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Cholestasis with Normal Gammaglutamyl Transferasis from Management to Molecular Investigation with Novel Mutations in ATP8B1 and ABCB11: Tunisian Study

Barkaoui E1,4*, Alobaidy H2, Darragi I4, Ayadi A3, and Abbes S4

1Protection of Maternal and Infantil Center Ezzouhour Tunis, Tunisia

2El Khadra Hospital Tripoli, Libya

3Pediatric Center Mahdia Hospital, Tunisia

4Laboratory of Cellular Hematology and Molecular Biology, Pasteur Institute Tunis, Tunisia

*Corresponding Author:
Emna Barkaoui
Head of Protection of Maternal and Infantil Center Ezzouhour Tunis Tunisia
Laboratory of Hematology And Molecular Biology
Pasteur Institute Tunis, Tunisia
Tel: 00216 53 73 15 16
E-mail: [email protected]

Received Date: January 01, 2017; Accepted Date: January 18, 2017; Published Date: January 25, 2017

Citation: Emna B, Hanna A, Imen D, Abdelkarim A, et al. (2018) Cholestasis with Normal Gammaglutamyl Transferasis from Management to Molecular Investigation with Novel Mutations in ATP8B1 and ABCB11: Tunisian Study. J Genet Disord. 2:2.

 
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Abstract

Introduction: Cholestasis with normal gamma-glutamyltransferasis (GGT) is a heterogeneous group. ATP8B1 and ABCB11 two genes implicated on the respectively phenotypes PFIC1, BRIC1 and PFIC2 BRIC2, lithiasis, and drug cholestasis.

Aims:
The aim of this study is to report the phenotype, the outcome and to determine the profile of mutations.

Methods & Materials:
Eleven patients with persistent normal GGT cholestasis were studied from 93 patients with liver disease referred to us from 2007 to 2014. Eight patients were studied for ATP8B1, five for ABCB11, one family, with two patients, was studied for both genes. Polymerase chain reaction (PCR) was performed using a set of selected primers and direct sequencing by Applied Biosystems. Intronic variants were analyzed by the Human Splicing Finder program.

Results:

Patients phenotype:
The age at the diagnosis was ranged from neonatal to 12 months. Consanguinity was found in 10/11 of the patients. The constant features were pruritus and hepatomegaly. No one developed portal hypertension (3 to 22 years). Two patients presented asthma with high level of IgE with negative sweat test. Two patients developed intense pruritus despite ursodesoxycholic acid (UDCA) and rifadin with severe lichenification of the skin, they were successfully treated by sertraline with a considerable improve of the skin. In one patient, we observed a relapse of the pruritus after two months requiring biliary derivation and in one patient, frequent crisis of crazy laughs, sertraline was stopped. Two patients developed liver failure respectively at the age of six months (hepatocarcinoma was found in explant liver) and six years old, received liver transplant.

Molecular Study Results:
For ATP8B1, we found two novel exonic mutations, the V310F and D379RfsX51 and two known variants the F305I and R952Q, seven novel intronic mutations, three silent mutations: two new mutations one of them D234D was near splicing site. For ABCB11, we found three exonic mutations : Y354X , two novel mutations N372YfsX23, A986P and two novel intronic variants. The analysis of the seven intronic variants by splicing Finder Program showed the possible role of four of them in the disease.

Conclusions:
This study is the first to be performed in Tunisia for patients with normal GGT cholestasis. The molecular results were preliminary but did not find founder effect. We report both novel exonic and intronic mutations. Most patients have more than two related variants, which may be due to high consanguinity. This study should be completed by the transcriptional study for the novel intronic and silent mutations.

