Role of ABCC2 common variants in intrahepatic cholestasis of pregnancy

The pathogenesis of intrahepatic cholestasis of pregnancy （ICP）, a disorder that adversely affects maternal wellbeing and fetal outcome, is unclear. However, multiple factors probably interact along with a genetic predisposition. We would like to add some comments on a paper recently published concerning the role of ABCB11 and ABCC2 polymorphisms in both ICP and contraceptive-induced cholestasis, especially in the light of our recently published findings about a positive association between ICP and ABCC2 common variants.     

ABCB4 mutations underlie hormonal cholestasis but not pediatric idiopathic gallstones

AIM:To investigate the contribution of ABCB4 mutations to pediatric idiopathic gallstone disease and the potential of hormonal contraceptives to prompt clinical manifestations of multidrug resistance protein 3 deficiency.METHODS:Mutational analysis of ABCB4,screening for copy number variations by multiplex ligation-dependent probe amplification,genotyping for low expression allele c.1331T>C of ABCB11 and genotyping for variation c.55G>C in ABCG8 previously associated with cholesterol gallstones in adults was performed in 35 pediatric subjects with idiopathic gallstones who fulfilled the clinical criteria for low phospholipid-associated cholelithiasis syndrome(LPAC,OMIM#600803)and in 5young females with suspected LPAC and their families(5 probands,15 additional family members).The probands came to medical attention for contraceptiveassociated intrahepatic cholestasis.RESULTS:A possibly pathogenic variant of ABCB4was found only in one of the 35 pediatric subjects with idiopathic cholesterol gallstones whereas 15 members of the studied 5 LPAC kindreds were confirmed and another one was highly suspected to carry predictably pathogenic mutations in ABCB4.Among these 16,however,none developed gallstones in childhood.In 5index patients,all young females carrying at least one pathogenic mutation in one allele of ABCB4,manifestation of LPAC as intrahepatic cholestasis with elevated serum activity of gamma-glutamyltransferase was induced by hormonal contraceptives.Variants ABCB11c.1331T>C and ABCG8 c.55G>C were not significantly overrepresented in the 35 examined patients with suspect LPAC.CONCLUSION:Clinical criteria for LPAC syndrome caused by mutations in ABCB4 cannot be applied topediatric patients with idiopathic gallstones.Sexual immaturity even prevents manifestation of LPAC.     

Promoter polymorphism of transforming growth factor-β1 gene and ulcerative colitis

AIM： To elucidate the possible difference in two promoter polymorphisms of the transforming growth factor-β1 （TGF-β1） gene （-800G 〉 A, -509C 〉 T） between ulcerative colitis （UC） patients and normal subjects.
METHODS： A total of 155 patients with established ulcerative colitis and 139 normal subjects were selected as controls. Two single nucleotide polymorphisms within the promoter region of TGF-β1 gene （-509C 〉 T and -800G 〉 A） were genotyped using PCR-RFLP. RESULTS： There was a statistically significant difference in genotype and allele frequency distributions between UC patients and controls for the -800G 〉 A polymorphism of the TGF-β1 gene （P 〈 0.05）. The frequency of the TGF-β1 gene polymorphism at position -800 showed that the AA genotype and the allele A frequencies significantly differed between the patients and healthy controls （P 〈 0.05）. At position -509, there was no statically significant difference in genotype and allele frequency between the patients and control subjects.
CONCLUSION： The results of our study indicate that there is a significant difference in both allele and genotype frequency at position -800G 〉 A of TGF-β1 gene promoter between Iranian patients with UC and normal subjects.     

