- Email: [email protected]
771 Mitochondria-Targeting Antioxidant Attenuates Cholestasis-Induced Liver Injury in Mice
AASLD Abstracts
incompletely characterized. The heparin-degrading endosulfatase 2, SULF2, affects the activity of a number of heparin-binding growth factors by removing 6-O-sulfates from heparan sulfate proteoglycans (HSPGs). In our previous study, we showed that SULF2 can increase HCC growth and that high SULF2 expression predicts a worse prognosis after resection. HSPGs are known to mediate angiogenesis. Therefore, we investigated whether SULF2 can promote angiogenesis of HCC and explored its mechanism. Methods: The correlation between SULF2 mRNA and angiogenesis associated gene mRNAs was assessed in 139 resected HCCs by cDNA microarray analysis. Angiogenesis associated gene expression and microvascular density were assessed in diethyl nitrosamine (DEN)-induced HCC tumors in SULF2 transgenic mice as well as in xenografts established from SULF2-transfected Hep3B HCC cells. Cell viability, cell migration and tube formation ability of human hepatic sinusoidal endothelial cells (HHSEC) and human umbilical vein endothelial cells (HUVEC) were assessed after co-culture with SULF2-negative Hep3B cells transfected with SULF2 or SULF2-expressing Huh7 cells transfected with shRNA targeting SULF2. Angiogenic signaling pathways were assayed by Western blotting. Results: SULF2 mRNA expression was positively correlated with key angiogenesis associated genes which include CD31, VACAM1, VEGF B, and SDF1 in 139 resected HCCs. Microvascular density was increased in tumor tissues from both SULF2 up-regulated mouse models. Upregulation of SULF2 in Hep3B cells increased cell viability, cell migration and tube formation ability of HHSEC and HUVEC in a paracrine fashion; conversely knockdown of SULF2 in Huh7 cells reversed these morphologic features. Assessment of angiogenic pathways showed that the periostin pathway was activated in HHSEC cells after co-culture with SULF2 up-regulated Hep3B cells. The downstream factors p-AKT and p-FAK were also increased. The changes in p-AKT and p-FAK were further enhanced by activation of the TGF- β1 signaling pathway. The same association of TGF- β1, periostin, and p-FAK signaling changes was observed in both SULF2 up-regulated mouse HCC tumor models. Conclusions: Together, these findings suggest that SULF2 promotes angiogenesis of HCCs by increasing the angiogenic potency of HHSEC in a paracrine fashion. The TGFβ1/periostin signaling pathway plays an important role in regulating SULF2mediated angiogenesis of HCC. The SULF2/TGF β1/periostin pathway may be a rational target for anti-angiogenic therapy of HCC.
PLINK was used for statistical analysis. Results: Sequencing of ABCB4 identified 1 splice site, 9 Synonymous and 9 NS variants. Of note, one NS variant, p.A934T, described in LPAC patients, was present in 4 subjects in the ET group and none in the SNL group. All 4 affected individuals were of African American (AA) race (20 AA in the ET and 13 AA in the SNL group). Population analyses indicate that p.A934T is differentially represented in US racial subgroups, with an AA heterozygote frequency of 2.45%, compared with <0.01% among European Americans. Armitage trend test demonstrated p.A934T is significantly associated with ET outcome when examining all races (p=0.04). Other variants reported in LPAC or PFIC3 patients (p.T34M, p.T175A, p.R590Q, p.R652G, p.R788Q, p.E1058K) were similarly distributed among ET and SNL groups. Conclusions: This is the first WES study to discover genetic determinants of clinical outcomes in BA and identified ABCB4 as a biologically plausible candidate gene modifier. Heterozygosity for p.A934T is overrepresented among AA subjects with BA requiring early transplantation post-HPE. This finding underscores the utility of WES and subsequent validation studies of one candidate gene in a large, well-phenotyped cohort of racially diverse subjects with BA. With further application, this approach is poised to discover additional genetic contributors to BA outcomes. Support: NIH (TL1TR000456, U01DK062456, U01DK062470) & the Bauer, Spain & Alpard Foundations 846 Mutations in Tight Junction Protein 2 Underlie a Spectrum of Cholestatic Liver Disease Melissa Sambrotta, Sandra Strautnieks, Laura Brett, Suzanne Davison, Richard J. Thompson Background. Tight Junction Protein 2 is a cytoplasmic protein essential for the assembly of cell-cell junctional structures. It is encoded by TJP2. In 2003 a single, incompletely penetrant, missense mutation was identified in TJP2 in Amish patients