Heterogeneous group; Gammaglutamyl transferasis; Cholestasis; Genetic analysis

Abbreviations

ATPase: Adenosin Tri phosphate; DNA: Deoxyribo Nucleic Acid; HC: Hepatocarcinoma; MCT: Medium Chain Triglycerides; PCR: Polymerase Chain Reaction; PFIC: Progressive Familial Intra Hepatic Cholestasis; UDCA: Ursodesoxycholic Acid

Introduction

Progressive familial intra hepatic cholestasis (PFIC) is a heterogeneous group of biliary acids disorders divided into two groups distinguished by value of gammaglutamyl transferasis (GGT) with three types (PFIC3, PFIC2, PFIC1) [1-3]. The group with high GGT (PFIC3) due to mutation in ABCB4 gene encodes floppies for phosphatidyl choline (PC) [4]. The group with normal GGT is due to two genes [3]. ATP8B1 for PFIC1 and ABCB11 for PFIC2, characterized by jaundice, intense pruritis, hepatomegalia, splenomegalia with normal GGT and cholesterol rates. PFIC1 patients could have extrahepatic symptoms as deafness, pancreatitis and tubulopathy. ATP8B1 plays in translating phosphatidyl serine (PS) from the outer to the inner leaflet cell membrane [5,6] and could affect the membrane integrity [7]. ABCB11 codes for bile salt export pumpprotein (BSEP). The estimated incidence varies between 1/50 000 and 1/100 000 births.

The present research purports to report the preliminary results of the first molecular study for ATP8B1 and ABCB11.

Materials and Methods

From June 2007 to March 2013, 93 patients with liver diseases were referred to our centre (among them18 patients from Libya, three from Algeria, one from Mali).

Patients: Fourteen patients have cholestasis with normal GGT, eleven were genetically investigated. The parents of all patients gave written informed consent for the genetic analysis. The phenotype diagnosis was performed by a combination of clinical (jaundice, hepatomegaly, splenomegaly with pruritus), biological (cholestasis (conjugated bilirubin>17 micromole/L) with normal or low GGT (<40 UI/l) and normal cholesterol rates (<2.50 mmol/l)), high level of serum primary bile acids (>15 ng/l) and familial history. All patients had alpha-foetoprotein (AFP) (normal<10 ng/ml), ear (BER: Brainstem Evoked Responses), pancreatic (amylasemia (normal<100 UI/l), lipasemia (normal<60 UI/l), echography +/-tomodensitometry) and tubular (phosphor reabsorption rate), explorations. The management was standardized and treatment was based on: 1-special nutrition (rich milk medium chain triglycerides (MCT), Malto dextrin), 2-Ursodesoxycholic acid UDCA, 3-Parenteral vitamins ADEK, 4-Rifampycin for persistent pruritus, 5-Sertraline (Zoloft) used in refractory pruritus (as known protocol), 6-Bilary diversion for one patient, 7-Liver transplantation in two patients for developing liver failure respectively at the age of 6 months (with hepatocarcinoma in liver explant) and 6 years. Six unrelated families were screened for ATP8B1 and four families for ABCB11. One family was studied for both ATP8B1 and ABCB11.

Molecular study: DNA was extracted from peripheral blood leucocytes with a standard phenol chloroform technique. Polymerase chain reaction (PCR) was performed using a set of selected primers for coding exon according to http://pharmagenetics.ucsf.edu (from 2 to 28 but considered from 1 (exon 2) to 27 (exon 28) as coding exons). PCR products were purified using the High Pure PCR purification system before direct sequencing by version 2.0 Big Dye Terminator cycle sequencing kit (Applied Biosystems). Bioinformatic software was used for genetic analysis (« Sequencer» and « Human splicing Finder site»). For ATP8B1: First, we analyzed common variations previously described in PFIC, for coding exons 6, 7, 8, 9, 15, 17, 18 and 22. The total exons were screened for only two patients (P1, P6). For ABCB11, the only exons bearing the common mutations were screened: exon7, exon9, exon11, exon14 and exon23.