Endotoxin receptor CD14 gene variants and histological features in chronic HCV infection

AIM： To analyze the correlation between CD14 rs2569190/C-159T single nucleotide polymorphism （SNP） and disease progression in chronic hepatitis C. METHODS： Liver biopsy specimens from a total of 137 and 349 patients with chronic hepatitis C were separately evaluated with respect to necroinflammatory activity （grading） and architectural changes （staging）. In one group, further histological lesions characteristic for hepatitis C, hepatitis C virus subtypes, and biochemical parameters of liver disease were also investigated. Samples of genomic DNA were genotyped for the respective SNP by 5＇-nuclease assays using fluorescent dye-labeled allele-specific probes. RESULTS： Genotype distribution did not deviate from the Hardy-Weinberg equilibrium. In the first group, patients homozygous for the variant allele T were found to be younger than C allele carriers （39.6 ±12.5 vs 45.7 ± 11.5, P = 0.008）. Among the histological lesions studied, portal lymphoid aggregates were more frequently observed among TT homozygotes than among C carriers （21/37 vs 32/100, P = 0.008）. The presence of portal lymphoid aggregates was closely correlated with hepatic inflammation （P = 0.003） and with bile duct damage （P 〈 0.001）. The degree of fibrosis, in contrast, was not found to be related to the CD14 gene C-159T polymorphism.CONCLUSION： The data suggest a possible relationship between CD14 C-159T polymorphism and the formation of portal lymphoid aggregates, but not liver fibrosis progression in chronic hepatitis C.     

Predictors of premature delivery in patients with intrahepatic cholestasis of pregnancy

AIM： To evaluate the predictive value of clinical symptoms and biochemical parameters for prematurity in intrahepatic cholestasis of pregnancy （ICP）. METHODS： Sixty symptomatic patients with ICP were included in this retrospective analysis. Preterm delivery was defined as delivery before 37 wk gestation. Predictors of preterm delivery were disclosed by binary multivariate logistic regression analysis. RESULTS： Mean time of delivery was 38.1 ± 1.7 wk. No stillbirths occurred. Premature delivery was observed in eight （13.3%） patients. Total fasting serum bile acids were higher （47.8 ±15.2 vs 41.0 ± 10.0 μmol/L, P 〈 0.05）, and pruritus tended to start earlier （29.0 ± 3.9 vs 31.6 ± 3.3 wk, P = 0.057） in patients with premature delivery when compared to those with term delivery. Binary multivariate logistic regression analysis revealed that early onset of pruritus （OR 1.70, 95% CI 1.23-2.95, P = 0.038） and serum bile acid （OR 2.13, 95% CI 1.13-3.25, P = 0.013） were independent predictors of preterm delivery. CONCLUSION： Early onset of pruritus and high levels of serum bile acids predict preterm delivery in ICP, and define a subgroup of patients at risk for poor neonatal outcome.     

Clinical and ABCB11 profiles in Korean infants with progressive familial intrahepatic cholestasis

AIM: To investigate clinical profiles and mutations of ABCB11 in Koreans with progressive familial intrahepatic cholestasis 2 and review the differences between Koreans and others.METHODS: Of 47 patients with neonatal cholestasis, five infants had chronic intrahepatic cholestasis with normal γ-glutamyl transpeptidase. Direct sequencing analyses of ABCB11, including exons and introns, were performed from peripheral blood.RESULTS: Living donor-liver transplantation was performed in four patients because of rapidly progressive hepatic failure and hepatocellular carcinoma. Three missense mutations were found in two patients: compound heterozygous 677CT(S226L)/3007GA(G1003R) and heterozygous 2296GA(G766R). The mutations were located near and in the transmembranous space. CONCLUSION: Alterations in the transmembrane of the bile salt export pump in the Korean infants were different from those previously reported in Chinese, Japanease, Taiwanese, and European patients.     