with hypercholanaemia, in the absence of significant liver disease. We have recently shown that homozygous protein-truncating mutations in TJP2 are present in patients with severe early-onset cholestatic liver disease. Most of these patients required liver transplantation. Aim and Methods. In order to explore the wider phenotypic spectrum associated with mutations in TJP2, a cohort of 53 patients with severe early-onset of normal GGT cholestasis was selected. No mutations in the known progressive familial intrahepatic cholestasis genes ( ABCB11 and ATP8B1) had been identified. Genetic testing was undertaken using a combination of next-generation sequencing technology and Sanger DNA sequencing. Pathogenicity of missense mutations was determined with in silico analysis. Results. Genetic analysis revealed homozygous mutations in 8 patients. Three deletions and 2 nonsense mutation were identified, all predicted to lead to the truncation of the protein. In three families missense mutations were found. p.Gly737Arg was present in one patient who required liver transplantation at 13 months. However p.His788Leu was found in 2 unrelated families. Both patients had severe earlyonset cholestasis, which subsequently remitted. One of these patients relapsed at 5 years, but remitted again at age 7 years. Conclusion. We have previously described 8 families, including 12 individuals, with 8 different protein truncating mutations. We now report a further 8 families. Six manifest persistent cholestasis: five had protein-truncating mutations, and one had a homozygous missense mutation. The other 2 individuals had remitting cholestasis and were both homozygous for the same missense mutation. TJP2 was previously shown to underlie a condition with minimal liver disease. In addition to our recent identification of mutations in patients with progressive familial intrahepatic cholestasis, we now show that it can also lead to an intermediate phenotype. It is therefore clear that TJP2 deficiency represents a spectrum of liver disease phenotypes.
771 Mitochondria-Targeting Antioxidant Attenuates Cholestasis-Induced Liver Injury in Mice Yanjun Shi, Hasibur Rehman, Yasodha Krishnasamy, Venkat K. Ramshesh, Rick G. Schnellmann, John J. Lemasters, Michael P. Murphy, Zhi Zhong Background: Our previous study showed that the mitochondrial permeability transition (MPT) occurs in the liver after cholestasis, leading to cell death. Aim: Because reactive oxygen species (ROS) are a known stimulator of the MPT, we investigated whether MitoQ, an orally active antioxidant that accumulates in mitochondria, attenuates cholestatic liver injury caused by bile duct ligation (BDL) in mice. Methods: MitoQ (500 μM) was added to drinking water. Three days after starting MitoQ feeding, mice were subjected to BDL, and livers were harvested 1 and 3 days later. Mitochondrial depolarization and cell death were detected in living mice by intravital confocal/multiphon microscopy of rhodamine 123 (Rh123) and propidium iodide (PI), respectively, and onset of the MPT was assessed by entry of calcein into mitochondria. Results: 4-Hydroxynonenal adducts (4-HNE) increased markedly after BDL, indicating ROS formation, and MitoQ blunted increases in 4-HNE. Rh123 fluorescence was punctate in virtually all hepatocytes in control mice, indicating mitochondrial polarization. At 6 h after BDL, Rh123 fluorescence disappeared in many hepatocytes (~18/hpf) whereas PI-positive non-viable hepatocytes were <1/hpf, indicating mitochondrial depolarization prior to cell death. Calcein resided in the cytosol in hepatocytes of sham-operated mice but entered mitochondria 6 h after BDL, confirming the onset of the MPT in vivo. MitoQ decreased the number of depolarized hepatocytes after BDL by 72%. Serum ALT increased from 46 U/L before surgery to 3300 and 1700 U/L at 1 and 3 days, respectively, after BDL. ALT was blunted by 85% 1 day after BDL in mice given MitoQ. Focal necrosis occurred in 17% of liver tissue 1 day after BDL, and MitoQ attenuated necrosis to 5%. TUNEL-positive cells were barely detectable in livers after sham operation but increased to 4% and 2% at 1 and 3 days, respectively, after BDL and MitoQ decreased TUNEL-positive cells to 0.5%. Cleaved caspase-3 was barely detectable in livers from shamoperated mice, increased markedly after BDL, and was decreased by MitoQ. Conclusion. Taken together, we suggest that MitoQ decreases liver injury after BDL by blocking ROSinduced MPT onset. This mitochondria-targeting antioxidant may be a promising therapy to prevent early stage cholestatic liver injury (NIDDK).
