ATP-binding Cassette Subfamily B Member 4 Research Articles

Expanding the spectrum of progressive familial intrahepatic cholestasis: A report of 3 cases.

Progressive familial intrahepatic cholestasis (PFIC) is a group of autosomal recessive disorders caused by defects in bile secretion or transport usually presenting as cholestasis in pediatric age. Herewith, we describe 3 PFIC cases with diagnostic challenges and highlight the role of genetic analysis. The clinical history, laboratory data, liver biopsy, and molecular analysis for each case were reviewed. Case 1, a Hispanic male from Puerto Rico with hepatomegaly since age 2 months, was eventually diagnosed with PFIC3 following identification of a homozygous splice site variant in ATP binding cassette subfamily B member 4 (ABCB4) (c.2784-12T>C) at age 17 years by whole-exome sequencing (WES). Case 2 was a 37-year-old man with a history of alcoholism, abnormal liver function tests, and ductopenia on biopsy. Molecular testing revealed a pathogenic heterozygous ABCB4 mutation (c.1633C>T) variant leading to a diagnosis of PFIC3. Case 3 was a 2-year-old female initially presenting as a drug-induced liver injury but was diagnosed with PFIC10 following identification of a heterozygous frameshift mutation (p.Asp300Trpfs*19) and a heterozygous missense mutation (c.1357T>C) in myosin VB (MYO5B) by WES. These PFIC cases highlight the heterogenous presentation and diagnostic challenges, and they emphasize the role of next-generation sequencing, particularly the utility of WES.

Clinical phenotype and genotype analysis of progressive familial intrahepatic cholestasis type 3 caused by novel ABCB4 gene mutation

Objective: To investigate the pathogenic mechanism and clinical characteristics of the novel splicing variant of ATP-binding cassette subfamily B member 4 (ABCB4) and provide a basis for subsequent genetic diagnosis. Methods: The clinical data of a 5-year-old child with cholestatic liver disease admitted to the Beijing Children's Hospital of Capital Medical University was retrospectively analyzed. The pathogenic variations were detected by whole exome sequencing and verified by Sanger sequencing, and bioinformatics was used to predict the pathogenicity of the mutation sites. Possible pathogenic variations were verified in vitro by Minigene assay. The clinical outcome was followed after discharge from hospital. Results: The 5-year-old boy had developed cholestasis at the age of 11 months. His physical examination showed obvious enlargement of the liver and spleen. Cholestatic cirrhosis was diagnosed by liver function tests, abdominal ultrasonography, liver biopsy and pathology. The results of genetic analysis showed that the patient was a complex heterozygote of the ABCB4 gene, with a pathogenic mutation c.2860G>A and a novel mutation c.2065-8T>G, derived from the mother and father respectively. The conservative prediction of the c.2065-8T>G site showed that this region was highly conserved and may affect splicing. Minigene assay results confirmed that the c.2065-8T>G mutation resulted in a 7 bp retention of intron 16 in the mature mRNA. In the absence of nonsense-mediated mRNA decay, the amino acid frameshift forms a truncated protein, which is represented by p.Glu689ValfsTer19. The patient was diagnosed as progressive familial intrahepatic cholestasis type 3 (PFIC3) and treated with ursodeoxycholic acid (UDCA). His clinical symptoms improved during 18 months of follow-up. Conclusions: The c.2065-8T>G variant is confirmed to affect the splicing process and exhibits complex heterozygosity with c.2860G>A, which is identified as the cause of the disease. PFIC3 children with this variant showed cholestatic liver disease as the main manifestation with a slow progression and was sensitive to treatment with UDCA.

Ivacaftor-Mediated Potentiation of ABCB4 Missense Mutations Affecting Critical Motifs of the NBDs: Repositioning Perspectives for Hepatobiliary Diseases.

