A homozygous R148W mutation in Semaphorin 7A causes progressive familial intrahepatic cholestasis

Article29 September 2021Open Access Source DataTransparent process A homozygous R148W mutation in Semaphorin 7A causes progressive familial intrahepatic cholestasis Qiong Pan orcid.org/0000-0003-1033-3098 Cholestatic Liver Diseases Center, Department of Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China These authors contributed equally to this work Search for more papers by this author Gang Luo Cholestatic Liver Diseases Center, Department of Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China These authors contributed equally to this work Search for more papers by this author Jiaquan Qu orcid.org/0000-0002-0204-5646 Cholestatic Liver Diseases Center, Department of Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China These authors contributed equally to this work Search for more papers by this author Sheng Chen Department of Pediatrics, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China These authors contributed equally to this work Search for more papers by this author Xiaoxun Zhang orcid.org/0000-0002-3892-7464 Cholestatic Liver Diseases Center, Department of Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China Search for more papers by this author Nan Zhao Cholestatic Liver Diseases Center, Department of Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China Search for more papers by this author Jingjing Ding Cholestatic Liver Diseases Center, Department of Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China Search for more papers by this author Hong Yang Cholestatic Liver Diseases Center, Department of Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China Search for more papers by this author Mingqiao Li Cholestatic Liver Diseases Center, Department of Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China Search for more papers by this author Ling Li Cholestatic Liver Diseases Center, Department of Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China Search for more papers by this author Ying Cheng Cholestatic Liver Diseases Center, Department of Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China Search for more papers by this author Xuan Li Cholestatic Liver Diseases Center, Department of Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China Search for more papers by this author Qiaoling Xie Cholestatic Liver Diseases Center, Department of Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China Search for more papers by this author Qiao Li Cholestatic Liver Diseases Center, Department of Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China Search for more papers by this author Xueqian Zhou Cholestatic Liver Diseases Center, Department of Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China Search for more papers by this author Huiling Zou Department of Pediatrics, Changsha Hospital for Maternal & Child Health Care, Changsha, China Search for more papers by this author Shijun Fan Medical Research Center, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China Search for more papers by this author Lingyun Zou Bao'an Maternal and Child Health Hospital, Jinan University, Shenzhen, China Search for more papers by this author Wei Liu Institute of Immunology, Third Military Medical University (Army Medical University), Chongqing, China Search for more papers by this author Guohong Deng Department of Infectious Diseases, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China Search for more papers by this author Shi-Ying Cai Department of Internal Medicine and Liver Center, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author James L Boyer orcid.org/0000-0002-8959-6036 Department of Internal Medicine and Liver Center, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Jin Chai Corresponding Author [email protected] orcid.org/0000-0002-8543-4566 Cholestatic Liver Diseases Center, Department of Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China Search for more papers by this author Qiong Pan orcid.org/0000-0003-1033-3098 Cholestatic Liver Diseases Center, Department of Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China These authors contributed equally to this work Search for more papers by this author Gang Luo Cholestatic Liver Diseases Center, Department of Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China These authors contributed equally to