847 The Types of Inflammatory Bowel Disease (IBD) Predispose to Distinct Clinical Phenotypes of Primary Sclerosing Cholangitis (PSC) in Children Keaton R. Jones, Philip Bufler, Alexander G. Miethke Background: PSC is a chronic progressive cholangiopathy which is commonly associated with IBD. Since the gut-liver axis is considered to play an important role in the pathogenesis of PSC we hypothesized that the type of IBD determined the disease course of pediatric onset PSC. Methods: In this retrospective study, medical records of 52 patients who received medical care for PSC at a single institution between 2009 and 2013 were reviewed. Results: The clinical diagnosis of PSC was confirmed in all 52 subjects based on review of reports of hepatobiliary imaging and liver biopsy. A diagnosis of IBD was established by endoscopy in 43 (83%), comprising of Crohn disease (CD) in 16 (31%) and ulcerative colitis (UC) in 27 (52%) patients. Ethnicity was primarily Caucasian in both groups (81 vs 77% in CD vs UC), but males predominated in the CD group (62 vs 46% males in CD vs UC). Mean age of diagnosis of PSC was 10.8 years in CD-PSC and 12.0 in UC-PSC patients. CD and PSC were often diagnosed simultaneously whereas the diagnosis of UC preceded detection of abnormal liver enzymes by a mean of 7 months. Maintenance therapy of colitis differed between both groups in regards to use of immunomodulator and corticosteroid therapy. Infliximab was used in 43% of CD- and 27 % of UC-PSC subjects. 9 patients (7 with PSCUC) with end-stage liver disease were referred for liver transplantation and were excluded from subsequent analysis. Of 34 patients with PSC, IBD and native liver, 6 (18%) were found to have autoimmune sclerosing cholangitis based on positive ANA serology and liver histomorphology with interface hepatitis (5 in UC- group). For 18 subjects with laboratory values available at the time of diagnosis of PSC, serum GGT levels and white blood cell counts (WBC) were significantly higher in CD- than in UC patients (see table). During follow up, peak serum total bilirubin levels and those obtained at the most recent visit were higher in CD- PSC patients. Importantly, elevated levels of biomarkers of cholestasis were associated with a higher prevalence of extrahepatic biliary disease in CD-PSC patients. Narrowing or dilatation with caliber change affecting the common hepatic or common bile ducts were detected by ERCP or MRCP in 9/12 PSC-CD (75%) compared with 7/ 21 PSCUC patients (33%; p=0.03). Conclusion: Compared with UC, concomitant CD predisposes in pediatric onset PSC to extrahepatic bile duct injury and greater degree of cholestasis, as gauged by serum biochemistries. How disease pathogenesis and treatment of colitis are linked to short and long-term outcomes in CD- and UC-PSC patients, especially progression of bile duct epithelial injury and biliary fibrosis, requires further investigation.
845 Whole Exome Sequencing Identifies ABCB4 Gene Variants As Modifiers of Biliary Atresia Outcomes Anya Mezina, Khanjan Gandhi, Aniko Sabo, Donna Muzny, Richard Gibbs, Madhuri Hegde, Saul J. Karpen Background: The role of genetic factors in determining clinical outcomes in biliary atresia (BA) after Kasai hepatoportoenterostomy (HPE) are unknown. To begin to address this, we performed a pilot study of whole exome sequencing (WES) in 20 BA patients from the NIDDK-supported ChiLDREN dataset to identify variants in genes that stratify between two disparate outcome groups: 10 who underwent early transplant prior to age 2 (ET) compared to 10 who survived beyond age 4 with native liver (SNL) and normal platelet count (>150,000). WES identified ~ 50 candidate genes with disproportionately more non-synonymous (NS) variants in the ET compared to SNL group. Among this list was the ABCB4 phospholipid floppase gene, which, if verified, would provide a pathophysiological link to worsening cholangiopathy in BA since ABCB4 gene mutations are implicated in liver diseases such as Low Phospholipid Associated Cholelithiasis (LPAC) and Progressive Familial Intrahepatic Cholestasis 3 (PFIC3). We hypothesized that deleterious variants in ABCB4 would modulate phenotypic severity and sought to validate this in a larger cohort of ET and SNL ChiLDREN subjects. Methods: Sanger DNA sequencing was performed for all 27 coding exons and the promoter region of the ABCB4 gene from 195 ChiLDREN subjects stratified as SNL (n=97) or ET (n=98). Variants were called by comparison to reference NM_018849.2.
