ORIGINAL ARTICLE—LIVER, PANCREAS, AND BILIARY TRACT
Cryptogenic cholestasis in young and adults: ATP8B1, ABCB11,ABCB4, and TJP2 gene variants analysis by high-throughputsequencing
Giovanni Vitale1 • Stefano Gitto1 • Francesco Raimondi3,4 • Alessandro Mattiaccio2 •
Vilma Mantovani2 • Ranka Vukotic1 • Antonietta D’Errico5 • Marco Seri1 •
Robert B. Russell3,4 • Pietro Andreone1,6
Received: 25 October 2017 / Accepted: 4 December 2017
� Japanese Society of Gastroenterology 2017
Abstract
Background Mutations in ATP-transporters ATPB81,
ABCB11, and ABCB4 are responsible for progressive
familial intrahepatic cholestasis (PFIC) 1, 2 and 3, and
recently the gene for tight junction protein-2 (TJP2) has
been linked to PFIC4.
Aim As these four genes have been poorly studied in
young people and adults, we investigated them in this
context here.
Methods In patients with cryptogenic cholestasis, we
analyzed the presence of mutations by high-throughput
sequencing. Bioinformatics analyses were performed for
mechanistic and functional predictions of their conse-
quences on biomolecular interaction interfaces.
Results Of 108 patients, 48 whose cause of cholestasis was
not established were submitted to molecular analysis.
Pathogenic/likely pathogenic mutations were found in ten
(21%) probands for 13 mutations: two in ATP8B1, six in
ABCB11, two in ABCB4, three in TJP2. We also identified
seven variants of uncertain significance: two in ATP8B1,
one in ABCB11, two in ABCB4 and two in TJP2. Finally,
we identified 11 benign/likely benign variants. Patients
with pathogenic/likely pathogenic mutations had higher
levels of liver stiffness (measured by FibroScan�) and bile
acids, as well as higher rates of cholestatic histological
features, compared to the patients without at least likely
pathogenic mutations. The multivariate analysis showed
that itching was the only independent factor associated
with disease-causing mutations (OR 5.801, 95% CI
1.244–27.060, p = 0.025).
Conclusions Mutations in the genes responsible for PFIC
may be involved in both young and adults with cryptogenic
cholestasis in a considerable number of cases, including in
heterozygous status. Diagnosis should always be suspected,
particularly in the presence of itching.
Keywords Progressive familial intrahepatic cholestasis �Cryptogenic disease � Pathogenic mutations � Geneticvariants � Bioinformatics analysis
Abbreviations
PFIC Progressive familial intrahepatic cholestasis
TJP2 Tight junction protein-2
FIC1 Familial intrahepatic cholestasis 1
BSEP Bile salt export pump
MDR Multidrug resistance P-glycoprotein 3
Electronic supplementary material The online version of thisarticle (https://doi.org/10.1007/s00535-017-1423-1) contains supple-mentary material, which is available to authorized users.