this work Search for more papers by this author Jiaquan Qu orcid.org/0000-0002-0204-5646 Cholestatic Liver Diseases Center, Department of Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China These authors contributed equally to this work Search for more papers by this author Sheng Chen Department of Pediatrics, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China These authors contributed equally to this work Search for more papers by this author Xiaoxun Zhang orcid.org/0000-0002-3892-7464 Cholestatic Liver Diseases Center, Department of Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China Search for more papers by this author Nan Zhao Cholestatic Liver Diseases Center, Department of Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China Search for more papers by this author Jingjing Ding Cholestatic Liver Diseases Center, Department of Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China Search for more papers by this author Hong Yang Cholestatic Liver Diseases Center, Department of Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China Search for more papers by this author Mingqiao Li Cholestatic Liver Diseases Center, Department of Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China Search for more papers by this author Ling Li Cholestatic Liver Diseases Center, Department of Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China Search for more papers by this author Ying Cheng Cholestatic Liver Diseases Center, Department of Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China Search for more papers by this author Xuan Li Cholestatic Liver Diseases Center, Department of Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China Search for more papers by this author Qiaoling Xie Cholestatic Liver Diseases Center, Department of Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China Search for more papers by this author Qiao Li Cholestatic Liver Diseases Center, Department of Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China Search for more papers by this author Xueqian Zhou Cholestatic Liver Diseases Center, Department of Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China Search for more papers by this author Huiling Zou Department of Pediatrics, Changsha Hospital for Maternal & Child Health Care, Changsha, China Search for more papers by this author Shijun Fan Medical Research Center, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China Search for more papers by this author Lingyun Zou Bao'an Maternal and Child Health Hospital, Jinan University, Shenzhen, China Search for more papers by this author Wei Liu Institute of Immunology, Third Military Medical University (Army Medical University), Chongqing, China Search for more papers by this author Guohong Deng Department of Infectious Diseases, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China Search for more papers by this author Shi-Ying Cai Department of Internal Medicine and Liver Center, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author James L Boyer orcid.org/0000-0002-8959-6036 Department of Internal Medicine and Liver Center, Yale University School of Medicine, New Haven, CT, USA Search for more papers by this author Jin Chai Corresponding Author [email protected] orcid.org/0000-0002-8543-4566 Cholestatic Liver Diseases Center, Department of Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China Search for more papers by this author Author Information Qiong Pan1, Gang Luo1, Jiaquan Qu1, Sheng Chen2, Xiaoxun Zhang1, Nan Zhao1, Jingjing Ding1, Hong Yang1, Mingqiao Li1, Ling Li1, Ying Cheng1, Xuan Li1, Qiaoling Xie1, Qiao Li1, Xueqian Zhou1, Huiling Zou3, Shijun Fan4, Lingyun Zou5, Wei Liu6, Guohong Deng7, Shi-Ying Cai8, James L Boyer8 and Jin Chai *,1 1Cholestatic Liver Diseases Center, Department of Gastroenterology, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China 2Department of Pediatrics, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China 3Department of Pediatrics, Changsha Hospital for Maternal & Child Health Care, Changsha, China 4Medical Research Center, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China 5Bao'an Maternal and Child Health Hospital, Jinan University, Shenzhen, China 6Institute of Immunology, Third Military Medical University (Army Medical University), Chongqing, China 7Department of Infectious Diseases, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China 8Department