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Investigation of Common Variations of ABCB4, ATP8B1 and ABCB11 Genes in Patients with Progressive Familial Intrahepatic Cholestasis


1 MD, PhD Candidate, Gastroentrohephatology Research Center, Shiraz University of Medical Sciences, Shiraz, IR Iran
2 MD, Gastroentrohephatology Research Center, Shiraz University of Medical Sciences, Shiraz, IR Iran
3 Master in Genetic, Department of Medical Genetics, School of Medicine, Shiraz University of Medical Sciences, Shiraz, IR Iran
4 PhD, Department of Medical Genetics, Bioinformatics and Computational Biology Research Center, School of Medicine, Shiraz University of Medical Sciences, Shiraz, IR Iran
5 PhD, Department of Medical Genetics, School of Medicine, Shiraz University of Medical Sciences, Shiraz, IR Iran
*Corresponding Author: Majid Fardaei, PhD, Department of Medical Genetics, School of Medicine, Shiraz University of Medical Sciences, Shiraz, IR Iran, E-mail: mfardaei@sums.ac.ir.
Hepatitis Monthly. 17(2): e43500 , DOI: 10.5812/hepatmon.43500
Article Type: Research Article; Received: Nov 1, 2016; Revised: Dec 19, 2016; Accepted: Jan 29, 2017; epub: Feb 4, 2017; collection: Feb 2017

Abstract


Background: Progressive familial intrahepatic cholestasis (PFIC) is a heterogeneous group of hepatic disorders that can progress rapidly, leading to cirrhosis and death due to liver failure. Mutations and variations in three genes, including ATP8B1, ABCB11, and ABCB4, have been reported to be the main genetic cause of three subtypes of this disorder including PFIC1, PFIC2, and PFIC3, respectively.

Objectives: Therefore, the aim of this study was to investigate more common mutations and variations associated with PFIC considering clinical and Para-clinical features of the disease.

Methods: Thirty-five unrelated patients with PFIC from the south of Iran were selected randomly among all PFIC patients referring to Namazi hospital, affiliated to Shiraz University of Medical Sciences. Genomic DNA was extracted from the peripheral blood lymphocytes. Sequences related to these variations were then amplified by PCR in the 35 cholestasis patients and analyzed by Sanger® sequencing.

Results: The results showed that there was no variation in interest exon of ABCB4. Moreover, in ATP8B1, there was no prevalent mutation and only an unknown significant variation (c.*1101 + 366G > A) was found. However, in the ABCB11 gene, different variations were found including c.1434 + 174G > A, c.1434 + 70C > T, c.1331T > C (p.Val444Ala, a common variant proposed to be associated with cholestasis), c.1309-93G > A, c.1309-165C > T. Also, 11 and 13 cases showed heterozygote and homozygote, respectively, for V444A variation of the ABCB11 gene.

Conclusions: The allele frequency of V444A in this study was 52.8%. This variation has been previously implicated with higher frequencies in ICP and DIC than normal subjects, suggesting that this variation may become disease-relevant in certain conditions.

Keywords: Intrahepatic Cholestasis; Mutation; ABCB4; ATP8B1; ABCB11

1. Background


Progressive familial intrahepatic cholestasis (PFIC) is a heterogeneous class of autosomal recessive hepatic disorders that begins in the neonatal period or first year of life and usually progresses to cirrhosis within the first decade of life (1, 2). PFIC can progress rapidly and result in cirrhosis during infancy, leading to death due to liver failure at ages usually between infancy and adolescence (1, 2).


