G320R variant found in two patients, one with emphysema and the other with lung cancer. This variant results from a single base-pair substitution in exon 4 of the A1AT gene, and has been characterized as P by isoelectric focusing. Functional evaluation of the A1AT p.G320R variant was through comparing specific trypsin inhibitory activity in two patients with pulmonary disorders, carriers of the p.G320R variant, and 19 healthy individuals, carriers of normal A1AT M variants. Results showed that specific trypsin inhibitory activity was lower in both emphysema (2.45 mU/g) and lung cancer (2.07 mU/g) patients than in carriers of the normal variants (range 2.51-3.71 mU/g). This rare A1AT variant is associated with reduced functional activity of A1AT protein. Considering that it was found in patients with severe pulmonary disorders, this variant could be of clinical significance.     

Stratification by CARD15 variant genotype in a genome-wide search for inflammatory bowel disease susceptibility loci

Previously we have conducted a genome-wide search for inflammatory bowel disease susceptibility loci in a large European cohort. Results from this study demonstrated suggestive evidence of linkage to loci at chromosomes 1q, 6p, and 10p and replicated linkages on chromosomes 12 and 16. Recently, NOD2/CARD15 on chromosome 16q12 has been found to be strongly associated with Crohn's disease. In order to determine if there are other loci in the genome that interact with the three associated functional variants in CARD15 (R702W, G908R, 1007fs), we have stratified our large inflammatory bowel disease genome scan cohort by dividing pedigrees into two groups stratified by CARD15 variant genotype. The two pedigree groups were analysed using non-parametric allele sharing methods. The group of pedigrees that contained one of the three CARD15 variants had two suggestive linkage results occurring in 6p (lod = 3.06 at D6S197, IBD phenotype) and 10p (lod=2.29 at D10S197, CD phenotype). In addition, at 16q12 where CARD15 is located, the original genome scan had a peak lod score of 2.18 at D16S415 (CD phenotype). The stratified pedigree cohort containing one of three CARD15 variants had a peak lod score of 0.90 at D16S415 (CD phenotype), accounting for approximately less than half of the genetic evidence for linkage at this locus. This result is in agreement with the existence of a substantial number of private variants at the NOD2/CARD15 locus. Interaction with NOD2/CARD15 needs to be considered in future gene identification efforts on chromosomes 6 and 10.     

Population frequencies of single nucleotide polymorphisms (SNPs) in immuno-modulatory genes

Inherited polymorphisms in immuno-modulatory genes may contribute to variations in immune function and genetic susceptibility for complex diseases, including cancer. We report results from a comprehensive study to discover novel single nucleotide polymorphisms (SNPs) and to estimate allelic frequency for both novel and known coding and regulatory region SNPs in genes encoding proteins that have been implicated in the immune response to tumors. We identified 12 novel nucleotide substitution variants and one deletion variant in 17 genes analyzed (TGFBETA;R, BETA;2M, IFNGAMMA;, TNFALPHA;, TNFALPHA;R, LTALPHA;, IL-6, IL-12, IL-2, IL-1ALPHA;, IL-1BETA;, IL-1RN, IL-10, CTLA4, CD40L, FAS and FASL). We determined the frequency of these novel polymorphisms, as well as 17 previously identified polymorphisms, in a control sample of 158 individuals, approximately half of which were Caucasian (n = 74) and half of which were African American (n = 84). Significant differences in allele frequencies were observed between the two racial groups for 13/17 genes tested. These allelic variations maybe associated with alterations in immune function and thus susceptibility to a number of complex disease states such as cancer.     