Genetic variations of beta 2-adrenergic receptor gene are associated with essential hypertension in Xinjiang Kazakans

Objective The aims of the present study were to investigate the associations of 46 A〉G, 79 C〉G, 491 C〉T and 659 C〉G genetic variants of the human beta 2-adrenergic receptor （β2-AR）, ADRB2, gene with essential hypertension （EH） in Xinjiang Kazakans population.Methods A gender-matched case-control （271 hypertensive cases and 267 normotensive controls） study was used to investigate the associations of the four variations in the coding region of ADRB2 with EH. The genotypes of the variants were identified by the polymerase chain reaction and restriction fragment length polymorphism （PCR-RFLP） methods. Results 46 A〉G, 79 C〉G and 659 C〉G polymorphisms were common in the Kazakan population, but 491 C〉T was a mutation （frequency ofT allele was only 0.003） and only found in EH group. The fxequency distributions of genotypes and alleles for 659 C〉G between the EH and control groups was significantly different （P〈0.05）, while those for 46 A〉G and 79 C〉G polymorphisms were not statistically different. Logistic regression analysis suggested that the G allele of 659 C〉G polymorphism was a risk factor for hypertension （minor allele vs common homo; odds ratio, 13.240, 95% CI, 4.052-43.274; P〈0.05）. Covariance analysis showed that systolic and diastolic blood pressure levels in GG＋CG group of 659 C〉G were significantly higher than those in the CC group, but no significant difference of blood pressure were found between common homo and minor allele for 46 A〉G and 79C〉G polymorphisms. Haplotype analysis showed that two hyplotypes, HI： 46A-79C-491C-523C（48%）and H5：46A-79C-491C-659G, were associated with EH.Conelusion ADRB2 genetic variants may play independent roles in the molecular genetic mechanism of EH in Xinjiang Kazakans population （d Geriatr Cardio12010; 7：52-57）.     

Prolonged cholestasis following successful removal of common bile duct stones： Beware patients on estrogen therapy

There are various well described forms of chronic cholestatic jaundice in adults, such as autoimmune cholangitis, drug-induced cholangitis and intrahepatic cholestasis of pregnancy. We present two cases of prolonged cholestasis following removal of gallstones at endoscopic retrograde cholangiopancreatography （ERCP） and subsequent clear cholangiography. Both patients were taking oral estrogens at the time of presentation, which were subsequently withdrawn. The first case responded rapidly to corticosteroid treatment, and the second case had a much slower resolution with ursodeoxycholic acid. Both cases highlighted the significance of estrogen-induced cholestasis in female patients with protracted jaundice following ERCP and removal of intra-ductal stones. After oral estrogens are discontinued, a short course of steroids needs to be considered.     

Liver stiffness in the hepatitis B virus carrier： A non-invasive marker of liver disease influenced by the pattern of transaminases

AIM： To investigate the usefulness of transient elastography by Fibroscan （FS）, a rapid non-invasive technique to evaluate liver fibrosis, in the management of chronic hepatitis B virus （HBV） carriers. METHODS： In 297 consecutive HBV carriers, we studied the correlation between liver stiffness （LS）, stage of liver disease and other factors potentially influencing FS measurements. In 87 chronic hepatitis B （CriB） patients, we monitored the FS variations according to the spontaneous or treatment-induced variations of biochemical activity during follow-up. RESULTS： FS values were 12.3 ± 3.3 kPa in acute hepatitis, 10.3 ± 8.8 kPa in chronic hepatitis, 4.3 ± 1.0 kPa in inactive carriers and 4.6 ± 1.2 kPa in blood donors. We identified the cut-offs of 7.5 and 11.8 kPa for the diagnosis of fibrosis ≥S3 and cirrhosis respectively, showing 93.9% and 86.5% sensitivity, 88.5% and 96.3% specificity, 76.7% and 86.7% positive predictive value （PPV）, 97.3% and 96.3% negative predictive value （NPV） and 90.1% and 94.2% diagnostic accuracy. At multivariate analysis in 171 untreated carriers, fibrosis stage （t = 13.187,P 〈 0.001）, active vs inactive HBV infection （t = 6.437, P 〈 0.001）, alanine aminotransferase （ALT） （t = 4.740, P 〈 0.001） and HBV-DNA levels （t = -2.046, P = 0.042） were independently associated with FS. Necroinflammation score （t = 2.158, 〉 10/18 vs ≤ 10/18, P = 0.035） and ALT levels （t = 3.566, P =0.001） were independently associated with LS in 83 untreated patients without cirrhosis and long-term biochemical remission （t = 4.662, P 〈 0.001） in 80 treated patients. During FS monitoring （mean followup 19.9 ± 7.1 mo） FS values paralleled those of ALT in patients with hepatitis exacerbation （with 1.2 to 4.4-fold increases in Crib patients） and showed a progressive decrease during antiviral therapy. CONCLUSION： FS is a non-invasive tool to monitor liver disease in chronic HBV carriers, provided that the pattern of biochemical ac     