Until now, genetic and molecular studies have identified three subtypes of PFIC, which include PFIC1 (the former Byler disease), PFIC2, and PFIC3 as a result of mutations in genes involved in bile formation. PFIC1 is due to ATP8B1 gene mutations, which can cause the milder phenotype (3). PFIC2 is caused by mutations in the ABCB11 gene (4, 5). This gene which is located on human chromosome 2q24 encodes the ATP-dependent canalicular bile salt export pump (BSEP) in liver and the loss of BSEP function responsible for the decreased biliary bile salt secretion resulting in the accumulation of bile salts inside the hepatocyte and finally severe hepatocellular cholestasis (6). PFIC3 results from diseases causing variants in the ABCB4 gene located on chromosome 7q21. This gene is translated into the protein MDR3 with an essential role in biliary phospholipid (phosphatidylcholine) excretion across the canalicular membrane (7-9). Bile from patients with PFIC3 is not inactivated by phospholipids (with very low concentrations of phospholipid) that results in bile canaliculi and biliary epithelium injuries, leading to cholangitis and cholestasis cholestatic liver disease (10). Given the fact that different ATP8B1, ABCB11, and ABCB4 variants have been associated with cholestasis, we made an attempt to investigate the most common variants reported in these genes among 35 patients with cholestasis.

2. Methods


2.1. Patients

Thirty-five unrelated patients with cholestasis from the south of Iran were enrolled in this study. All cholestasis patients were diagnosed by a group of gastroenterohepatology specialists in gastroenterohepatology ward of Namazi hospital, affiliated to Shiraz University of Medical Sciences, on the basis of characteristic symptoms such as jaundice, dark urine, light-colored stools, and generalized itchiness. The patients were selected randomly. All the patients gave informed consent before undergoing genetic analysis for common variations of ATP8B1, ABCB4, and ABCB11. Three-milliliter whole-blood samples were collected from the patients, drawn into EDTA tubes, and stored at -20ºC until use.


2.2. Preparation of Genomic DNA

Genomic DNA was extracted from the peripheral blood lymphocytes by QIAamp DNA Blood Mini Kit (QIAGEN, Germany) according to the manufacturer’s instructions. The genomic DNA concentration was measured by NanoDrop (ND1000, USA) and stored at - 20ºC until use.


2.3. PCR and Sequencing

Sequences covering the variations of ATP8B1 (I661T, exon 18), ABCB4 (p.R652G, exon 16), and ABCB11 (p.V444A, exon 13) in 35 cholestasis patients were amplified by PCR primers given in Table 1. These primer pairs were designed and evaluated on the basis of the ATP8B1, ABCB4, and ABCB11 reference genomic sequences (ENSEMBLE) using NCBI-BLAST, UCSC (BLAT and In Silico PCR) and Allele ID 7.5 (Table 1). The total volume of the PCR was 50 μL containing 1 μL of each primer (20 pmol/μL), 1 μL DNA template (50 - 200 ng), 25 μL TEMPase Hot Start 2x Master Mix Blue (Ampicon, A290806), and 22 μL dH2O. The PCRs were carried out according to Amplicon TEMPase Hot Start protocol and programs given in Table 1. Ten microliters of the PCR products were visualized on 2% agarose gel containing SYBR Safe.


Table 1.
Primers and PCR Protocols Used for Amplification of the Interest Exons of ATP8B1 (NI661T), ABCB4 (R652G) and, ABCB11 (V444A) Genes

2.4. DNA Sequencing

PCR products of interest exons and exon-intron boundaries of three mentioned genes in our 35 patients were analyzed by DNA sequencing. Sanger sequencing data were analyzed using NCBI BLAST and Codon Code Aligner software.

3. Results


A total of 35 patients (70 alleles) with progressive intrahepatic cholestasis were studied for detection of common variants associated with this disorder in interest exons of ATP8B1 (I661T), ABCB4 (p.R652G), and ABCB11 (p.V444A). There was not identified any variation in interest exon of ABCB4. Also, in ATP8B1, a prevalent mutation was not identified, and only an unknown significant variation (c.*1101 + 366G > A) was identified in patients 4, 9, 15, 21, 26, and 29 as heterozygote and patient 14 as homozygote. However, in ABCB11 gene, different variations were found including c.1434 + 174G > A, c.1434 + 70C > T, c.1331T > C (p.Val444Ala, common variant), c.1309-93G > A, and c.1309-165C > T. Since among these variations, only p.Val444Ala is important due to its susceptibility and association with intrahepatic cholestasis, its distribution in our patients are given in Table 2.