Results

Patient’s phenotype profile (Table 1): The Consanguinity was found in 10/11 of the patients. The constant features were pruritus and hepatomegaly. No one developed portal hypertension. Two patients (P1, P6) developed intense pruritus despite UDCA and rifadin with severe lichenifaction of the skin were treated by sertraline. On sertraline the two patients’ skin and pruritus improved considerably with disappearance of the lichenifactions but P1 relapsed after two months requiring biliary derivation and P6 developed frequent crisis of crazy laughs, the sertraline was then stopped. The skin improvement remained constant after cessation of the treatment. Patient P1 presented also abdominal pain with episodes of pancreatitis treated by low fat diet. Two patients developed liver failure respectively at the age of 6 months and 6 years old and received liver transplant in France (P5, P11). Patient (P11) was referred to us for neonatal cholestasis, the investigations at 1.5 months showed positive cytomegalovirus (CMV) PCR with high level of alphafoetoproteine (AFP) (>200 000 ng/ml) with persistent normal GGT level, he developed sudden and early liver failure after normalisation of liver parameters on UDCA, he received liver transplant at the age of 6 months, hepatocarcinoma was found in explants liver.

Patient/
Sample
Origin/ Region Consanguinity/
Family history
Age
at onset
First phenotype classification Outcome/
Particularity
P1/10 Sfax
Hancha
Center
+/ Brother dead with normal GGT PFIC Neonatal PFIC1
Pancreatic crisis
Bilary diversion
14 years Old/
Elevated GGT after bilary diversion
P2/13 SfaxHancha
Center
+ Neonatal PFIC1 PFIC1 to BRIC1
Good outcome
22 years Old
*P3/16 Kef
North west
+ 6 months PFIC1
PFIC2
Ataxia by vitamin E defect
Short stature
Normal liver parameters
12 years Old
*P4/17 Kef
North west
+ Neonatal
screening
PFIC1
PFIC2
High level of AFP
Normal liver parameters
10 years Old
P5/9 Medenine
South
+ 6 months PFIC1 Liver transplantation at 6 years old
Good outcome
8 years Old
P6/34  Kairouan
Center
+ 7 months PFIC1 Stable
P7/40   Mahdia
Center
+ NP PFIC1 Stable
P8/43 Mahdia
Center
+ NP PFIC1 Stable
P9/64 Guebelli
South
+ Neonatal PFIC2
High AFP
Indication of liver transplant
3 years Old
P10/71 Mali - Neonatal PFIC2
High AFP
Returned to Mali
?
P11/87 Medenine
South
+ Neonatal PFIC2
High AFP
Liver transplant
HCC
Good outcome
6 years Old

Table 1: Phenotype characteristics of patients.

Molecular results: In ATP8B1 (Figure 1) we found three exonic mutations, two of them were novel: V310F (p.310G>T) in one patient (P2) and D379RFsX51 in two patients (P1, P6), the F305I (p.305T>A) mutation was found in patient P6, one polymorphism R952Q, three silent mutations, and seven intronic variants (Table 2 and Table 3). The Three silent mutations were R271R (homozygote in all studied Tunisian patients) and two novel mutations the D232D mutation located near the splicing site and the P634P. The polymorphisms R952Q was found in 9/24 alleles (P4, P8). In ABCB11, We found two novel exonic mutations (N372YfsX23, A986P) and two novel intronic variants (IVS13-43 T>C, IVS13-24 T>C). One patient (P11) had p.Tyr354X mutation.