& Pietro [email protected]
1 Department of Medical and Surgical Sciences, University of
Bologna, Bologna, Italy
2 Center for Applied Biomedical Research (CRBA), University
Hospital, Bologna, Italy
3 CellNetworks, Bioquant, Heidelberg University, Im
Neuenheimer Feld 267, 69120 Heidelberg, Germany
4 Bioochemie Zentrum Heidelberg (BZH), Heidelberg
University, Im Neuenheimer Feld 328, 69120 Heidelberg,
Germany
5 Addari Institute of Oncology and Transplant Pathology,
Policlinico S. Orsola-Malpighi, University of Bologna,
Bologna, Italy
6 Department of Medical and Surgical Sciences and Research
Center for the Study of Hepatitis, University of Bologna,
Italy, Via Massarenti 9, 40138 Bologna, Italy
123
J Gastroenterol
https://doi.org/10.1007/s00535-017-1423-1
http://orcid.org/0000-0002-4794-9809https://doi.org/10.1007/s00535-017-1423-1http://crossmark.crossref.org/dialog/?doi=10.1007/s00535-017-1423-1&domain=pdfhttp://crossmark.crossref.org/dialog/?doi=10.1007/s00535-017-1423-1&domain=pdfhttps://doi.org/10.1007/s00535-017-1423-1
GGT Gamma-glutamyl-transpeptidase
AP Alkaline phosphatase
BRIC Benign intrahepatic cholestasis
LPAC Low-phospholipid-associated cholelithiasis
ICP Intrahepatic cholestasis of pregnancy
DIC Drug-induced cholestasis
HTS High-throughput sequencing
NGS Next-generation sequencing
PSC Primary sclerosing cholangitis
BA Bile acids
MAF Minor allele frequency
SIFT Sorting Intolerant From Tolerant
HGMD Human Gene Mutation Database
ACMG American College of Medical Genetics and
Genomics
P Pathogenic
LP Likely pathogenic
VUS Variants of uncertain significance
LB Likely benign
B Benign
SD Standard deviation
CI Confidence interval
SNP Single-nucleotide polymorphism
ALT Alanine aminotransferase
OR Odds ratio
Introduction
Progressive familial intrahepatic cholestasis (PFIC) is a
group of autosomal recessive cholestatic diseases that
affects especially newborns and children, and represents a
consolidated indication for liver transplantation [1]. These
disorders are rare and unavoidably progressive to liver
cirrhosis and portal hypertension. However, their incidence
is hard to establish because of difficulties in diagnosis [1].
Mutations in four genes have been linked to PFIC. For
instance, mutations in ATP8B1, which encodes the
aminophospholipid flippase familial intrahepatic cholesta-
sis-1 protein (FIC1), are responsible for PFIC1 [2]. Muta-
tions in ABCB11, encoding a protein that functions as a bile
salt export pump (BSEP), are responsible for the PFIC2 [3].
Mutations in ABCB4, which codes for the multidrug
resistance P-glycoprotein 3 (MDR3), a flippase that medi-
ates the outflow of phosphatidylcholine into the bile, are
linked to PFIC3 [4]. Here, levels of gamma-glutamyl-
transferase (GGT) are always increased [5], in contrast to
very low values that characterize PFIC1 and PFIC2, in
which high levels of alkaline phosphatase (AP) are instead
present. Mutations in a fourth gene, TJP2 (coding for tight
junction protein-2) were linked to intrahepatic cholestasis
with low GGT (PFIC4) [6].
Variants in the same genes cause several other liver
diseases. Benign intrahepatic cholestasis (BRIC) is linked
to mutations in ATP8B1 and ABCB11, characterized by
intermittent cholestasis without progression to cirrhosis.
Low-phospholipid-associated cholelithiasis (LPAC) is
related to mutations in ABCB4 with symptomatic intra-
hepatic lithiasis. Intrahepatic cholestasis of pregnancy
(ICP) is a reversible pregnancy-specific cholestasis char-
acterized by pruritus, elevated liver enzymes, and increased
serum bile acids involving mutations in ATP8B1, ABCB11,
and ABCB4. Mutations in ABCB11 and ABCB4 are asso-
ciated with drug-induced cholestasis (DIC), a disorder
induced by certain drugs. These liver diseases are often
associated to heterozygous mutations [7, 8].
Only heterozygous status for ABCB4 mutations has been
well studied in children with cholestatic liver disease
[9, 10] while Dröge and colleagues [11] recently revealed a
high number of different genetic variants by sequencing
FIC1, BSEP, and MDR3 in a large cohort of patients with
supposed genetic cholestasis.
To date, no authors have investigated the four genes
linked to FIC in young persons and adults, with and
without progressive forms of liver failure.
High-throughput sequencing (HTS), including next-
generation sequencing (NGS), is a technology proposed for
the molecular diagnosis of PFIC, based on the massive
parallel sequencing of specific genomic loci, whole exome
or genomes. Compared to classic Sanger, NGS allows rapid
sequencing with more information at lower costs
[6, 12, 13].