AASLD Abstracts
S-928
incompletely characterized. The heparin-degrading endosulfatase 2, SULF2, affects the activity of a number of heparin-binding growth factors by removing 6-O-sulfates from heparan sulfate proteoglycans (HSPGs). In our previous study, we showed that SULF2 can increase HCC growth and that high SULF2 expression predicts a worse prognosis after resection. HSPGs are known to mediate angiogenesis. Therefore, we investigated whether SULF2 can promote angiogenesis of HCC and explored its mechanism. Methods: The correlation between SULF2 mRNA and angiogenesis associated gene mRNAs was assessed in 139 resected HCCs by cDNA microarray analysis. Angiogenesis associated gene expression and microvascular density were assessed in diethyl nitrosamine (DEN)-induced HCC tumors in SULF2 transgenic mice as well as in xenografts established from SULF2-transfected Hep3B HCC cells. Cell viability, cell migration and tube formation ability of human hepatic sinusoidal endothelial cells (HHSEC) and human umbilical vein endothelial cells (HUVEC) were assessed after co-culture with SULF2-negative Hep3B cells transfected with SULF2 or SULF2-expressing Huh7 cells transfected with shRNA targeting SULF2. Angiogenic signaling pathways were assayed by Western blotting. Results: SULF2 mRNA expression was positively correlated with key angiogenesis associated genes which include CD31, VACAM1, VEGF B, and SDF1 in 139 resected HCCs. Microvascular density was increased in tumor tissues from both SULF2 up-regulated mouse models. Upregulation of SULF2 in Hep3B cells increased cell viability, cell migration and tube formation ability of HHSEC and HUVEC in a paracrine fashion; conversely knockdown of SULF2 in Huh7 cells reversed these morphologic features. Assessment of angiogenic pathways showed that the periostin pathway was activated in HHSEC cells after co-culture with SULF2 up-regulated Hep3B cells. The downstream factors p-AKT and p-FAK were also increased. The changes in p-AKT and p-FAK were further enhanced by activation of the TGF- β1 signaling pathway. The same association of TGF- β1, periostin, and p-FAK signaling changes was observed in both SULF2 up-regulated mouse HCC tumor models. Conclusions: Together, these findings suggest that SULF2 promotes angiogenesis of HCCs by increasing the angiogenic potency of HHSEC in a paracrine fashion. The TGFβ1/periostin signaling pathway plays an important role in regulating SULF2mediated angiogenesis of HCC. The SULF2/TGF β1/periostin pathway may be a rational target for anti-angiogenic therapy of HCC.
PLINK was used for statistical analysis. Results: Sequencing of ABCB4 identified 1 splice site, 9 Synonymous and 9 NS variants. Of note, one NS variant, p.A934T, described in LPAC patients, was present in 4 subjects in the ET group and none in the SNL group. All 4 affected individuals were of African American (AA) race (20 AA in the ET and 13 AA in the SNL group). Population analyses indicate that p.A934T is differentially represented in US racial subgroups, with an AA heterozygote frequency of 2.45%, compared with <0.01% among European Americans. Armitage trend test demonstrated p.A934T is significantly associated with ET outcome when examining all races (p=0.04). Other variants reported in LPAC or PFIC3 patients (p.T34M, p.T175A, p.R590Q, p.R652G, p.R788Q, p.E1058K) were similarly distributed among ET and SNL groups. Conclusions: This is the first WES study to discover genetic determinants of clinical outcomes in BA and identified ABCB4 as a biologically plausible candidate gene modifier. Heterozygosity for p.A934T is overrepresented among AA subjects with BA requiring early transplantation post-HPE. This finding underscores the utility of WES and subsequent validation studies of one candidate gene in a large, well-phenotyped cohort of racially diverse subjects with BA. With further application, this approach is poised to discover additional genetic contributors to BA outcomes. Support: NIH (TL1TR000456, U01DK062456, U01DK062470) & the Bauer, Spain & Alpard Foundations 846 Mutations in Tight Junction Protein 2 Underlie a Spectrum of Cholestatic Liver Disease Melissa Sambrotta, Sandra Strautnieks, Laura Brett, Suzanne Davison, Richard J. Thompson Background. Tight Junction Protein 2 is a cytoplasmic protein essential for the assembly of cell-cell junctional structures. It is encoded by TJP2. In 2003 a single, incompletely penetrant, missense mutation was identified in TJP2 in Amish patients with hypercholanaemia, in the absence of significant liver