Table 2.
Clinical and Para Clinical Sings of the Patients and Distribution of c.1331T > C (p.Val444Ala) in ABCB11 Gene among Our Patients

It was found that there were 11 and 13 cases of heterozygote and homozygote, respectively, for V444A variation of ABCB11 gene. As mentioned above, this change results in a reduction in the quantity of BSEP protein in liver cells and may be associated with cholestasis in certain conditions, such as pregnancy and the use of ethinylestradiol and levonorgestrel. Among 35 patients involved in this study, 10 cases showed no mutation or variation in the investigated exons of the three genes and other exons should be investigated for their genetic origin. All data are given in Table 2.


Clinical presentation and signs observed in our patients were jaundice, itching, bleeding tendency, fever, tea-color urine, pruritus, ascites, gastrointestinal bleeding, encephalopathy, and itching wounds. Their Para clinical findings on the basis of ultrasonography and histopathological examination indicated enlarged liver, inhomogeneous liver echo, spleen prominent size ascites pleural effusion, cirrhosis, chronic cholecystitis, fibrosis, splenomegaly, hepatomegaly, giant cell formation, and intracalcinular cholestasis (Table 2).

4. Discussion


Molecular genetic testing to identify mutations and variations associated with cholestasis in ATP8B1, ABCB4, and ABCB11 genes is important to confirm PFIC2 diagnosis. Different mutations in these genes have been identified in Asian population (11, 12). The majority of them are different from those identified in other populations. Prevalent mutations that have been reported in Europe were not detected in other regions such as China. Therefore, mutations in these genes may be ethnicity-specific (13)


V444A is a highly prevalent variant of ABCB11 and its allele frequency has been reported in Japanese and Caucasian populations (14). The allele frequency of V444A in the current study was 52.8%. This variation has been previously implicated with higher frequencies in ICP and DIC than normal subjects, suggesting that this variation may become disease relevant in certain conditions (15, 16). However, further larger-scale studies are required to fully uncover the role of V444A variant.


Bile salt export pump (BSEP) is highly conserved during vertebrate evolution, and its expression is inhibited by PFIC type II mutations (17). PFIC type 2 is due to mutations in ABCB11, the gene encoding the BSEP protein. In our study, most of the patients (62.85%) had homozygote mutations in ABCB11 gene. Jaundice, itching, bleeding tendency, fever, tea-color urine, enlarged liver, normal echo, intracellular and intracalcinular cholestasis, cholestasis, pruritus, ascites, GI bleeding, encephalopathy, increased liver echo, cholestasis, giant cell formation fibrosis, and cirrhosis were some of the clinical and Para clinical features of the PFICII in our patients.


ATP8B1 deficiency is a severe autosomal recessive liver disease resulting from mutations in the ATP8B1 gene characterized by a spectrum from intermittent (benign recurrent intrahepatic cholestasis; BRIC) to progressive familial intrahepatic cholestasis (PFIC) (18).


About 20% of our cases had homozygote and heterozygote mutations in their ATP8B1 genes. However, most of them had also mutations in their ABCB11 gene. Jaundice, poor growth, pruritus, encephalopathy, ascites, GI bleeding, enlarged liver, coarse echo, chronic hepatic parenchymal damage, focal steatosis and ballooning degeneration, fibrosis as well as elevated liver enzymes, heterogeneous echo, relative destruction of lobular and vascular architecture, fibrosis, splenomegaly, hepatomegaly, relative destruction of lobular and vascular architecture were clinical and Para clinical findings of the disease in our patients confirmed by the detection of the mutations.


Different molecular methods have been used to investigate mutations in genes associated with cholestasis such as single-strand conformation polymorphisms (SSCP) (19), denaturing high performance liquid chromatography (DHPLC), and DNA sequencing (20). The gold standard is DNA sequencing since it can reveal mutations and variations across three genes with 28 exons for each. However, this method is very expensive. Therefore, the current study tried to investigate common mutations and variations associated with cholestasis in three specific genes and their main exons.


Our study showed that while two common mutations were not identified in our patients, the other common variant, V444A, that previously has been proposed to be associated with cholestasis in certain condition such as pregnancy and the use of ethinylestradiol and levonorgestrel, was identified in a high frequency (37 chromosomes out of 70, 52.8%). The common V444A variant can be investigated in control samples to be concluded for its susceptibility and association with intrahepatic cholestasis in our patients since this variant leads to a reduction in the quantity of BSEP protein in liver cells and can be a good indicator in these patients (15).