Intrahepatic cholestasis of pregnancy： When should you look further？

Pruritis with abnormal liver function tests is the classical presentation of intrahepatic cholestasis of pregnancy （ICP）, a condition associated with significant fetal complications. Although the etiology of ICP is unclear in many cases, certain features of the clinical presentation should alert the practitioner to the possibility of an underlying metabolic defect, which may not only affect subsequent pregnancies, but may be an indicator of more serious subsequent liver disease. We report a kindred of Anglo-Celtic descent, among whom many members present with ICP, gallstones or cholestasis related to use of oral contraception. Genetic studies revealed a novel mutation in the ABCB4 gene, which codes for a phospholipid transport protein. The clinical significance of this mutation and the importance of identifying such patients are discussed.     

Adult progressive intrahepatic cholestasis associated with genetic variations in ATP8B1 and ABCB11

Severe intrahepatic cholestasis with low serum γ-glutamyltranspeptidase (γ-GT) activity is exceptionally rare in adult patients, and its association with multi-genetic alterations of bile salt transporters has not been reported. We investigated a 25-year-old man presenting with a four-year history of jaundice. Laboratory and radiographic examinations revealed clinical pictures of progressive intrahepatic cholestasis with low γ-GT. Serial liver histopathology demonstrated cirrhosis resulting from progressive persistent cholestatic injury. Genetic sequencing studies for the entire coding exons of ATP8B1 and ABCB11 uncovered a heterozygous missense mutation 1798 C->T (R600W) in ATP8B1 , and a homozygous nucleotide substitution 1331 T->C (V444A) in ABCB11 . In conclusion, this is a rare case of adult onset progressive intrahepatic cholestasis with low γ-GT associated with heterozygous ATP8B1 mutation and homozygous ABCB11 polymorphism. Further studies are necessary to investigate the impact of heterozygous R600W mutation and whether other cholestatic disorders are multi-genetic.     

Association of the multidrug-resistance-associated protein gene (ABCC2) variants with intrahepatic cholestasis of pregnancy

BACKGROUND/AIMS: We hypothesized that common genetic variation at ABCC2 influences ICP susceptibility. Hence we studied the association of single nucleotide polymorphisms (SNPs) of promoter, coding and non-coding regions of ABCC2 and intrahepatic cholestasis of pregnancy (ICP). METHODS: 70 ICP patients and 112 healthy pregnant women in the third trimester of their pregnancies were included in a cross sectional study. Four tag SNPs (rs717620 A/G; rs2756105 C/T; rs2002042 C/T; rs3740066 A/G) encompassing 70 kb in chr.10 and representing 46 polymorphic sites (r(2) > 0.8) were genotyped. Besides, 2 additional SNPs (rs17222723 A/T and rs8187710 G/A) were included. RESULTS: In univariate analysis, rs2002042 and rs3740066 were significantly associated with ICP (p < 0.04 and 0.01, respectively) but after multiple testing correction, only rs3740066 remained significantly associated with disease status (p < 0.03). We also observed a positive association between the rs3740066 and ALT, AST, alkaline phosphatase and total and conjugated bilirubin concentrations. Consistent with the analysis of individual markers, we observed that haplotype frequency of the ABCC2 gene in ICP patients significantly differed from controls (p < 0.03). CONCLUSIONS: We found an association between the rs3740066 in exon 28 of ABCC2 gene and ICP. The risk of disease for homozygous AA carriers is 4-fold higher (OR 4.44 CI 95% 1.83-10.78, p < 0.001) in comparison with GG carriers.     