Patient/
Sample
Region Genomic position DNA
change
Protein change Zygosity Predicted effect
P1/10 Exon7
+
Exon9
+
Exon11 Exon16         
g.105677 T>C
+
g.107994 A>C
+
g.111405InsA g.131799 T>C
c.696 T>C
+
c.811 A>C
+
c.1134_1135inA c.1902 T>C
p.D232D
+
p.R271R
+
p.D379RFsX51 p.P634P
Homozygote Homozygote Homozygote Heterozygote Silent mutation Silent mutation Truncated protein Silent mutation
P2/13 Exon9
+
Exon9
g.107994 A>C
+
g.108610 G>T
c.811 A>C
+
c.928 G>T
p.R271R
+
p.V310F
Homozygote Heterozygote Silent mutation Missense
*P3/16 Exon9
+
Exon22
g.107994  A>C
+
g.148026 G>A
c.811 A>C
+
c.2855 G>A
p.R271R
+
p.R952Q
Homozygote
+
Homozygote
Silent mutation Polymorphism
*P4//17 Exon9
+
Exon22
g.107994 A>C
+
g.148026 G>A
c.811 A>C
+
c.2855 G>A
p.R271R
+
p.R952Q
Homozygote
+
Heterozygote
Silent mutation Polymorphism
P5/9 Exon9 g.107994 A>C c.811 A>C p.R271R homozygote Silent mutation
P6/34 Exon7
+
Exon9
+
Exon11 + Exon16
g.105677 T>C
+
g.107994 A>C
+
g.108598 T>A
+
g.111405InsA
+
g.131799 T>C
c.696 T>C
+
c.811 A>C c.913 T>A
+
c.1134_1135inA
+
c1902 T>C
p.D232D
+
p.R271R p.F305I
+
D379RFsX51
+
p.P634P
Homozygote Homozygote Heterozygote Homozygote Heterozygote Silent mutation Silent mutation Missense Truncated protein Silent mutation
P7//40 Exon9 g.107994 A>C c.811 A>C p.R271R homozygote Silent mutation
P8/43 Exon9
+
Exon22
g.107994 A>C
+
g.148026 G>A
c.811 A>C
+
c.2855 G>A
p.R271R
+
p.R952Q
Homozygote Heterozygote Silent mutation Polymorphism

Table 2: Variants in ATP8B1.

genetic-disorders-Mutations

Figure 1: Mutations in ATP8B1.

Discussion

Many mutations were published from many ethnic groups (more than 100) and in 2004, Klomp described 54 mutations [8-12]. Our study was the first in Tunisia and cholestasis with normal GGT constitutes 15% (14/93). The patients were from different regions but most of them from the center. This ascertainment was found for other diseases. One patient was from Mali. The parental consanguinity was found in all Tunisian patients. In Tunisia, the consanguinity was high and estimated at 32% and could be elevated in each disease separately from 80% to 100% [13]. Four patients had permanent jaundice with high level of AFP (P4, P9, P10, and P11) and studied for ABCB11. Spraul et al. [9] found that elevated serum AFP concentrations were above normal limit value in 12/15 PFIC2 patients however authors considered that the combination of alanine aminotransferasis activity (ALT) ≤ 5x upper limit of normal range (ULN) and normal AFP value was present in all PFIC1 patients and (ALT)>5x ULN range and high AFP value was present in 73% of PFIC2 patients [9].

Patient P11 was referred to us for neonatal cholestasis with positive cytomegalovirus (CMV) PCR, high levels of alphafoetoproteine (AFP) (>200 000 ng/ml) and persistent normal GGT levels. For this case the treatment by UDCA normalized all liver parameters and the AFP was rapidly decreased. CMV infection was interpreted as post natal undercurrent infection. For this patient the first diagnosis of PFIC2 was confirmed with Y354X mutation. None of the patients studied developed portal hypertension after long time of outcome (Table 1) in our study.