disease. We have recently shown that homozygous protein-truncating mutations in TJP2 are present in patients with severe early-onset cholestatic liver disease. Most of these patients required liver transplantation. Aim and Methods. In order to explore the wider phenotypic spectrum associated with mutations in TJP2, a cohort of 53 patients with severe early-onset of normal GGT cholestasis was selected. No mutations in the known progressive familial intrahepatic cholestasis genes ( ABCB11 and ATP8B1) had been identified. Genetic testing was undertaken using a combination of next-generation sequencing technology and Sanger DNA sequencing. Pathogenicity of missense mutations was determined with in silico analysis. Results. Genetic analysis revealed homozygous mutations in 8 patients. Three deletions and 2 nonsense mutation were identified, all predicted to lead to the truncation of the protein. In three families missense mutations were found. p.Gly737Arg was present in one patient who required liver transplantation at 13 months. However p.His788Leu was found in 2 unrelated families. Both patients had severe earlyonset cholestasis, which subsequently remitted. One of these patients relapsed at 5 years, but remitted again at age 7 years. Conclusion. We have previously described 8 families, including 12 individuals, with 8 different protein truncating mutations. We now report a further 8 families. Six manifest persistent cholestasis: five had protein-truncating mutations, and one had a homozygous missense mutation. The other 2 individuals had remitting cholestasis and were both homozygous for the same missense mutation. TJP2 was previously shown to underlie a condition with minimal liver disease. In addition to our recent identification of mutations in patients with progressive familial intrahepatic cholestasis, we now show that it can also lead to an intermediate phenotype. It is therefore clear that TJP2 deficiency represents a spectrum of liver disease phenotypes.
771 Mitochondria-Targeting Antioxidant Attenuates Cholestasis-Induced Liver Injury in Mice Yanjun Shi, Hasibur Rehman, Yasodha Krishnasamy, Venkat K. Ramshesh, Rick G. Schnellmann, John J. Lemasters, Michael P. Murphy, Zhi Zhong Background: Our previous study showed that the mitochondrial permeability transition (MPT) occurs in the liver after cholestasis, leading to cell death. Aim: Because reactive oxygen species (ROS) are a known stimulator of the MPT, we investigated whether MitoQ, an orally active antioxidant that accumulates in mitochondria, attenuates cholestatic liver injury caused by bile duct ligation (BDL) in mice. Methods: MitoQ (500 μM) was added to drinking water. Three days after starting MitoQ feeding, mice were subjected to BDL, and livers were harvested 1 and 3 days later. Mitochondrial depolarization and cell death were detected in living mice by intravital confocal/multiphon microscopy of rhodamine 123 (Rh123) and propidium iodide (PI), respectively, and onset of the MPT was assessed by entry of calcein into mitochondria. Results: 4-Hydroxynonenal adducts (4-HNE) increased markedly after BDL, indicating ROS formation, and MitoQ blunted increases in 4-HNE. Rh123 fluorescence was punctate in virtually all hepatocytes in control mice, indicating mitochondrial polarization. At 6 h after BDL, Rh123 fluorescence disappeared in many hepatocytes (~18/hpf) whereas PI-positive non-viable hepatocytes were <1/hpf, indicating mitochondrial depolarization prior to cell death. Calcein resided in the cytosol in hepatocytes of sham-operated mice but entered mitochondria 6 h after BDL, confirming the onset of the MPT in vivo. MitoQ decreased the number of depolarized hepatocytes after BDL by 72%. Serum ALT increased from 46 U/L before surgery to 3300 and 1700 U/L at 1 and 3 days, respectively, after BDL. ALT was blunted by 85% 1 day after BDL in mice given MitoQ. Focal necrosis occurred in 17% of liver tissue 1 day after BDL, and MitoQ attenuated necrosis to 5%. TUNEL-positive cells were barely detectable in livers after sham operation but increased to 4% and 2% at 1 and 3 days, respectively, after BDL and MitoQ decreased TUNEL-positive cells to 0.5%. Cleaved caspase-3 was barely detectable in livers from shamoperated mice, increased markedly after BDL, and was decreased by MitoQ. Conclusion. Taken together, we suggest that MitoQ decreases liver injury after BDL by blocking ROSinduced MPT onset. This mitochondria-targeting antioxidant may be a promising therapy to prevent early stage cholestatic liver injury (NIDDK).
