Intra-Hepatic Cholestasis of Pregnancy and Its Consequences: A Review

Background: The most common cause of liver illness in pregnancy is intrahepatic cholestasis (IHCP). It has a varying incidence due to geographic variance; factors such as advanced age, multiple pregnancy, family history, and previous pregnancy cholestasis have demonstrated a higher prevalence in these patients. Cholestasis in pregnancy has an aetiology that is currently unknown. It usually occurs after ovarian hyperstimulation syndrome in early pregnancy and coincides with growing oestrogen levels in the second half of pregnancy [1]. The ABCB4 gene mutation is largely associated in a subtype of progressive familial intrahepatic cholestasis, where disease clustering in first-degree relatives increases hereditary predisposition. Itchy palms and soles with elevated liver enzymes and bile acids are the most common symptoms. Some of the reported maternal problems in these patients include preterm labour, HELLP syndrome, acute fatty liver of pregnancy, and postpartum haemorrhage [2]. There are no precise antenatal foetal monitoring tests that can predict foetal fatalities in the womb. To reduce perinatal death with expectant treatment beyond this gestation, it is recommended that a pregnancy be terminated near 36–37 weeks of pregnancy.

A215 MDR3 DEFICIENCY MIMICKING WILSON DISEASE

Background The chronic phenotype of ALF includes a broad differential diagnosis. Class III multi-drug resistance P-glycoprotein 3 (MDR3) deficiency, also referred to as progressive familial intrahepatic cholestasis type 3, is an autosomal recessive genetic disorder. It is caused by a defect on the ABCB4 gene located on chromosome 7, which encodes MDR3. MDR3 is responsible for transporting phosphatidylcholine across the canalicular membrane, thereby allowing it to be incorporated into bile micelles. MDR3 deficiency results in increased levels of free bile acids and detergent bile. Progressive cholangiopathy ensues from this detergent bile and indirectly leads to cholestasis and liver failure in severe cases. Significantly increased urinary and hepatic copper (Cu), which are hallmarks of Wilson disease, have also been reported in patients with acute hepatitis and cholestasis including patients with MDR3 deficiency Aims We report a case of a girl who presented with a chronic phenotype of PALF, who had multiple features of Wilson disease and so was treated as such until genetic analysis confirmed MDR3 deficiency Methods Results A 6 year old girl presented to the ED with a 1mth history of epistaxis and a 1wk history of abdominal pain and distension, facial edema, pallor and fever. Her family history was significant for parental consanguinity and maternal itch during pregnancy. On examination she had clubbing, scleral icterus and a distended abdomen with hepatosplenomegaly. Her bloodwork showed bicytopenia (HGB 53 & Plts 63) along with liver dysfunction (INR 2.9, albumin 25, conjugated bilirubin 9) and raised liver enzymes (transaminases & GGT >10xULN). Her total serum bile acids were raised at 134. An US showed hepatosplenomegaly with multiple hyperechoic nodules and perisplenic varices. She was extensively worked up for malignancy, autoimmune and metabolic disease. Serum ceruloplasmin was reduced, ophthalmology examination showed no KF rings and her 24hr urinary Cu was 10xULN. Liver Cu quantification was markedly raised at 40xULN. Liver biopsy showed cirrhosis with fibrosis related minimal non-specific portal and septal inflammation. Additionally, complete loss of canalicular staining on immunohistochemistry for MDR3 protein was noted, suggestive of MDR3 deficiency. Based on the Cu levels, a provisional diagnosis of Wilson disease was made and Cu chelation therapy was commenced pending genetic testing. A cholestatic gene panel subsequently showed homozygous pathogenic variant for the ABCB4 gene. Trientine was stopped and she was commenced on ursodeoxycholic acid. Though biochemically she remains largely unchanged, she is clinically stable whilst awaiting a liver transplant Conclusions This case highlights the diagnostic difficulties associated with Cu test result interpretation in patients with chronic cholestatic liver disease and urges a thorough consideration of alternative diagnoses of PALF Funding Agencies None

Clinical profile and screening of exon 6 and 14 of ABCB4 gene in obstetric cholestasis patients at a tertiary care hospital in Rawalpindi, Pakistan

Objective: Intrahepatic Cholestasis of Pregnancy (ICP) is a rare pregnancy specific disorder. Genetic variants of ABCB4 gene increase ICP risk. This study was conducted to determine frequency of ICP cases presented at a tertiary care hospital in Rawalpindi, Pakistan and to screen for genetic variants of exon 6 and 14 of ABCB4 gene inICP cases. Methods: This analytical study included ICP patients presented at Department of Gynaecology and Obstetrics, Holy Family Hospital Rawalpindi, from February 2017 to May 2017. Sanger’s sequencing was performed using genomic DNA extracted from blood samples of patients and controls. Results: Twenty pregnant women out of 1150 (1.74%) had ICP and enrolled during study period. Overall 95% patients had pruritus and among them 40%, 20% and 10% had a history of miscarriages, stillbirths and familial ICP respectively. Genetic analysis revealed an already reported variant i.e., c.504C>T in exon 6 in thirteen patients and a novel variant i.e., c.1686A>G in exon 14 in five patients. Both variants were not present in controls. In silico analysis suggested that both variants might affect pre-mRNA splicing of ABCB4 transcript. Conclusion: ICP had a frequency of 1.74% among pregnant women. Identification of a novel heterozygous variant in five patients and an already reported variant in thirteen patients reaffirms genetic heterogeneity and role of ABCB4 in ICP etiology. Keywords: intrahepatic cholestasis of pregnancy, ABCB4 gene, Single Nucleotide Polymorphism (SNP), Continuous...

Mutagenesis Analysis of ABCB4 Gene Promoter of Danio rerio

Zebrafish abcb4 gene (ortholog to human ABCB1 gene) serves primarily in multidrug resistance (MDR) mechanism by effluxing chemotherapeutic agents, chemicals, xenobiotics, and numerous anti-cancer drugs out of the cells. This study aims to identify the specific transcription factor binding sites (TFBS) within the promoter region of zebrafish abcb4 gene and determine the functional roles of these factors in abcb4 gene expression regulation via mutagenesis analysis. First, primers were designed to target and amplify the promoter region of zebrafish abcb4 gene through gradient PCR. The zebrafish abcb4 gene promoter was then cloned into pGL3.0 vector and sent for sequencing. The sequencing results revealed high similarity to zebrafish DNA sequence from clone DKEY-24I24 in linkage group 16, indicating a successful cloning of targeted gene. Thereafter, consensus sequence of zebrafish abcb4 gene promoter was generated with the length of 1,392 bp which was close to its expected size during primer design (1,500 bp). Using MATCH tool, 155 TFBSs were found within zebrafish abcb4 gene promoter region. Activator protein 1 (AP-1) TFBS at 1,255 bp was chosen to be mutated through site-directed mutagenesis. Mutagenic primers (forward primer: 5’ GGG CAA GGC AGT ATA AAC GTG 3’ and reverse primer: 5’ TTA TGT TTC TAG GGA TTA CGT CAC 3’) were designed to substitute AGT with GGG to remove the AP-1 TFBS. By mutating the zebrafish abcb4 gene promoter, the MDR phenomenon driven by zebrafish abcb4 gene can be elucidated and this might provide clues to the development of tumor and malignancy in human. The results from this study may enrich the knowledge in chemotherapy and cancer treatments.