Intrahepatic cholestasis of pregnancy: the severe form is associated with common variants of the hepatobiliary phospholipid transporter ABCB4 gene

BACKGROUND: Intrahepatic cholestasis of pregnancy (ICP) is characterised by troublesome maternal pruritus, raised serum bile acid levels and increased fetal risk. Mutations of the ABCB4 gene encoding the hepatobiliary phospholipid transporter have been identified in a small proportion of patients with cholestasis of pregnancy. In a recent prospective study on 693 patients with cholestasis of pregnancy, a cut-off level for serum bile acid (> or =40 micromol/l) was determined for increased risk of fetal complications. OBJECTIVES: To investigate whether common combinations of polymorphic alleles (haplotypes) of the genes encoding the hepatobiliary ATP-binding cassette (ABC) transporters for phospholipids (ABCB4) and bile acids (ABCB11) were associated with this severe form of cholestasis of pregnancy. METHODS: For genetic analysis, 52 women with bile acid levels > or =40 micromol/l (called cases) and 52 unaffected women (called controls) matched for age, parity and geographical residence were studied. Gene variants tagging common ABCB4 and ABCB11 haplotypes were genotyped and haplotype distributions were compared between cases and controls by permutation testing. RESULTS: In contrast with ABCB11 haplotypes, ABCB4 haplotypes differed between the two groups (p = 0.019), showing that the severe form of cholestasis of pregnancy is associated with the ABCB4 gene variants. Specifically, haplotype ABCB4_5 occurred more often in cases, whereas haplotypes ABCB4_3 and ABCB4_7 were more common in controls. These associations were reflected by different frequencies of at-risk alleles of the two tagging polymorphisms (c.711A: odds ratio (OR) 2.27, p = 0.04; deletion intron 5: OR 14.68, p = 0.012). CONCLUSION: Variants of ABCB4 represent genetic risk factors for the severe form of ICP in Sweden.     

Recurrent intrahepatic cholestasis of pregnancy and chain-like choledocholithiasis in a female patient with stop codon in the ABDC4-gene of the hepatobiliary phospholipid transporter

We report the case of a 40-years-old female patient with recurrent cholestatic liver disease who presented twice with severe intrahepatic cholestasis of pregnancy and pronounced choledocholithiasis between pregnancies. Bile duct stones were removed endoscopically and a laparoscopic cholecystectomy was performed after the second pregnancy. Liver histology revealed intrahepatic cholestasis with portal inflammation and fibrosis, resembling progressive familial intrahepatic cholestasis (PFIC). Molecular genetic studies identified the heterozygous mutation c.957C > T in the ABCB4 gene encoding the hepatobiliary phospholipid transporter. This is the first report of this mutation that introduces a stop codon in an index patient with intrahepatic cholestasis of pregnancy and multiple bile duct stones. In addition, we detected the ABCB11 polymorphism V 444A, which is associated with a decreased expression of the bile salt export pump. Whereas homozygous carriers of the ABCB4 mutation develop PFIC type 3, the heterozygous ABC transporter mutations represent genetic risk factors for cholelithiasis and recurrent cholestatic hepatitis upon challenge with oral contraceptives or during pregnancy. Of note, the patient presented with normal serum gamma-glutamyltranspeptidase activities during pregnancy-associated cholestatic episodes but normal liver enzymes after delivery, whereas choledocholithiasis was associated with high gamma-glutamyl transpeptidase levels. It is unknown whether ursodeoxycholic acid prevents cholestasis or gallstones in patients with ABCB4 deficiency.     