of Internal Medicine and Liver Center, Yale University School of Medicine, New Haven, CT, USA *Corresponding author. Tel: +86 23 68765346; Fax: +86 23 65410853; E-mail: [email protected] EMBO Mol Med (2021)13:e14563https://doi.org/10.15252/emmm.202114563 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Semaphorin 7A (SEMA7A) is a membrane-bound protein that involves axon growth and other biological processes. SEMA7A mutations are associated with vertebral fracture and Kallmann syndrome. Here, we report a case with a mutation in SEMA7A that displays familial cholestasis. WGS reveals a SEMA7AR148W homozygous mutation in a female child with elevated levels of serum ALT, AST, and total bile acid (TBA) of unknown etiology. This patient also carried a SLC10A1S267F allele, but Slc10a1S267F homozygous mice exhibited normal liver function. Similar to the child, Sema7aR145W homozygous mice displayed elevated levels of serum ALT, AST, and TBA. Remarkably, liver histology and LC-MS/MS analyses exhibited hepatocyte hydropic degeneration and increased liver bile acid (BA) levels in Sema7aR145W homozygous mice. Further mechanistic studies demonstrated that Sema7aR145W mutation reduced the expression of canalicular membrane BA transporters, bile salt export pump (Bsep), and multidrug resistance-associated protein-2 (Mrp2), causing intrahepatic cholestasis in mice. Administration with ursodeoxycholic acid and a dietary supplement glutathione improved liver function in the child. Therefore, Sema7aR145W homozygous mutation causes intrahepatic cholestasis by reducing hepatic Bsep and Mrp2 expression. SYNOPSIS A new type of progressive familial intrahepatic cholestasis (PFIC) was caused by the homozygous R148W mutation in SEMA7A. Preliminary mechanistic studies revealed that the mutation reduced hepatic expression of canalicular membrane bile acid (BA) efflux transporters Bsep and Mrp2, resulting in intrahepatic cholestasis. A female child patient presented with elevated levels of serum ALT, AST, and total bile acid (TBA) of unknown etiology. A SEMA7AR148W homozygous mutation was identified in the child patient. The Sema7aR145W (human R148W) homozygous mice displayed elevated levels of serum ALT, AST, and TBA, as well as hepatocyte hydropic degeneration and intrahepatic accumulation of conjugated BAs. The Sema7aR145W homozygous mutation reduced the expression of canalicular membrane Bsep and Mrp2 in mouse livers and sandwich-cultured primary hepatocytes. Administration with ursodeoxycholic acid (UDCA) and a dietary supplement glutathione (GSH) were effective for this new type of PFIC. The paper explained Problem Semaphorin 7A (SEMA7A) is crucial for axonal growth, T-cell responses, and other biological processes. Mutations in SEMA7A are associated with increased risks of vertebral fracture and Kallmann syndrome. However, whether SEMA7A mutations involve in liver diseases remains unknown. Results We identified a homozygous R148W mutation in SEMA7A in a female child, who developed cholestatic liver injury with elevated levels of serum ALT, AST, and total bile acid (TBA) without known etiology. Although this patient carried SLC10A1S267F allele, Slc10a1S267F homozygous mice exhibited normal liver function. In contrast, Sema7aR145W homozygous mice displayed elevated levels of serum ALT, AST, and TBA, recapitulating the patient's phenotypes. The homozygous mice also presented the accumulation of conjugated bile acid in the liver and reduced protein expression of canalicular membrane BA transporters, bile salt export pump (BSEP), and multidrug resistance-associated protein-2 (MRP2). Administration with ursodeoxycholic acid (UDCA) and a dietary supplement glutathione (GSH) improved liver function in the child. Impact Our findings uncover the clinical features of a novel form of progressive familial intrahepatic cholestasis (PFIC), caused by the SEMA7AR148W homozygous mutation. Administration with UDCA and GSH may be an effective therapy for this new type of PFIC. Introduction Bile acid (BA) transporters are crucial for maintaining BA homeostasis by driving bile flow (Boyer, 2013; Boyer & Soroka, 2021). The deficiency or reduction of canalicular membrane BA efflux transporters, including bile salt export pump (BSEP/ABCB11) and multidrug resistance-associated protein-2 (MRP2/ABCC2), can impair bile flow in the liver (Boyer, 2013; Boyer & Soroka, 2021). Impaired bile