Influence of interleukin-12 receptor β1 polymorphisms on tuberculosis

Host genetic factors may be important determinants of susceptibility to tuberculosis, and several candidate gene polymorphisms have been shown to date. A series of recent reports concerning rare human deficiencies in the type-1 cytokine pathway suggest that more subtle variants of relevant genes may also contribute to susceptibility to tuberculosis at the general population level. To investigate whether polymorphisms in the interleukin-12 receptor (IL-12R) gene predispose individuals to tuberculosis, we studied these genes by single-strand conformational polymorphism analysis and direct sequencing. Although no common polymorphisms could be identified in the IL-12R beta 2 gene ( IL-12RB2), we confirmed four single nucleotide polymorphisms (SNPs; 641A-->G, 684C-->T, 1094T-->C, and 1132G-->C) causing three missense variants (Q214R, M365T, G378R) and one synonymous substitution in the extracellular domain of the IL-12R beta 1 gene ( IL12RB1). All SNPs were in almost perfect linkage disequilibrium (D'=0.98), and two common haplotypes of IL12RB1(allele 1: Q214-M365-G378; allele 2: R214-T365-R378) were revealed. Polymerase chain reaction/restriction fragment length polymorphism and sequence analyses were used to type IL12RB1polymorphisms in 98 patients with tuberculosis and 197 healthy controls in Japanese populations. In our case-control association study of tuberculosis, the R214-T365-R378 allele (allele 2) was over-represented in patients with tuberculosis, and homozygosity for R214-T365-R378 (the 2/2 genotype) was significantly associated with tuberculosis (odds ratio: 2.45; 95% CI: 1.20-4.99; P=0.013). In healthy subjects, homozygotes for R214-T365-R378 had lower levels of IL-12-induced signaling, according to differences in cellular responses to IL-12 between two haplotypes. These data suggest that the R214-T365-R378 allele, i.e., variation in IL12RB1, contribute to tuberculosis susceptibility in the Japanese population. This genetic variation may predispose individuals to tuberculosis infection by diminishing receptor responsiveness to IL-12 and to IL-23, leading to partial dysfunction of interferon-gamma-mediated immunity.     

Characterization of common BRCA1 and BRCA2 variants

Many missense variants identified in BRCA1 and BRCA2, two genes responsible for the majority of hereditary breast and ovarian cancer, are of unclear clinical significance. Characterizing the significance of such variants is important for medical management of patients in whom they are identified. The aim of this study was to characterize eight of the most common reported missense mutations in BRCA1 and BRCA2 occurring in patients tested for hereditary risk of breast and ovarian cancers. The prevalence of each variant in a control population, co-segregation of the variant with cancer within families, location of the variant within the gene, the nature of the amino acid substitution and conservation of the wild-type amino acid among species were considered. In a control population, the BRCA1 variants M1652I, R1347G, and S1512I, were each observed at a frequency of 4.08%, 2.04%, and 2.04%, respectively, and the BRCA2 variants A2951T, V2728I, and D1420Y, were seen at 1.02%, 0.68%, and 0.34%, respectively. Although the BRCA2 variants T598A and R2034C were not seen in this group of controls, other clinical and published observations indicate that these variants are not deleterious. Based on epidemiological and biological criteria, we therefore conclude that the BRCA1 missense mutations R1347G, S1512I and M1652I, and the BRCA2 missense mutations T598A, D1420Y, R2034C, V2728I, and A2951T, are not deleterious mutations.     

APOBEC3G genetic variants and their influence on the progression to AIDS

The cytosine deaminase APOBEC3G, in the absence of the human immunodeficiency virus type 1 (HIV-1) accessory gene HIV-1 viral infectivity factor (vif), inhibits viral replication by introducing G-->A hypermutation in the newly synthesized HIV-1 DNA negative strand. We tested the hypothesis that genetic variants of APOBEC3G may modify HIV-1 transmission and disease progression. Single nucleotide polymorphisms were identified in the promoter region (three), introns (two), and exons (two). Genotypes were determined for 3,073 study participants enrolled in six HIV-AIDS prospective cohorts. One codon-changing variant, H186R in exon 4, was polymorphic in African Americans (AA) (f = 37%) and rare in European Americans (f < 3%) or Europeans (f = 5%). For AA, the variant allele 186R was strongly associated with decline in CD4 T cells (CD4 slope on square root scale: -1.86, P = 0.009), The 186R allele was also associated with accelerated progression to AIDS-defining conditions in AA. The in vitro antiviral activity of the 186R enzyme was not inferior to that of the common H186 variant. These studies suggest that there may be a modifying role of variants of APOBEC3G on HIV-1 disease progression that warrants further investigation.     