We aimed to develop a targeted NGS panel to investi-
gate the presence of mutations in ATP8B11, ABCB11,
ABCB4, and TJP2 in a population with cryptogenic
cholestasis and related them to the corresponding pheno-
types and risk factors.
Materials and methods
Patients
From May 2013 to November 2016, all outpatients with
cholestatic disease aged[ 6 years were enrolled consecu-tively at the Department of Medical and Surgical Sciences,
a tertiary Italian referral center. We excluded other causes
of cholestasis as follows; primary biliary cholangitis, pri-
mary sclerosing cholangitis (PSC), overlap syndromes,
IgG4-cholangiopathy and obstructive jaundice were
excluded by demonstration of a normal anatomy of the
biliary tree and negative specific serological tests. Viral
hepatitis, alcohol abuse, hemochromatosis, Wilson disease,
J Gastroenterol
123
alfa1-antitrypsin deficiency were excluded too. Chronic
idiopathic cholestasis was defined as GGT e/o AP persis-
tently C 1.5-fold the upper normal values in at least two
tests or as history of itching combined with elevated serum
bile acids (BA) concentration ([ 10 lmol/l) for more than6 months. This study was conducted in accordance with
ethical guidelines of the World Medical Association’s
Declaration of Helsinki and patients or their legal guar-
dians provided written informed consent. Subjects affected
by cryptogenic cholestasis underwent laboratory analysis,
including BA serum concentration, liver fibrosis evaluation
by transient elastography (FibroScan�) and, if indicated,
liver biopsy within 6 months from the execution of the
genetic tests. Histological bile duct alteration was defined
as lobular cholestasis (ductal hepatocyte metaplasia, ductal
proliferation and immunohistochemistry for bile duct
cytokeratin 7, anti-BSEP, and anti-MDR3 antibodies). An
expert senior pathologist performed the liver histology.
Clinical variables considered were history of DIC or
itching, family history of cholestasis, personal or family
history of ICP, neonatal jaundice, juvenile cholelithiasis
(defined as history of gallstones\ 40 years).We compared patients with disease-causing variants
with the remaining population with idiopathic cholestasis.
NGS analysis
We extracted DNA from peripheral blood using the Max-
well 16 blood DNA purification kit (Promega, Madison,
WI, USA). Comprehensive molecular analysis of ABCB11,
ATP8B1, ABCB4, and TJP2 genes was performed by
multiplex targeted amplicon-based sequencing approach
using the Ion Torrent technology (Thermo Fisher Scien-
tific, Waltham, MA, USA). Primers were designed by Ion
AmpliSeqTM Designer tool to cover the entire coding
region of the four genes plus 50 bp of intronic flanking
regions. Target coverage of 98.9% was obtained through
194 amplicons for 42 kb. Libraries were performed by
AmpliseqTM Library Kit 2.0 and concentrations were
evaluated by the Ion Library TaqManTM quantitation kit
using real-time PCR. Emulsion-PCR was performed by Ion
PGMTM Hi-Q OT2 kit and the enrichment was realized by
Ion OneTouchTM ES. NGS was performed by the Ion
Torrent PGMTM System according to the manufacturer’s
procedures (Life Technologies, CA, USA). Molecular
analysis was simultaneously carried out in 12 barcoded
samples in an Ion 318 Chip v2 for each run.
The read files obtained from sequencing were mapped to
the GRCh37/hg19 assembly and the sequence variants
were identified by Variant Caller and Ion Reporter
software.
Allelic variants were reported according to Human
Genome Variation Society guidelines (http://www.hgvs.
org/content/guidelines). NGS uncovered regions as well as
the potential pathogenic variants were confirmed by Sanger
sequencing using ABI PRISM 3730 Genetic Analyzer
(Thermo Fisher Scientific, Waltham, MA, USA).