flow leads to cholestasis, resulting in liver injury characterized by elevated levels of bile acid (BA) and liver enzymes in the serum and liver (Wagner & Trauner, 2016; Pan et al, 2018; Amirneni et al, 2020). Cholestasis is attributed to multiple pathogenic factors, including hereditary and acquired impairment of bile formation (Wagner & Trauner, 2016). Several cholestatic disorders have been characterized as mutations in genes involved in bile formation, including ATP8B1 (a phospholipid flippase), BSEP, multidrug resistance protein 3 (MDR3/ABCB4), tight junction protein-2 (TJP2), farnesoid X receptor (FXR/NR1H4), and myosin VB (MYO5B), leading to progressive familial intrahepatic cholestasis (PFIC) 1–6, respectively (Amirneni et al, 2020). Here, we present a mutation in Semaphorin 7A (SEMA7A) for an additional cause of PFIC. Semaphorins are extracellular signaling proteins mediated through their membrane receptors plexins and integrins, contributing to tissue morphogenesis and homeostasis (Alto & Terman, 2017). Semaphorin 7A (SEMA7A), also known as the John Milton Hagen (JMH) antigen or CD108, is the only membrane-bound semaphoring linked to glycophosphatidylinositol (GPI) (Yamada et al, 1999). SEMA7A is expressed in multiple tissues, including the liver, intestine, lung, bone, and brain (Yamada et al, 1999; Liu et al, 2010; Song et al, 2021). It is crucial for axon growth, immune cell activation, pulmonary fibrosis, cancer metastasis, and other biological processes (Yamada et al, 1999; Suzuki et al, 2007; Liu et al, 2010; Song et al, 2021). Mutations in SEMA7A are associated with decreased bone mineral density (BMD) and Kallmann syndrome in humans (Koh et al, 2006; Zhao et al, 2020). However, the functional role of SEMA7A mutations in human liver diseases, including cholestasis, remains unclear. In this report, we identified a female infant patient with a homozygous mutation in SEMA7A (p.R148W) and elevated levels of serum alanine transaminase (ALT), aspartate transaminase (AST), and total bile acids (TBA). A genetic mouse model with the homozygous mutation recapitulated the clinical phenotypes. Administration with ursodeoxycholic acid (UDCA) and a dietary supplement glutathione (GSH) improved liver function in the child. Our findings describe the clinical features and possible therapy for a novel form of PFIC, caused by the SEMA7AR148W homozygous mutation in humans. Results Clinical features of the child patient and her family An infant patient (Patient IV.4, Fig 1A) presented with elevated levels of serum ALT, AST, and TBA, but normal levels of serum alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), direct bilirubin (DBIL), and total bilirubin (TBIL) (AppendixTable S1). Further laboratory and radiological examinations excluded any of the known liver diseases (Appendix Fig S1 and Appendix Tables S4–S8), leading to a speculation of mutations in genes involved in bile formation, such as those PFIC genes (Amirneni et al, 2020). However, the whole-genome sequencing (WGS) analysis of the studied family members did not show any mutation in known PFIC genes (i.e., ATP8B1, BSEP, MDR3, TJP2, FXR, and MYO5B), whereas a homozygous mutation (p.S267F) in sodium/taurocholate cotransporting polypeptide (NTCP/SLC10A1) was found in the patient. NTCP is a BA uptake transporter at the basolateral membrane of hepatocytes, and its loss-of-function mutation (p.R252H) causes hypercholanemia without liver injury (Vaz et al, 2015). Sanger sequencing analysis evidenced the homozygous p.S267F mutation in SLC10A1 (Appendix Fig S2), with an allele frequency of 8–12% in populations of Southern China, which is significantly associated with hypercholanemia in Han Chinese (Liu et al, 2017). To determine whether the SLC10A1S267F mutation was important for her liver injury, we generated Slc10a1S267F homozygous mice, a point mutation of c.800C>T (Ser267Phe) in murine Slc10a1 (Fig EV1). The Slc10a1S267F homozygous mice exhibited normal levels of serum ALT, AST, and TBA (Appendix Table S2) and liver histology. Therefore, the SLC10A1S267F homozygous mutation was unlike to cause liver injury and hypercholanemia in our patient, suggesting that her abnormal liver function may be attributed to other genetic mutations. Figure 1. Identification of a new case with a rare homozygous p.R148W mutation in SEMA7A A. Pedigrees of the studied family. A child patient (Patient: IV.4) with