As far as the exonic mutations are concerned we detected three in ATP8B1 and two in ABCB11. Two novel mutations were found for ATP8B1, D379RFsX51 and V310F; D379RFsX51 is predicted to lead to truncation of synthesis at amino acid 379 (of 1251) and significantly change the structure of ATP8B1 protein and could be associated with PFIC1. The V310F mutation changes valine, aliphatic amino acid and apolar to phenylalanine aromatic and apolar amino acid without predictive effect on structure but its implication for PFIC needs a screen of controls and a functional study. The F305I, a missense mutation was described before the present study in one patient with ICP from a cohort of 182 women [14], in one of 120 healthy control pregnancies [14], in one patient with chronic pancreatitis [15] and in our study it is the first time found in PFIC patient. This mutation results in the substitution of a phenylalanine by isoleucine and creates an EcoRV site and could play a role for PFIC1 [14]. The V310F and F305I mutations, each one is specific to one family. The R952Q (c.2855G>A) known as polymorphism (rs12968116) was equally frequent among ICP patients and Finnish controls (respectively 14.7 and 14% of samples), twice as frequent among CEPH controls (28%) [16], we found this variant in nine of 24 alleles and homozygous in two individuals (P4 and his father). Most of the patients of this study had more than two variants and P6 have five variants two of them with possible association for PFIC1. The intronic mutations and after analysis by « Human splicing Finder site», they showed the possible implication of four variants in splicing site (IVS1+45T>G, IVS7+20C>T, IVS22+53T>A, IVS11+46G>A). These variants are novel. Patients P1 and P7 are homozygote respectively for the IVS7+20C>T and IVS22+53T>A. The patient P1 was homozygote for two mutations exonic and intronic both, patient P7 was only homozygote for intronic mutation. For all the other intronic variants, patients are heterozygote. For the three other intronic mutations, they could be polymorphisms and some of them were specific to the family (Table 3). Additional studies are required, functional and transcriptional study should be completed to confirm the role of these variants in relation to the disease.

Exon/Intron Sample/Patient Nucleotide
change
Zygosity Predicted effect
1/1 34(P6) IVS1+45T>G Heterozygote Potential alteration of splicing site
8/7 12(P1)
9(P5),
13(P2), 
IVS7-8T>A
IVS7-8T>A
IVS7-8T>A
Heterozygote
Heterozygote
Heterozygote
Polymorphism
Not specific of a family
7/7 34(P6)
10(P1)
IVS7+20C>T
IVS7+20C>T New Donor site
Heterozygote
Homozygote
Activation of an intronic cryptic donor site.
Potential alteration of splicing site.
11/11 10(P1),
34(P6)
IVS11+46G>A ESS Site broken Heterozygote
Heterozygote
Alteration of an intronic ESS site.
Potential alteration of splicing
22/21 17(P4), IVS22-8C>T Heterozygote Polymorphism?
Specific of the family
No significant splicing Motif alteration detected
22/22 34(P6), 40(P7) IVS22+53T>A New ESE site Heterozygote Homozygote Creation of an intronic ESE site.
Potential alteration of splicing
22/22 34(P6), IVS22+59A >G Heterozygote Specific of the family
Polymorphism

Table 3: Intronicvariations in ATP8B1.

For ABCB11, the insertion mutation N372YfsX23 was responsible for truncated protein. For the A986P mutation, we must screen it in controls to confirm its association with PFIC2. Intronic variants (intron13: IVS13-43 T>C; IVS13-24 T>C) were novel and their analysis with Splice Site Finder program found that they could play a role in the splicing site. One patient P11 had a p.Tyr354X mutation, which was found by French laboratory [9]. This nonsense mutation creates a premature stop codon and a truncated protein. This patient was transplanted with early liver failure associated with hepatocarcinoma. Two maternal uncles have developed early hepatocarcinoma associated to hepatitis B. Up to the present day there is no correlation between hepatocarcinoma and mutations. Our case represents a rare example of early hepatocellular carcinoma complicating PFIC2 that need evaluation of clinic symptoms and we highlight that the first presentation with high level of AFP with unexplained early liver failure after response to treatment may be related to high risks of hepatocarcinoma in PFIC2. Knisely et al. reported 7 PFIC2 patients with hepatocarcinoma at age under 5 years [17].

Conclusion

This study is the first to be performed in Tunisia for patients with normal GGT cholestasis. The preliminary molecular results report both novel exonic and intronic mutations with their possible implication for the disease. Most patients have more than two related variants, which may be due to high consanguinity. This study should be completed by the transcriptional study for the novel intronic and silent mutations and all of the patients examined should be screened for all exons and for other genes for negative patients.

Acknowledgement

This article has been financially supported by the Ministry of Higher Education and Scientific Research of Tunisia (LR 05 SP 04).

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