ABCB4 (ATP-binding cassette subfamily B member 4) is a hepatocanalicular floppase involved in biliary phosphatidylcholine (PC) secretion. Variations in the ABCB4 gene give rise to several biliary diseases, including progressive familial intrahepatic cholestasis type 3 (PFIC3), an autosomal recessive disease that can be lethal in the absence of liver transplantation. In this study, we investigated the effect and potential rescue of ten ABCB4 missense variations in NBD1:NBD2 homologous positions (Y403H/Y1043H, K435M/K1075M, E558K/E1200A, D564G/D1206G and H589Y/H1231Y) all localized at the conserved and functionally critical motifs of ABC transporters, six of which are mutated in patients. By combining structure analysis and in vitro studies, we found that all ten mutants were normally processed and localized at the canalicular membrane of HepG2 cells, but showed dramatically impaired PC transport activity that was significantly rescued by treatment with the clinically approved CFTR potentiator ivacaftor. Our results provide evidence that functional ABCB4 mutations are rescued by ivacaftor, paving the way for the repositioning of this potentiator for the treatment of selected patients with PFIC3 caused by mutations in the ATP-binding sites of ABCB4.

A common variant in the hepatobiliary phospholipid transporter ABCB4 modulates liver injury in PBC but not in PSC: prospective analysis in 867 patients

BackgroundThe ATP-binding cassette subfamily B member 4 (ABCB4) gene encodes the hepatic phospholipid transporter. Variants in the ABCB4 gene are associated with various cholestatic phenotypes, some of which progress to liver fibrosis and cirrhosis. The aim of our study was to investigate the role of the cholestasis-associated variant ABCB4 c.711A > T (p.I237I, rs2109505) in patients with primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC).ResultsTwo cohorts of Polish patients took part in this study. The Szczecin cohort comprised 196 patients with PBC (174 females, 38% with cirrhosis) and 135 patients with PSC (39 females, 39% with cirrhosis). The Warsaw cohort consisted of 260 patients with PBC (241 females, 44% with cirrhosis) and 276 patients with PSC (97 females, 33% with cirrhosis). Two control cohorts—150 healthy blood donors and 318 patients without liver disease, were recruited in Szczecin and in Warsaw, respectively. The ABCB4 c.711A > T polymorphism was genotyped using TaqMan assay. In both PBC cohorts, carriers of the risk variant presented more frequently with cirrhosis (Szczecin: OR = 1.841, P = 0.025; Warsaw: OR = 1.528, P = 0.039). The risk allele was associated with increased serum AST, GGT and ALP (all P < 0.05) at inclusion. During the follow-up, patients in both cohorts significantly improved their laboratory results, independently of their ABCB4 c.711A > T genotype (P > 0.05). During 8 ± 4 years follow-up, a total of 22 patients in the Szczecin PBC group developed cirrhosis, and this risk was higher among carriers of the risk variant (OR = 5.65, P = 0.04). In contrast to PBC, we did not detect any association of ABCB4 c.711A > T with a liver phenotype in PSC cohorts.ConclusionsThe frequent pro-cholestatic variant ABCB4 c.711A > T modulates liver injury in PBC, but not in PSC. In particular, carriers of the major allele are at increased risk of progressive liver scarring.

ABCB4 variant is associated with hepatobiliary MR abnormalities in people with low-phospholipid-associated cholelithiasis syndrome

Background & AimsThe low-phospholipid-associated cholelithiasis (LPAC) syndrome is a recently described peculiar form of cholelithiasis associated with the ATP-binding-cassette subfamily B, member 4 (ABCB4) gene deficiency. The purpose of our study was to analyse the relationship between magnetic resonance (MR) features and the genetic status of ABCB4 in people with LPAC syndrome.MethodsA total of 233 individuals with proven LPAC syndrome were enrolled between January 2003 and June 2018 in a retrospective single-centre study. Inclusion criteria included availability of clinical files, MR images, and genetic data. MR images were analysed by consensus among 3 senior radiologists blinded to the status of ABCB4 gene mutation.ResultsA total of 125 individuals (mean age at first MR imaging 40.8 years; 66% females; 48% ABCB4 variant) were included. MR abnormalities were found in 61 (49%) of the 125 individuals. Forty (67%) of the 60 individuals with an ABCB4 gene variant had MR abnormalities as compared with 21 (33%) of the 65 individuals without an ABCB4 gene variant (odds ratio [OR] 4.1, 95% CI 1.9–9.5, p = 0.0001). Compared to individuals with no variant, individuals with an ABCB4 variant were more likely to show intrahepatic macrolithiasis (56 vs. 17%; OR 6.3, 95% CI 2.6–16.2, p <0.0001), bile duct dilatation (60 vs. 18%; OR 6.5, 95% CI 2.7–16.3, p <0.0001), and at least 1 MR feature of complication (35 vs. 15%; OR 2.9, 95% CI 1.1–7.8, p <0.05).ConclusionsABCB4-related LPAC syndrome is associated with more frequent and severe hepatobiliary MR abnormalities. This finding strongly supports the major role of the ABCB4 gene in the pathogenesis of LPAC syndrome and highlights a genotype–phenotype association in this inherited disease with genetic heterogeneity.Lay summaryABCB4-related LPAC syndrome associated with an ABCB4 gene variant demonstrates more frequent and severe hepatobiliary MR abnormalities. This finding supports the major role of the ABCB4 gene in the pathogenesis of LPAC syndrome.