In conclusion, the current study investigated common mutations and variations of ATP8B1, ABCB4, ABCB11 genes. V444A was a highly prevalent variation found in ABCB11 in the study. However, it is not fully clear that if they are associated with pediatric cholestatic diseases. More studies should be conducted to investigate this variation, identify the molecular mechanism of ABCB11 product and BSEP and therefore, determine its usefulness in the personalized management of special patients.


There are so many studies that have evaluated the variations in ABCB4, ABCB11 and ATP8B1 genes separately. However, no study investigated the mutations of three genes in each study patient. This is considerable because we found that a large number of our cases (17.14%) had mutations in both ABCB11 and ATP8B1 genes.


One of the limitations of the study was to find patients who voluntarily agreed to participate in this study and donate blood samples. On the other hand, more developed sequencing of related genes, for example whole exon sequencing, was needed to find underlying mutations in cases with no detected mutation using sanger sequencing. In our study, some of the patients had no mutation in interest areas of three mentioned genes, so they should be evaluated by further molecular investigations to find molecular basis of the disease.

Acknowledgments

The authors of this study appreciate staff of Namazi hospital for their kind cooperation in data gathering and blood sampling. This study was supported by Shiraz University of Medical Sciences (grant No. of 93-7346).

Footnotes

Conflict of Interest: None declared.

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Table 1.

Primers and PCR Protocols Used for Amplification of the Interest Exons of ATP8B1 (NI661T), ABCB4 (R652G) and, ABCB11 (V444A) Genes

Gene (Interest Variant) Primer Sequence PCR Program PCR Product
ATP8B1 (NI661T) Forward: GGATGATAAAGCCAGACCTTGT 95°C for 15 min, 35 cycles for: 95°C, 30 sec, 64°C, 30 sec, 72°C, 20 sec, and final extension 72°C, 7 min 502 bp
Reverse: GTGCCAGTGTCAAATGCTGAA
ABCB4 (R652G) Forward: TCCTTGATTGAGAAGCAGTTAG 95°C, 15 min, 35 cycles for: 95°C for 30 sec, 59°C for 30 sec, 72°C for 30 sec, and final extension 72°C for 7 min 571 bp
Reverse: GCATCTCAGCGTAAAGACTAC
ABCB11 (V444A) Forward: TCTTGGTCATGGCTCTCA Touch Down PCR, 95°C for 15 min, 20 cycles for: 95°C for 30 sec, 67°C for 30 sec (-0.5°C per cycle), 72°C for 20 sec, 15 cycles for:95°C 30 sec, 58°C 30 sec, 72°C for 25 sec, and final extension 72°C for 7 min 626 bp
Reverse: ATCACTGACTGAAATGTTGC

Table 2.

Clinical and Para Clinical Sings of the Patients and Distribution of c.1331T > C (p.Val444Ala) in ABCB11 Gene among Our Patients