elevated serum ALT, AST, and TBA levels and her nuclear family members (III.4, III.5, and IV.3) were evaluated by WGS analysis. Mutated alleles are depicted as "a" for SEMA7A. Square symbols: male; circles: female; solid: the patient. The reference alleles are depicted by a plus sign. The arrow points to the proband. B. The homozygous p.R148W mutation in SEMA7A was further confirmed by Sanger sequencing. C. The protein domain architecture of SEMA7A and conservation of the R148 position in Vertebrata. D. Structures of WT SEMA7A and R148W mutant show local conformational changes, as a result of the replacement of an arginine (carbon atoms in yellow) with a tryptophan (pale) at position 148, which is highlighted in ball-and-stick models. E, F. The surface electrostatic potential map of the WT and R148W mutant proteins, respectively, in which positively and negatively charged resides are expressed in blue and red, respectively, and non-polar residues are denoted in white. Source data are available online for this figure. Source Data for Figure 1 [emmm202114563-sup-0003-SDataFig1.zip] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Generation and characterization of Slc10a1S267F mutant mice Schematic diagram for the generation of Slc10a1S267F mutant mice. Genotyping of Slc10a1S267F mutant mice. Genomic DNA was extracted from mouse tails, and the specific region covering the mutation was amplified by PCR. Identification of Slc10a1S267F homozygous mice by Sanger sequencing analysis. Source data are available online for this figure. Download figure Download PowerPoint SEMA7A is a candidate susceptibility gene To identify potential causative genetic factors, we screened the WGS data using a recessive inheritance model (Appendix Tables S9 and S10), a compound heterozygous inheritance model (Appendix Tables S11 and S12 (Schulten et al, 2016)), a de novo model (Appendix Table S13), and a dominant inheritance model (Appendix Table S14). The nonsynonymous, rare, and predicted to be damaging variants were further filtered to assess for further evidence of disease causation (Appendix Tables S9–S14). Notably, a predicted missense variant in SEMA7A gene (homozygous p.R148W) was identified and was prioritized for further analysis (Appendix Tables S9–S14). Sanger sequencing confirmed that the patient (Patient IV.4) had homozygous variant of c.C442T in SEMA7A, a missense mutation of p.R148W (Fig 1B and Appendix Fig S3). The R148 in SEMA7A is conserved in vertebrata (Fig 1C) and is located in a β-hairpin loop at an edge of the second β-sheet (Liu et al, 2010). This amino acid is partially exposed to solvent with slight restraints from two neighboring β-hairpin loops. Structure modeling indicated that the replacement of R148 with a Trp residue changed its local conformation due to the bulky indole side chain of tryptophan (Fig 1D) and altered the electrostatic property of local milieu by replacing a positively charged amino acid with an aromatic residue (Fig 1E and F). These findings suggest that the mutation may affect the function of the SEMA7A protein. Sema7aR145W homozygous mutation induces elevated serum ALT, AST, and TBA levels in mice Similar to our patient with the homozygous SEMA7AR148W mutation, Sema7aR145W homozygous mice (Fig EV2) displayed elevated levels of serum ALT, AST, and TBA in both 4 and 8 weeks of ages, compared to the age-matched WT and heterozygotes (Fig EV3A–C and Table 1 & Appendix Table S15). These homozygotes also had slightly elevated levels of serum TBIL and DBIL (Table 1). Moreover, liver histological assessments in Sema7aR145W homozygotes exhibited striking hydropic degeneration in hepatocytes, substantially higher than that of WT and heterozygotes (Fig EV3D). Together, the Sema7aR145W homozygous mutation in mice caused liver injury, characterized by elevated levels of serum ALT, AST, and TBA and striking hydropic degeneration in hepatocytes. Click here to expand this figure. Figure EV2. Generation and characterization of Sema7aR145W mutant mice Schematic diagram for the generation of Sema7aR145W mutant mice. Genotyping Sema7aR145W mutant mice. Genomic DNA was extracted from mouse tails, and the specific region was amplified by PCR. Identification of Sema7aR145W homozygous mice by Sanger sequencing analysis. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. A Sema7aR145W homozygous mutation causes the elevated serum ALT, AST, and TBA levels and remarkable hydropic degeneration in mouse livers A–C. Serum ALT, AST, and TBA in Sema7aR145W WT (n = 6, two male/four female), heterozygous (n = 10, six male/four female) and homozygous mice (n = 12, seven male/five female). Data are shown as means ± SD. D. Representative images of H&E staining, Sirius Red staining, and IHC analysis of CK19 expression in WT, Sema7aR145W heterozygous and homozygous mice. The Sema7aR145W homozygous mutation caused significant liver injury with elevated levels of serum ALT, AST, and TBA and striking hydropic degeneration in hepatocytes. Scale bar, 50 µm. Data information: The data were analyzed by the Mann–Whitney U-test. Source data are available online for this figure. Download figure Download PowerPoint Table 1. Serum biochemistry of Sema7aR145W mutant mice (8-week-old). Wild type (n = 6) Heterozygote (n = 10) Homozygote (n = 12) Gender (male/female) 2/4 6/4 7/5 Serum ALT (IU/l) 21.00 ± 2.69 27.14 ± 8.24 75.23 ± 99.65a,b, a,b a P < 0.05 versus the WT mice. b P < 0.05 versus the heterozygous mutant mice. The data were analyzed by the independent-samples Student's t-test or the Mann–Whitney U-test when applicable. Serum AST (IU/l) 83.26 ± 19.91 97.81 ± 34.87 244.40 ± 185.40a,b, a,b a P < 0.05 versus the WT mice. b P < 0.05 versus the heterozygous mutant mice. The data were analyzed by the independent-samples Student's t-test or the Mann–Whitney U-test when applicable. Serum ALP (IU/l) 98.80 ± 57.10 86.45 ± 36.04 93.91 ± 82.04 Serum TBA (μmol/l) 1.86 ± 0.51 2.28 ± 1.05 90.03 ± 121.39a,b, a,b a P < 0.05 versus the WT mice. b P < 0.05 versus the heterozygous mutant mice. The data were analyzed by the independent-samples Student's t-test or the Mann–Whitney U-test when applicable. Serum TBIL (μmol/l) 0.98 ± 1.15 3.65 ± 7.64 7.28 ± 5.18a,b, a,b a P < 0.05 versus the WT mice. b P < 0.05 versus the heterozygous mutant mice. The data were analyzed by the independent-samples Student's t-test or the Mann–Whitney U-test when applicable. Serum DBIL (μmol/l) 0.39 ± 0.61 2.91 ± 6.59 4.63 ± 4.38a a P < 0.05 versus the WT mice. Values are mean ± SD. ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; TBA, total bile acids; TBIL, total bilirubin; DBIL, direct bilirubin. a P < 0.05 versus the WT mice. b P < 0.05 versus the heterozygous mutant mice. The data were analyzed by the independent-samples Student's t-test or the Mann–Whitney U-test when applicable. Sema7aR145W homozygous mutation causes intrahepatic BA accumulation by reducing the expression of canalicular membrane BA efflux transporters Bsep and Mrp2 LC-MS/MS analysis of mouse liver extracts indicated that hepatic levels of major murine conjugated BAs, taurocholic acid (TCA), tauromuricholic acid (TMCA), taurochenodeoxycholic acid (TCDCA), and taurohyodeoxycholic acid (THDCA), were markedly higher in Sema7aR145W homozygous mice than WT mice (P < 0.05, Appendix Table S16), hallmarks of impaired hepatic excretion of BA. Furthermore, hepatic gene expression revealed that the mRNA transcripts and/or protein expression of BA synthetic enzymes Cyp7a1, Cyp8b1, and Cyp2c70 decreased while BA efflux transporters Mrp3, Mrp4, and Ostα/β each increased in homozygous mice, when compared to WT controls (Fig 2A–D). Most importantly, the levels of hepatic Bsep and Mrp2 protein expression in the Sema7aR145W homozygous mice were markedly reduced whereas their mRNA transcripts were not down-regulated (Fig 2A and D). Multiplex immunofluorescent (IF) analysis further revealed that reduced levels of Bsep and Mrp2 expression were observed in the livers and primary hepatocytes in sandwich cultures from Sema7aR145W homozygous mice (Fig 2E and F), suggesting that these two proteins decreased by the post-translational regulation in the livers of Sema7aR145W mutant mice. Interestingly, the levels of canalicular membrane Bsep and Mrp2 proteins were also markedly reduced in primary mouse hepatocytes in collagen sandwich cultures following transfection with SEMA7A_WT or SEMA7A_R148W construct in a dose-dependent manner (Fig 2G). Furthermore, when the same amount of DNA was transfected into these cells, we observed greater reduction in SEMA7A_R148W construct-transfected cells than in SEMA7A_WT construct-transfected cells (Fig 2G). Together, these findings indicated that the Sema7aR145W mutation reduced the protein expression of hepatic Bsep and Mrp2, resulting in intrahepatic cholestasis