Targeting Mitochondrial-Nuclear Apoptotic Signaling Pathways, Metabolic Pathways and Reversal of Doxorubicin (DOX) Cell Resistance in MCF-7 and MDA‑MB-231 Cancer Cell Lines by Sambucus Nigra (SNA)

Background: The study aimed to assess the effect of anti-tumor and anti-proliferative properties of Sambucus nigra (SNA) on MCF-7 and MDA MB-231 breast tumor cell lines.Methods: The cytotoxicity of SNA was assessed based on dose/time by the MTT assay. Also, the influence of SNA on apoptotic pathways, cellular and metabolic resistance in these cell lines was examined by real-time PCR, lipid peroxidase was measured by malondialdehyde (MDA) and the effect of apoptosis and necrosis was determined by flow cytometry.Results: Our data showed that DOX, SNA, and DOX + SNA treatment induced the expression of p53, Bax, Bcl-2, Caspase-3, and 8 levels involved in the apoptotic pathways. ATP binding cassette subfamily B member 4 (ABCB4) gene expression was decreased in MDA‑MB-231 breast cancer cells compared to MCF-7. Also, we observed that DOX, SNA, and DOX + SNA treatment induced expression of Monocarboxylate transporters (MCTs) metabolic pathways such as MCT1 and MCT4. Conclusion: Overall, the outcomes of this investigation show that the combination of SNA-Doxorubicin (DOX) in different groups of these cancer cells, especially in the MDA‑MB-231 cell lines synergistically intensified the induction of apoptosis in them. SNA enhances the anti-cancer effects of DOX to induce cellular apoptosis, alter metabolic pathways, and reduce cellular resistance. The research highlights the promising use of SNA as a chemosensitizer in the chemotherapy.

RAB10 Interacts with ABCB4 and Regulates Its Intracellular Traffic.

ABCB4 (ATP-binding cassette subfamily B member 4) is an ABC transporter expressed at the canalicular membrane of hepatocytes where it ensures phosphatidylcholine secretion into bile. Genetic variations of ABCB4 are associated with several rare cholestatic diseases. The available treatments are not efficient for a significant proportion of patients with ABCB4-related diseases and liver transplantation is often required. The development of novel therapies requires a deep understanding of the molecular mechanisms regulating ABCB4 expression, intracellular traffic, and function. Using an immunoprecipitation approach combined with mass spectrometry analyses, we have identified the small GTPase RAB10 as a novel molecular partner of ABCB4. Our results indicate that the overexpression of wild type RAB10 or its dominant-active mutant significantly increases the amount of ABCB4 at the plasma membrane expression and its phosphatidylcholine floppase function. Contrariwise, RAB10 silencing induces the intracellular retention of ABCB4 and then indirectly diminishes its secretory function. Taken together, our findings suggest that RAB10 regulates the plasma membrane targeting of ABCB4 and consequently its capacity to mediate phosphatidylcholine secretion.

Case report: progressive familial intrahepatic cholestasis type 3 with compound heterozygous ABCB4 variants diagnosed 15\u2009years after liver transplantation

