ABCB11 Gene Mutations Are Associated With Primary Intrahepatic Stone

ABCB11 gene plays an essential role in bile transport through its encoding protein bile salt export pump(BSEP),and its mutation has been found to closely associated with cholestasis and cholelithiasis.In our previous studies,ABCB11 mutation was frequently detected in patients with primary intrahepatic stone,and was demonstrated to affect the expression of BSEP.Therefore,we aim to investigate the relationship between ABCB11 mutation and the pathogenesis of primary intrahepatic stone,and to clarify the mechanism of ABCB11 mutation on the BSEP expression.Methods1.A total of 443 cases of primary intrahepatic stone(PIS)patients confirmed by preoperative imaging diagnosis were collected from the Southwest Hospital.And a total of 560 cases of healthy people(excluded from chronic liver disease and cholelithiasis)in the hospital's physical examination center were collected as the control group.Five milliliters of peripheral blood from the PIS patients and control group were collected.Lymphocytes isolated from the peripheral blood were stored at-80 ? immediately.Clinical data of these cases were collected and sorted.2.Lymphocytes DNA were extracted and performed with all exons sequencing to detect ABCB11 mutation in the PIS and healthy cases.Association between ABCB11 mutation and the patients' clinical data were analyzed.3.Total mRNA and membrane proteins were extracted from fresh frozen liver tissues of patients with primary intrahepatic stones,and BSEP mRNA and protein level were detected by quantitative PCR and Western blot,repectively.Correlation between BSEP expression and ABCB11 mutation was analyzed.4.A wild-type plasmid containing the open reading frame of human ABCB11 was constructed,and a mutant plasmid was further constructed through site-directed mutagenesis.HEK293 cells were transfected with wild-type and mutant plasmid,respectively.Total m RNA and membrane proteins were extracted after 48 hours of transfection.BSEP m RNA and protein expression in mutant and wild-type plasmids were detected by quantitative PCR and Western blot.After 48 hours of incubation,MDCK cells transfected with the wild-type and mutant plasmids respectively were performed with immunostaining.Distribution of BSEP in MDCK cells was detected by confocal laser scanning microscopy.Results1.Two significant mutations in ABCB11,a missense mutation rs118109635(p=0.025)and a synonymous mutation rs497692(p=0.006),were found in patients with primary intrahepatic stone when compared with the healthy controls.And these mutations were significantly correlated with preoperative jaundice(p=0.026,p=0.011,respectively).2.The expression of BSEP in PIS patients with missense mutation(rs118109635)was decreased,while the expression of mRNA was not changed.The expression of m RNA and BSEP protein were decreased in PIS patients with synonymous mutation(rs497692).3.At the cellular level,we also found that the mutant rs118109635 reduced the BSEP expression on HEK293 cell membrane,reduced the distribution on MDCK cell membrane,but did not change the expression level of m RNA.ConclusionsThe mutation sites of ABCB11 gene,rs118109635 and rs497692,were associated with the primary intrahepatic stone,the mutation of which can decrease the expression and distribution of BSEP on cell membrane.Therefore,the two mutation sites of ABCB11 gene,rs118109635 and rs497692,may contribute to the pathogenesis of PIS.