Host genes related to paneth cells and xenobiotic metabolism are associated with shifts in human ileum-associated microbial composition

The aim of this study was to integrate human clinical, genotype, mRNA microarray and 16 S rRNA sequence data collected on 84 subjects with ileal Crohn's disease, ulcerative colitis or control patients without inflammatory bowel diseases in order to interrogate how host-microbial interactions are perturbed in inflammatory bowel diseases (IBD). Ex-vivo ileal mucosal biopsies were collected from the disease unaffected proximal margin of the ileum resected from patients who were undergoing initial intestinal surgery. Both RNA and DNA were extracted from the mucosal biopsy samples. Patients were genotyped for the three major NOD2 variants (Leufs1007, R702W, and G908R) and the ATG16L1T300A variant. Whole human genome mRNA expression profiles were generated using Agilent microarrays. Microbial composition profiles were determined by 454 pyrosequencing of the V3-V5 hypervariable region of the bacterial 16 S rRNA gene. The results of permutation based multivariate analysis of variance and covariance (MANCOVA) support the hypothesis that host mucosal Paneth cell and xenobiotic metabolism genes play an important role in host microbial interactions.     

A naturally occurring sequence variation that creates a YY1 element is associated with increased cystic fibrosis transmembrane conductance regulator gene expression

We have identified previously a novel complex mutant allele in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in a patient affected with cystic fibrosis (CF). This allele contained a mutation in CFTR exon 11 known to cause CF (S549R(T>G)), associated with the first alteration described so far in the minimal CFTR promoter region (-102T>A). Studies on genotype-phenotype correlations revealed striking differences between patients carrying mutation (S549R(T>G)) alone, who had a severe disease, and patients carrying the complex allele (-102(T>A)+S549R(T>G)), who exhibited milder forms of CF. We thus postulated that the sequence change (-102T>A) may attenuate the effects of the severe (S549R(T>G)) mutation through regulation of CFTR expression. Analysis of transiently transfected cell lines with wild-type and -102A variant human CFTR-directed luciferase reporter genes demonstrates that constructs containing the -102A variant (which creates a Yin Yang 1 (YY1) core element) increases CFTR expression significantly. Electrophoretic mobility shift assays indicate that the -102 site is located in a region of multiple DNA-protein interactions and that the -102A allele recruits specifically an additional nuclear protein related to YY1. The finding that the YY1-binding allele causes a significant increase in CFTR expression in vitro may allow a better understanding of the milder phenotype observed in patients who carry a severe CF mutation within the same gene.     

Intellectual-disability-associated mutations in the ceramide transport protein gene CERT1 lead to aberrant function and subcellular distribution

The lipid molecule ceramide is transported from the endoplasmic reticulum to the Golgi apparatus for sphingomyelin production via the ceramide transport protein (CERT), encoded by CERT1. Hyperphosphorylation of CERT’s serine-repeat motif (SRM) decreases its functionality. Some forms of inherited intellectual disability (ID) have been associated with a serine-to-leucine substitution in the SRM (S132L mutation) and a glycine-to-arginine substitution outside the SRM (G243R mutation) in CERT; however, it is unclear if mutations outside the SRM disrupt the control of CERT functionality. In the current investigation, we identified a new CERT1 variant (dupAA) in a patient with mild ID that resulted from a frameshift at the C-terminus of CERT1. However, familial analysis revealed that the dupAA variant was not associated with ID, allowing us to utilize it as a disease-matched negative control for CERT1 variants that are associated with ID. Biochemical analysis showed that G243R and S132L, but not dupAA, impair SRM hyperphosphorylation and render the CERT variants excessively active. Additionally, both S132L and G243R mutations but not dupAA caused the proteins to be distributed in a punctate subcellular manner. On the basis of these findings, we infer that the majority of ID-associated CERT variants may impair SRM phosphorylation-dependent repression, resulting in an increase in sphingomyelin production concurrent with CERT subcellular redistribution.     