BackgroundProgressive familial intrahepatic cholestasis (PFIC) type 3 is an autosomal recessive disorder arising from mutations in the ATP-binding cassette subfamily B member 4 (ABCB4) gene. This gene encodes multidrug resistance protein-3 (MDR3) that acts as a hepatocanalicular floppase that transports phosphatidylcholine from the inner to the outer canalicular membrane. In the absence of phosphatidylcholine, the detergent activity of bile salts is amplified and this leads to cholangiopathy, bile duct loss and biliary cirrhosis. Patients usually present in infancy or childhood and often progress to end-stage liver disease before adulthood.Case presentationWe report a 32-year-old female who required cadaveric liver transplantation at the age of 17 for cryptogenic cirrhosis. When the patient developed chronic ductopenia in the allograft 15 years later, we hypothesized that the patient’s original disease was due to a deficiency of a biliary transport protein and the ductopenia could be explained by an autoimmune response to neoantigen that was not previously encountered by the immune system. We therefore performed genetic analyses and immunohistochemistry of the native liver, which led to a diagnosis of PFIC3. However, there was no evidence of humoral immune response to the MDR3 and therefore, we assumed that the ductopenia observed in the allograft was likely due to chronic rejection rather than autoimmune disease in the allograft.ConclusionsTeenage patients referred for liver transplantation with cryptogenic liver disease should undergo work up for PFIC3. An accurate diagnosis of PFIC 3 is key for optimal management, therapeutic intervention, and avoidance of complications before the onset of end-stage liver disease.

Low-phospholipid-associated cholelithiasis syndrome: Prevalence, clinical features, and comorbidities

Background & AimsLow-phospholipid-associated cholelithiasis (LPAC) syndrome, a rare genetic form of intrahepatic cholelithiasis in adults, is still poorly understood. We report the results of the largest-ever case-control study of patients with LPAC syndrome aiming to assess the prevalence, clinical features, and comorbidities of the disease.MethodsWe included all LPAC cases diagnosed between 2001 and 2016 in 11 French centres. Controls consisted of all patients who underwent a cholecystectomy for common gallstone disease in a single non-academic centre over 1 year. A logistic regression analysis was used to identify the clinical features associated with LPAC syndrome across several patient strata with increasing levels of diagnostic confidence. The ratio between the incident cases of LPAC syndrome and the total number of cholecystectomies for gallstones was used to assess the relative prevalence of the disease.ResultsIn this study, 308 cases and 206 controls were included. LPAC syndrome accounted for 0.5–1.9% of all patients admitted with symptomatic gallstone disease. Age at first symptoms <40 years, absence of overweight, persistence of symptoms after cholecystectomy, intrahepatic micro- or macrolithiasis, common bile duct (CBD) lithiasis, and no history of cholecystitis were independently associated with LPAC diagnosis. ATP-binding cassette subfamily B member 4 (ABCB4) variants, present in 46% of cases, were associated with CBD lithiasis, chronic elevation of gamma-glutamyltransferase (GGT), and personal or family history of hepato-biliary cancer.ConclusionsIn this case-control study, LPAC syndrome accounted for approximately 1% of symptomatic cholelithiasis in adults. In addition to pre-established diagnostic criteria, normal weight, CBD lithiasis, and no history of cholecystitis were significantly associated with the syndrome. ABCB4 gene variations in patients with LPAC were associated with CBD lithiasis, chronic cholestasis, and a personal or family history of hepato-biliary cancer.Lay summaryIn the largest case-control study ever conducted in patients with LPAC syndrome, a rare genetic form of intrahepatic cholelithiasis in young adults, LPAC syndrome was found in approximately 1% of all patients admitted to the hospital for symptomatic gallstones and, in addition to the pre-established characteristics of the syndrome (age at first symptoms <40 years, recurrence of symptoms after cholecystectomy, and/or imaging evidence of intrahepatic microlithiasis), was associated with lower BMI, higher prevalence of common bile duct stones, and lower incidence of acute cholecystitis. ABCB4 gene variants, which were detected in about half of cases, were associated with common bile duct stones and a personal or family history of hepato-biliary cancer.

Senescent Stromal Cells Promote Cancer Resistance through SIRT1 Loss-Potentiated Overproduction of Small Extracellular Vesicles.