847 The Types of Inflammatory Bowel Disease (IBD) Predispose to Distinct Clinical Phenotypes of Primary Sclerosing Cholangitis (PSC) in Children Keaton R. Jones, Philip Bufler, Alexander G. Miethke Background: PSC is a chronic progressive cholangiopathy which is commonly associated with IBD. Since the gut-liver axis is considered to play an important role in the pathogenesis of PSC we hypothesized that the type of IBD determined the disease course of pediatric onset PSC. Methods: In this retrospective study, medical records of 52 patients who received medical care for PSC at a single institution between 2009 and 2013 were reviewed. Results: The clinical diagnosis of PSC was confirmed in all 52 subjects based on review of reports of hepatobiliary imaging and liver biopsy. A diagnosis of IBD was established by endoscopy in 43 (83%), comprising of Crohn disease (CD) in 16 (31%) and ulcerative colitis (UC) in 27 (52%) patients. Ethnicity was primarily Caucasian in both groups (81 vs 77% in CD vs UC), but males predominated in the CD group (62 vs 46% males in CD vs UC). Mean age of diagnosis of PSC was 10.8 years in CD-PSC and 12.0 in UC-PSC patients. CD and PSC were often diagnosed simultaneously whereas the diagnosis of UC preceded detection of abnormal liver enzymes by a mean of 7 months. Maintenance therapy of colitis differed between both groups in regards to use of immunomodulator and corticosteroid therapy. Infliximab was used in 43% of CD- and 27 % of UC-PSC subjects. 9 patients (7 with PSCUC) with end-stage liver disease were referred for liver transplantation and were excluded from subsequent analysis. Of 34 patients with PSC, IBD and native liver, 6 (18%) were found to have autoimmune sclerosing cholangitis based on positive ANA serology and liver histomorphology with interface hepatitis (5 in UC- group). For 18 subjects with laboratory values available at the time of diagnosis of PSC, serum GGT levels and white blood cell counts (WBC) were significantly higher in CD- than in UC patients (see table). During follow up, peak serum total bilirubin levels and those obtained at the most recent visit were higher in CD- PSC patients. Importantly, elevated levels of biomarkers of cholestasis were associated with a higher prevalence of extrahepatic biliary disease in CD-PSC patients. Narrowing or dilatation with caliber change affecting the common hepatic or common bile ducts were detected by ERCP or MRCP in 9/12 PSC-CD (75%) compared with 7/ 21 PSCUC patients (33%; p=0.03). Conclusion: Compared with UC, concomitant CD predisposes in pediatric onset PSC to extrahepatic bile duct injury and greater degree of cholestasis, as gauged by serum biochemistries. How disease pathogenesis and treatment of colitis are linked to short and long-term outcomes in CD- and UC-PSC patients, especially progression of bile duct epithelial injury and biliary fibrosis, requires further investigation.
845 Whole Exome Sequencing Identifies ABCB4 Gene Variants As Modifiers of Biliary Atresia Outcomes Anya Mezina, Khanjan Gandhi, Aniko Sabo, Donna Muzny, Richard Gibbs, Madhuri Hegde, Saul J. Karpen Background: The role of genetic factors in determining clinical outcomes in biliary atresia (BA) after Kasai hepatoportoenterostomy (HPE) are unknown. To begin to address this, we performed a pilot study of whole exome sequencing (WES) in 20 BA patients from the NIDDK-supported ChiLDREN dataset to identify variants in genes that stratify between two disparate outcome groups: 10 who underwent early transplant prior to age 2 (ET) compared to 10 who survived beyond age 4 with native liver (SNL) and normal platelet count (>150,000). WES identified ~ 50 candidate genes with disproportionately more non-synonymous (NS) variants in the ET compared to SNL group. Among this list was the ABCB4 phospholipid floppase gene, which, if verified, would provide a pathophysiological link to worsening cholangiopathy in BA since ABCB4 gene mutations are implicated in liver diseases such as Low Phospholipid Associated Cholelithiasis (LPAC) and Progressive Familial Intrahepatic Cholestasis 3 (PFIC3). We hypothesized that deleterious variants in ABCB4 would modulate phenotypic severity and sought to validate this in a larger cohort of ET and SNL ChiLDREN subjects. Methods: Sanger DNA sequencing was performed for all 27 coding exons and the promoter region of the ABCB4 gene from 195 ChiLDREN subjects stratified as SNL (n=97) or ET (n=98). Variants were called by comparison to reference NM_018849.2.
AASLD Abstracts
S-928