Diagnosis and therapy of intrahepatic cholestasis of pregnancy

Intrahepatic cholestasis of pregnancy (ICP) is characterized by the occurrence of pruritus mostly in the third trimenon. Diagnosis is based on the presence of pruritus and elevated levels of serum bile acids in the absence of pruritic skin diseases. There is strong evidence of a genetic predisposition for ICP. Numerous studies have investigated the association of known cholestasis genes such as ABCB4 (also designated MDR3), ABCB11 ( BSEP) and ATP8B1 ( FIC1) with ICP. The results of these studies implicate a heterogeneous etiology of this syndrome. ICP increases the risk of preterm delivery and fetal loss. Furthermore, intense pruritus may necessitate premature induction of labor with its known higher frequency of complications for mother and child. Therefore, ICP pregnancies should be managed as high-risk pregnancies. Pharmaceuticals to alleviate pruritus or improve cholestasis like antihistamines, phenobarbital, anion exchange resins, dexamethasone or S-adenosylmethionine are not widely accepted because of questionable efficacy or side effects. Recent randomized studies have shown beneficial effects of ursodeoxycholic acid (UDCA) on laboratory data and pruritus in patients with ICP. Improved knowledge about the diagnostic classification of different types and pathophysiological mechanisms of ICP may allow for a more targeted treatment of this disease in future.     

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.     

Familial cholestasis: progressive familial intrahepatic cholestasis, benign recurrent intrahepatic cholestasis and intrahepatic cholestasis of pregnancy

Progressive familial intrahepatic cholestasis (PFIC) type 1, 2 and 3 are due to mutations in ATP8B1, ABCB11 and ABCB4, respectively. Each of these genes encodes a hepatocanalicular transporter, which is essential for the proper formation of bile. Mutations in ABCB4 can result in progressive cholestatic disease, while mutations in ATP8B1 and ABCB11 can result both in episodic cholestasis, referred to as benign recurrent intrahepatic cholestasis (BRIC) type 1 and 2, as well as in progressive cholestatic disease. This suggests a clinical continuum and these diseases are therefore preferably referred to as ATP8B1 deficiency and ABCB11 deficiency. Similarly PFIC type 3 is designated as ABCB4 deficiency. Heterozygous mutations in each of these transporters can also be associated with intrahepatic cholestasis of pregnancy. This review summarizes the pathophysiology, clinical features and current as well as future therapeutic options for progressive familial- and benign recurrent intrahepatic cholestasis as well as intrahepatic cholestasis of pregnancy.     

Linkage between a new splicing site mutation in the MDR3 alias ABCB4 gene and intrahepatic cholestasis of pregnancy

Intrahepatic cholestasis of pregnancy (ICP) is defined as pruritus and elevated bile acid serum concentrations in late pregnancy. Splicing mutations have been described in the multidrug resistance p-glycoprotein 3 (MDR3, ABCB4) gene in up to 20% of ICP women. Pedigrees studied were not large enough for linkage analysis. Ninety-seven family members of a woman with proven ICP were asked about pruritus in earlier pregnancies, birth complications and symptomatic gallstone disease. The familial cholestasis type 1 (FIC1, ATP8B1) gene, bile salt export pump (BSEP, ABCB11) and MDR3 gene were analyzed in 55 relatives. We identified a dominant mode of inheritance with female restricted expression and a new intronic MDR3 mutation c.3486+5G>A resulting in a 54 bp (3465-3518) inframe deletion via cryptic splicing site activation. Linkage analysis of the ICP trait versus this intragenic MDR3 variant yielded a LOD score of 2.48. A Bayesian analysis involving MDR3, BSEP, FIC1 and an unknown locus gave a posterior probability of >0.9966 in favor of MDR3 as causative ICP locus. During the episode of ICP the median gamma-glutamyl transpeptidase (gamma-GT) activity was 10 U/l (95% CI, 6.9 to 14.7 U/l) in the index woman. Four stillbirths were reported in seven heterozygous women (22 pregnancies) and none in five women (14 pregnancies) without MDR3 mutation. Symptomatic gallstone disease was more prevalent in heterozygous relatives (7/21) than in relatives without the mutation (1/34), (P = 0.00341). CONCLUSION: This study demonstrates that splicing mutations in the MDR3 gene can cause ICP with normal gamma-GT and may be associated with stillbirths and gallstone disease.