Cellular senescence is a potent tumor-suppressive program that prevents neoplastic events. Paradoxically, senescent cells develop an inflammatory secretome, termed the senescence-associated secretory phenotype, which is implicated in age-related pathologies including cancer. Here, we report that senescent cells actively synthesize and release small extracellular vesicles (sEV) with a distinctive size distribution. Mechanistically, SIRT1 loss supported accelerated sEV production despite enhanced proteome-wide ubiquitination, a process correlated with ATP6V1A downregulation and defective lysosomal acidification. Once released, senescent stromal sEVs significantly altered the expression profile of recipient cancer cells and enhanced their aggressiveness, specifically drug resistance mediated by expression of ATP-binding cassette subfamily B member 4 (ABCB4). Targeting SIRT1 with agonist SRT2104 prevented development of cancer resistance by restraining sEV production by senescent stromal cells. In clinical oncology, sEVs in peripheral blood of posttreatment cancer patients were readily detectable by routine biotechniques, presenting an exploitable biomarker to monitor therapeutic efficacy and predict long-term outcome. Together, this study identifies a distinct mechanism supporting pathologic activities of senescent cells and provides a potent avenue to circumvent advanced human malignancies by cotargeting cancer cells and their surrounding microenvironment, which contributes to drug resistance via secretion of sEVs from senescent stromal cells. SIGNIFICANCE: Senescent stromal cells produce a large number of sEVs to promote cancer resistance in therapeutic settings, a process driven by SIRT1 decline in stromal cells and ABCB4 augmentation in cancer cells.See related commentary by Wiley, p. 3193 GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/16/3383/F1.large.jpg.

Abstract 2969: Senescent stromal cells promote cancer resistance via Sirt1 loss-potentiated biogenesis of small extracellular vesicles

Abstract Cellular senescence is a potent tumor-suppressive program that prevents neoplastic events. Paradoxically, senescent cells develop an inflammatory secretome, termed the senescence-associated secretory phenotype (SASP) and implicated in age-related pathologies including cancer. Here we report that senescent cells actively synthesize and release small extracellular vesicles (sEVs) with a distinctive size distribution. Mechanistically, SIRT1 loss supports accelerated sEV production despite enhanced proteome-wide ubiquitination, a process correlated with ATP6V1A downregulation and defective lysosomal acidification. Senescent stromal cell-derived sEVs carry a special set of cargoes, including small RNA species such as microRNAs (miRNAs) whose spectrum markedly differ from that of proliferating cell-secreted sEVs. Once released, senescent stromal sEVs significantly alter the expression profile of recipient cancer cells and enhance their aggressiveness, specifically drug resistance mediated by expression of ATP binding cassette subfamily B member 4 (ABCB4). Targeting SIRT1 with an agonist SRT2104 prevents development of cancer resistance through restraining sEV production by senescent stromal cells. In clinical oncology, sEVs in peripheral blood of posttreatment cancer patients are readily detectable by routine biotechniques, presenting a novel biomarker to monitor therapeutic efficacy and predict long term outcome. Together, our study identifies a distinct mechanism supporting pathological activities of senescent cells, and provides a novel avenue to circumvent advanced human malignancies by co-targeting cancer cells and their surrounding microenvironment, which contributes to drug resistance via secretion of sEVs from senescent stromal cells. Citation Format: Liu Han, Qilai Long, Eric Lam, Yu Sun. Senescent stromal cells promote cancer resistance via Sirt1 loss-potentiated biogenesis of small extracellular vesicles [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2969.

Curcumin reverses doxorubicin resistance via inhibition the efflux function of ABCB4 in doxorubicin‑resistant breast cancer cells.

Doxorubicin is one of the most widely used chemotherapy agents for the treatment of breast cancer. However, the development of doxorubicin resistance limits the long-term treatment benefits in patients with breast cancer. Curcumin, a well-known dietary polyphenol derived from the rhizomes of turmeric (Curcuma longa), enhances the sensitivity of breast cancer cells to chemotherapeutic agents; however, the mechanisms underlying this phenomenon remain unclear. The aim of the present study was to evaluate the effect of curcumin on chemoresistance in doxorubicin-resistant breast cancerMCF-7/DOX and MDA-MB-231/DOX cell lines. Cell Counting Kit-8, monolayer transport, western blot and ATPase activity assays were performed during the study. The results revealed that curcumin significantly enhanced the effect of doxorubicin in doxorubicin-resistant breast cancer cells. The intracellular accumulation of doxorubicin was substantially increased following curcumin treatment in doxorubicin-resistant breast cancer cells, in a manner that was inversely dependent on the activity of ATP binding cassette subfamily B member 4 (ABCB4). Treatment with a combination of curcumin and doxorubicin decreases the efflux of doxorubicin in ABCB4-overexpressing cells. Furthermore, curcumin inhibited the ATPase activity of ABCB4 without altering its protein expression. In conclusion, curcumin reversed doxorubicin resistance in human breast cancer MCF-7/DOX and MDA-MB-231/DOX cells by inhibiting the ATPase activity of ABCB4. The study highlights the promising use of curcumin as a chemosensitizer in the treatment of breast cancer.