Cluster Analysis of Risk Factor Genetic Polymorphisms in Alzheimer’s Disease

Multiple genetic variants may contribute to the risk of developing Alzheimer’s disease. We have analyzed polymorphisms in 9 genes to determine whether particular combinations would contribute to this risk. The genes were APOE, LDLr, CST3, CTSD, TNF, BACE1, MAPT, STH, eNOS, and TFCP2. Three risk groups for the disease were identified. Risk group I was younger, was heterozygous for the CST3 (GA), CTSD2936 (AG), TNF -308 (AG) genetic variants. Risk group II was older, was homozygous for the −427 APOE promoter polymorphism (TT), and heterozygous for the MAPT deletion and for the STH variant (QR). Group III had both the youngest and oldest subjects, were heterozygous for the −863 (AC) and −1031 (CT) TNF promoter polymorphisms. All three groups carried the APOE 4 allele and were heterozygous for both BACE1 polymorphisms. The control groups were carriers of the APOE 3 allele and were homozygous for the BACE1 genetic variants. C. N. Randall, S. N. Morris, A. D. Winkie and G. R. Parker—STAR students. C. N. Randall, D. Strasburger, J. Prozonic, S. N. Morris, A. D. Winkie, G. R. Parker, D. Cheng and E. M. Fennell contributed equally to this study. Special issue article in honor of Dr. George DeVries.     

Functional studies on the IBD susceptibility gene IL23R implicate reduced receptor function in the protective genetic variant R381Q

Genome-wide association studies (GWAS) in several populations have demonstrated significant association of the IL23R gene with IBD (Crohn's disease (CD) and ulcerative colitis (UC)) and psoriasis, suggesting that perturbation of the IL-23 signaling pathway is relevant to the pathophysiology of these diseases. One particular variant, R381Q (rs11209026), confers strong protection against development of CD. We investigated the effects of this variant in primary T cells from healthy donors carrying IL23R(R381) and IL23R(Q381) haplotypes. Using a proprietary anti-IL23R antibody, ELISA, flow cytometry, phosphoflow and real-time RT-PCR methods, we examined IL23R expression and STAT3 phosphorylation and activation in response to IL-23. IL23R(Q381) was associated with reduced STAT3 phosphorylation upon stimulation with IL-23 and decreased number of IL-23 responsive T-cells. We also observed slightly reduced levels of proinflammatory cytokine secretion in IL23R(Q381) positive donors. Our study shows conclusively that IL23R(Q381) is a loss-of-function allele, further strengthening the implication from GWAS results that the IL-23 pathway is pathogenic in human disease. This data provides an explanation for the protective role of R381Q in CD and may lead to the development of improved therapeutics for autoimmune disorders like CD.     

Quantitative structure--activity relationship analysis and molecular dynamics simulation to functionally validate nonsynonymous polymorphisms of human ABC transporter ABCB1 (P-glycoprotein/MDR1)

Several preclinical and clinical studies suggest the importance of naturally occurring polymorphisms of drug transporters in the individual difference of drug response. To functionally validate the nonsynonymous polymorphisms of ABCB1 (P-glycoprotein/MDR1) in vitro, we generated SNP variant forms (i.e., S400N, R492C, R669C, I849M, A893P, A893S, A893T, M986V, A999T, P1051A, and G1063A) and expressed them in Sf9 cells. The kinetic properties (Km and Vmax) of those variants were analyzed by measuring the ATPase activity to obtain the ATPase profile for each variant toward structurally unrelated substrates. On the basis of the experimental data, we determined the substrate specificity of ABCB1 WT and its variants by the quantitative structure-activity relationship (QSAR) analysis method. While several SNP variants appeared to influence the substrate specificity of ABCB1, the nonsynonymous polymorphisms of 2677G > T, A, or C at amino acid position 893 (Ala > Ser, Thr, or Pro) have great impacts on both the activity and the substrate specificity of ABCB1. The A893P variant (2677G > C), a rare mutation, exhibited markedly high activity of ATPase toward different test compounds. Molecular dynamics (MD) simulation based on a three-dimensional structural model of human ABCB1 revealed that multiple kinks are formed in the intracellular loop between transmembrane domains 10 and 11 of the A893P variant (2677G > C) protein. The polymorphisms of 2677G, 2677T, and 2677A exhibit wide ethnic differences in the allele frequency, and these nonsynonymous polymorphisms are suggested to be clinically important because of their altered ATPase activity and substrate specificity toward different drugs.