Case No. ABCB11 ABCB4 ATP8B1 Clinical Signs Para Clinical Findings Treatment
1. 1 ND Jaundice, Itching, Bleeding Tendency, Fever, Tea Color Urine Enlarged Liver, Normal Echo, Intracellular and Intracalcinular Cholestasis, Fibrosis LT
2. Hom. ND ND pruritus, Ascites, GI bleeding, encephalopathy increased liver echo, cholestasis, giant cell formation, cirrhosis LT
3. ND ND ND Pruritus, clay stool, itching, jaundice, hepatomegaly, irritability heterogeneous echo of the liver, enlarged spleen, destruction of lobular and vascular architecture, nodule formation, cirrhosis LT
4. Het. ND Het. poor growth, pruritus, encephalopathy, ascites, GI bleeding, enlarged liver, coarse echo, chronic hepatic parenchymal damage, focal steatosis and ballooning degeneration, fibrosis LT
5. Hom. ND ND Jaundice increased echo, enlarged liver, enlarged spleen, cirrhosis LT
6. Het. ND ND pruritus, vomiting, diarrhea, fatty stool, tea color urine hepatomegaly, itching enlarged liver, enlarged spleen, fibrosis, cirrhosis LT
7. Hom. ND ND jaundice, icteric sclera, itching liver coarse echo LT
8. Het. ND ND Jaundice, pruritus, encephalopathy liver prominent size, enlarged spleen, cirrhosis, giant cell hepatitis LT
9. Het. ND Het. Jaundice Elevated liver enzymes, heterogeneous echo, relative destruction of lobular and vascular architecture, fibrosis LT
10. Hom. ND ND Jaundice Elevated liver enzymes, splenomegaly, hepatomegaly, hyperechoic lesion, spotty necrosis, infiltration of lymphocytes, fibrosis LT
11. ND ND ND Jaundice enlarged liver, increased echo of hepatic biliary tree, infiltration of inflammatory cells, fibrosis LT
12. Hom. ND ND Jaundice, Yellowish Skin, encephalopathy, GI bleeding, Ascites, Pruritus enlarged spleen, inhomogeneous echo, cirrhosis LT
13. Hom. ND ND Pruritus mucosal thickening of Gall bladder, Ascites, fibrosis LT
14. Hom. ND Hom. Pruritus, Pruritus Yellow Skin, And Sclera, Itching Wound Altered liver enzymes LT
15. Hom. ND Hom. Pruritus, Ascites Jaundice, Encephalopathy irregular outline of liver, coarse echo, spleen prominent size ascites pleural effusion, Cirrhosis, chronic cholecystitis LT
16. ND ND ND No data was available No data was available -
17. Hom. ND ND Jaundice enlarged liver, inhomogeneous echo LT
18. Het. ND ND Itching, Jaundice, anorexia, pruritus encephalopathy, GI bleeding enlarged liver, Gall stone, duct proliferation, inflammation, Rosette formation, Feathery changes LT
19. ND ND ND Itching, intractable pruritus Altered liver enzymes LT
20. ND ND ND Itching, severe pruritus Altered liver enzymes, increased liver echo LT
21. Hom. ND Het. Jaundice, icterus liver prominent size, hyperechoic liver, spleen prominent size, Extensive Fibrosis, portal fibrosis, ductular proliferation, cholestasis Liver biopsy
22. Hom. ND ND Jaundice, icterus Elevated liver enzymes, Liver coarse echo, enlarged spleen, destruction of lobular and vascular architecture, nodule formation, fibrosis LT
23. Hom. ND ND Jaundice, pruritus, encephalopathy, ascites, GI bleeding, palmar erythema, GI bleeding patent spleen, giant cell formation, feathery degeneration, fibrosis LT
24. ND ND ND Itching, Jaundice, generalized pruritus enlarged liver with homogenous echo BD, LT
25. ND ND ND Itching, pruritus cholestatic rosettes, fibrosis LT
26. ND ND Het. Altered liver enzymes irregular capsule, coarse echo, multifocal dilation of biliary ducts, cirrhosis LT
27. Het. ND ND Itching, Jaundice, fever, tea color urine, GI bleeding, Encephalopathy Enlarged Liver LT
28. Het. ND ND Itching, Jaundice, icteric, sever pruritus, irritability, jaundice, ecchymosis, bleeding tendency enlarged liver, inhomogeneous echo LT
29. Het. ND Het. Jaundice, bleeding tendency, splenomegaly, hepatomegaly, relative destruction of lobular and vascular architecture, fibrosis LT
30. ND ND ND Jaundice, yellowish skin, pruritus enlarged liver, coarse echo LT
31. Het. ND ND prolonged jaundice cholestatic rosettes, fibrosis LT
32. Hom. ND ND jaundice hepatomegaly, elevated LFT LT
33. ND ND ND No data was available No data was available -
34. ND ND ND No data was available No data was available -
35. Het. ND ND sever itching, encephalopathy, ascites, GI bleeding Cirrhosis, increased echo, enlarged spleen LT
Abbreviations: BD: Biliary diversion; LT, Liver transplantation; Het, heterozygote; Hom, homozygote; ND, Non-detected.