ABCB4 Gene Aberrations in Human Liver Disease: An Evolving Spectrum.

ATP-binding cassette subfamily B member 4 (ABCB4) is a phospholipid translocator at the canalicular membrane of the hepatocyte, which "flops" phosphatidylcholine into bile. Dysfunction of this transporter due to ABCB4 gene variants can cause liver diseases and has been called ABCB4 deficiency. Several diseases including progressive familial intrahepatic cholestasis type 3 (PFIC3), low phospholipid-associated cholelithiasis (LPAC), a subgroup of patients developing intrahepatic cholestasis of pregnancy (ICP), drug-induced liver injury and chronic cholangiopathy with biliary fibrosis and cirrhosis were attributed to ABCB4 deficiency and characterized in the past decade. LPAC and ICP are usually caused by monoallelic variants, whereas patients affected by PFIC3 are homozygous or compound heterozygous carriers of ABCB4 variants. Treatment with ursodeoxycholic acid is often effective, but as the more severe forms of ABCB4 deficiency progress, nevertheless, new diagnostic and therapeutic approaches are warranted. Current functional classifications for ABCB4 deficiency-associated mutations can guide the development of novel genotype-based targeted pharmacotherapies for these conditions. Recently, increasing evidence from genome-wide association studies is emerging on associations of ABCB4 variants with hepatobiliary malignancies.

Overexpression of ABCB4 contributes to acquired doxorubicin resistance in breast cancer cells in vitro.

Doxorubicin is one of the most active agents in the first-line therapy for metastatic breast cancer, but its utility is partially limited by the frequent emergence of doxorubicin resistance. In this study, we aimed to investigate the role of ATP-binding cassette sub-family B, member 4 (ABCB4) in acquired doxorubicin resistance in breast cancer cells, as well as its potential mechanism. In doxorubicin-sensitive and -resistant breast cancer cell lines MCF-7 and MDA-MB-231, the expression levels of ABCB4 were detected using real-time quantitative PCR and Western blot analysis, the DNA methylation and histone acetylation status of ABCB4 gene were investigated by bisulfite-sequencing PCR (BSP) and chromatin immunoprecipitation (ChIP) assays, and the doxorubicin sensitivity and intracellular doxorubicin accumulation were observed using cell cytotoxicity assay and flow cytometry. In Madin-Darby Canine Kidney (MDCKII) cells, In vitro transport assay was used to assess the ABCB4-mediated transport of doxorubicin. ABCB4 was overexpressed in doxorubicin-resistant breast cancer cells compared to their doxorubicin-sensitive counterparts, which was associated with reduced DNA methylation as well as increased histone acetylation at the ABCB4 promoter. ABCB4 could actively pump doxorubicin out of the cells, and knockdown of ABCB4 increased doxorubicin sensitivity and intracellular accumulation in doxorubicin-resistant breast cancer cells. Our results indicate that ABCB4 is overexpressed in breast cancer cells with acquired doxorubicin resistance, which could be attributed, at least partially, to the epigenetic modifications of ABCB4 gene. ABCB4 mediates the efflux transport of doxorubicin, and contributes to the acquired resistance of doxorubicin in breast cancer cells.

Exome sequencing of a family with lone, autosomal dominant atrial flutter identifies a rare variation in ABCB4 significantly enriched in cases.

BackgroundLone atrial flutter (AFL) and atrial fibrillation (AF) are common and sometimes consequential cardiac conduction disorders with a strong heritability, as underlined by recent genome-wide association studies that identified genetic modifiers. Follow-up family-based genetic analysis also identified Mendelian transmission of disease alleles. Three affected members were exome-sequenced for the identification of potential causative mutations, which were subsequently validated by direct sequencing in the other 3 affected members. Taqman assay was then used to confirm the role of any mutation in an independent population of sporadic lone AFL/AF cases.ResultsThe family cluster analysis provided evidence of genetic inheritance of AFL in the family via autosomal dominant transmission. The exome-sequencing of 3 family members identified 7 potential mutations: of these, rs58238559, a rare missense genetic variant in the ATP-binding cassette sub-family B, member 4 (ABCB4) gene was carried by all affected members. Further analysis of 82 subjects with sporadic lone AF, 63 subjects with sporadic lone AFL, and 673 controls revealed that the allele frequency for this variation was significantly higher in cases than in the controls (0.05 vs. 0.01; OR = 3.73; 95% CI = 1.16–11.49; P = 0.013).Conclusionsrs58238559 in ABCB4 is a rare missense variant with a significant effect on the development of AFL/AF.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-015-0177-0) contains supplementary material, which is available to authorized users.