Prediction of functional consequences of variants
and classification
The filtering step of sequence variants was carried out
according to default criteria for germline mutations:
insertions or deletions, non-sense variants, splicing-site
variants, and missense with minor allele frequency
(MAF) B 0.05 were considered. Three bioinformatics tools
were used to predict the role of missense variants. Sorting
Intolerant From Tolerant (SIFT) (http://www.jcvi.org/cms/
home/), PolyPhen-2 [14] and Mutation Taster [15] software
can evaluate whether an amino-acid substitution influences
protein structure and function, according to physical
modifications and the degree of conservation of protein
sequence among species. Variants that meet at least one of
the following criteria were also considered: ever described
before, reported in Human Gene Mutation Database
(HGMD), predicted not benign by at least one of the
bioinformatic tools. According to American College of
Medical Genetics and Genomics (ACMG) standards [16],
we classified the filtered variants into five categories:
pathogenic (P); likely pathogenic (LP); variants of uncer-
tain significance (VUS); likely benign (LB); and benign
(B).
Variants passing the above filtering process were map-
ped to Uniprot canonical sequences and subjected to
Mechismo [17] analysis to predict their functional conse-
quences at biomolecular interaction interfaces. The
approach matches protein sequence amino-acids to posi-
tions within structures and identifies sites affecting inter-
actions with other proteins, DNA/RNA or small molecules.
We considered low confidence predictions including
known structures or close (C 30% sequence identity)
homologs and only very confident, physical protein–pro-
tein interactions (as defined by Mechismo based on a
benchmark for the accuracy of perturbed interfaces). In
case of ATP8B1, no homolog template structure was
available in Mechismo, so we used a homology model rom
ModBase [18].
We annotated mutations and PTMs (phosphorylations
and acetylations) from Phosphosite [19] of the considered
genes on corresponding Uniprot canonical sequences using
lollipop diagrams [20].
Comparison with an international database
Allele frequency (AF) of common single-nucleotide poly-
morphism (SNPs) was matched with data reported in the
J Gastroenterol
123
http://www.hgvs.org/content/guidelineshttp://www.hgvs.org/content/guidelineshttp://www.jcvi.org/cms/home/http://www.jcvi.org/cms/home/
international Genome Aggregation Database (gnomAD),
Cambridge, MA (http://gnomad.broadinstitute.org/;37)
[allele frequencies accessed October 2017]. We compared
AF of SNPs in our cohort (with and without P/LP mutation)
to AFs of European (non-Finnish), East Asian, and
worldwide population.
Statistical analysis
Categorical variables are expressed as number (%), and
quantitative variables as mean ± standard deviation or as
median (range). Chi-square or Fisher’s exact test was used
to compare categorical variables, while for quantitative
variables the t test or Mann–Whitney’s test (unpaired data)
or the t test or Wilcoxon’s test (paired data) were used.
Binary logistic regression was performed for univariate and
multivariate analyses to identify predictors of causative
variants (variables were included if p\ 0.1 and removed ifp C 0.05). A p\ 0.05 was considered significant for alltests. The statistical software SPSS version 21.0 (�SPSS
Inc., Chicago, IL, USA) was used for statistical analyses.
Results
Patient characteristics
We evaluated consecutively 108 cholestatic patients and 48
fulfilled the established criteria (Supplementary Fig. 1).
We listed the main laboratory patterns in Table 1. There
was a slight bias for males (58%) and mean age at time of
the genetic test was 42 years; the adult population
(C 18 years) was 93.8%. A history of familial cholestatic
diseases and DIC was present in 35%, itching in 27%,
neonatal jaundice in 21%, juvenile cholelithiasis in 17%,
personal or family history of ICP in 13%. Histologic fea-
tures of cholestasis were present in 65% of cases (overall
44 patients agreed to a liver biopsy). Gene analysis
revealed the presence of P/LP mutations in about one-
fourth of subjects; clinical features and type of mutations
found in the 11 patients (ten unrelated probands and one
affected sister) were reported in Table 2.