ABCB4 is frequently epigenetically silenced in human cancers and inhibits tumor growth.

Epigenetic silencing through promoter hypermethylation is an important hallmark for the inactivation of tumor-related genes in carcinogenesis. Here we identified the ATP-binding cassette sub-family B member 4 (ABCB4) as a novel epigenetically silenced target gene. We investigated the epigenetic regulation of ABCB4 in 26 human lung, breast, skin, liver, head and neck cancer cells lines and in primary cancers by methylation and expression analysis. Hypermethylation of the ABCB4 CpG island promoter occurred in 16 out of 26 (62%) human cancer cell lines. Aberrant methylation of ABCB4 was also revealed in 39% of primary lung cancer and in 20% of head and neck cancer tissues. In 37% of primary lung cancer samples, ABCB4 expression was absent. For breast cancer a significant hypermethylation occurred in tumor tissues (41%) compared to matching normal samples (0%, p = 0.002). Silencing of ABCB4 was reversed by 5-aza-2'-deoxycytidine and zebularine treatments leading to its reexpression in cancer cells. Overexpression of ABCB4 significantly suppressed colony formation and proliferation of lung cancer cells. Hypermethylation of Abcb4 occurred also in murine cancer, but was not found in normal tissues. Our findings suggest that ABCB4 is a frequently silenced gene in different cancers and it may act tumor suppressivly in lung cancer.

ABCB4 and ABCB11 mutations in intrahepatic cholestasis of pregnancy in an Italian population

BackgroundGenetic alterations in the ATP-binding cassette subfamily B member 4 (ABCB4) and ATP-binding cassette subfamily B member 11 (ABCB11) have been associated to the onset of intrahepatic cholestasis of pregnancy (ICP) in predisposed women. AimsTo identify new and/or frequent ABCB4 and ABCB11 genes variants in a cohort of Italian patients with ICP and to evaluate the possible pathogenetic role for the novel mutations identified. MethodsDNA of 33 unrelated Italian women with obstetric cholestasis were screened for mutations in the entire coding sequence of ABCB4 and ABCB11 genes. Polymerase chain reaction and automated sequencing was performed on the 27 coding exons of both genes. ResultsGenotyping revealed 11 mutations, 5 of whom were novel variants: 2 localized on ABCB4 (p.I587DfsX603, p.I738LfsX744) and 3 on ABCB11 (p.V284D, p.Q558H, p.P731S). The most severe phenotypes were associated with the variants p.I587DfsX603, p.I738LfsX744 and p.V284D. Moreover, the already described mutation p.N510S found in ABCB4 seems to be strictly involved in the onset of ICP in that particular patient. ConclusionsOur data support the hypothesis of a significant involvement of ABCB4 mutations in the onset of ICP, but also confirm an important role for ABCB11 mutations in increasing the susceptibility to cholestasis of pregnancy.

Targeting the ABCB4 gene to control cholesterol homeostasis

Introduction:Multidrug resistance 3 (MDR3) P-glycoprotein is a lipid floppase that is encoded by the ATP-binding cassette sub-family B member 4 (ABCB4) gene and plays a crucial role in proper bile formation by transporting phosphatidylcholine across the canalicular plasma membrane of the hepatocyte into bile. The relevance of this function is underscored by the severe pathology that develops in patients with ABCB4 deficiency. This deficiency leads to the destruction of hepatocytes and cholangiocytes by bile salts, because their cytolytic action is not reduced by formation of mixed micelles with phospholipid.Areas covered:Evidence that phospholipid secretion into bile is also essential for biliary cholesterol secretion as cholesterol dissolves much better in mixed micelles of bile salts and phospholipid than in pure bile salt micelles. As a consequence, net biliary cholesterol secretion depends on the amount of phospholipid secreted and hence, the expression of ABCB4 indirectly determines biliary cholesterol output.Expert opinion:It can be argued that upregulation of the ABCB4 gene expression may not only be beneficial for liver pathology in patients with partial ABCB4 deficiency, but also for the prevention of gallstone formation and optimal cholesterol disposition in a much larger population.