NGS results and variants
NGS protocol provided an average sequencing depth
of * 10009, 98.78% of reads having 209 coverage, with99% reads on target and a coverage uniformity of 95.98%.
Thirty-one variants that satisfied our filtering criteria are
reported in Supplementary Table 1. Among these, 13 were
P/LP mutations, five were previously undescribed: three
were missense, one was a frameshift, and one was a non-
sense mutation.
Among P/LP variants, two were in ATP8B1, six in
ABCB11, two in ABCB4 and three in TJP2. We identified
seven VUS, two in each of ATP8B1, ABCB4 and TJP2, one
in ABCB11. Finally, we recorded 11 benign mutations, two
in each of ATP8B1, ABCB11 and ABCB4, five in TJP2.
ATP8B1 variants
Six variants were identified in the coding region of
ATP8B1, reported below; according to ACMG standards,
two LP: c.3655G[C (p.D1219H) and c.68C[T (p.P23L);the first was combined with heterozygous LP mutation
c.1057C[T on TJP2 gene in a 71-year-old woman (case 1).The second is a novel mutation found in a 31-year-old male
(case 2) who experienced neonatal jaundice and juvenile
cholelithiasis, with high GGT and bilirubin levels. We
classified the missense c.134A[C (p.N45T) and c.607A[G(p.K203E) as VUS and each was previously described as
risk allele in ICP [21]. We found these variants in com-
pound heterozygous in a 36-year-old female who showed a
Table 1 Baseline features of the entire study population
N 48
Male N (%) 28 (58.3)
Adult population C 18 years N (%) 45 (93.8)
Age (years, mean ± SD)
At time of genetic test 42 ± 15
At presentation 32 ± 15
Risk factor for cholestasis N (%)
DIC history 17 (35.4)
Neonatal jaundice 10 (20.8)
Itching history 13 (27.1)
ICP history 6 (12.5)
Juvenile cholelithiasis 8 (16.7)
Familiarity 17 (35.4)
Laboratory (median, range)
GGT (UI/l) 139 (5–597)
FA (UI/l) 283 (122–1012)
ALT (UI/l) 45 (9–387)
Bilirubin (mg/dl) 0.7 (0.4–11.2)
Bile acids (lmol/l) 11 (2.3–403)
Cholesterol (mg/dl) 211 (87–328)
Albumin (g/dl) 4.3 (2.9–5.2) 4.3 (2.9–5.2)
Platelets (n 9 103/ll) 229 (61–417)
FibroScan (kPa) (median, range) 5.3 (3.1–35.8)
Histologic features of cholestasis N (%) 31 (64.6)
N number, SD standard deviation, DIC drug-induced cholestasis, ICP
intrahepatic cholestasis of pregnancy, GGT gamma-glutamyltrans-
ferase, AP alkaline phosphatase, ALT alanine transaminases, kPa
kiloPascal
J Gastroenterol
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Table 2 Baseline features of the population with likely pathogenic (LP) and pathogenic (P) mutations
ID Sex LP and P mutations Additional variants Age
(years)
GGT
(U(L)
PA
(U/l)
Bilirubin
(mg/dl)
FibroScan
(kPa)
Notes
1_17421 F ATP8B1:
p.[D1219H]; [=]
TJP2:
p.[R322W]; [=]
ATP8B1:
p.[R952Q]; [=]
ABCB11:
p.[V444A(;)M677V]
TJP2:
p.[M668I]; [=]
71 121 394 1.2 5.5 No affected relatives
Segregation not
assessed
2_15710 M ATP8B1:
p.[P23L]; [=]
ABCB11:
p.[V444A(;)M677V]
31 325 138 3.3 7.4 Familiarity
Juvenile
cholelithiasis
Neonatal jaundice
The mutation was
inherited from the
mother and
segregates in both
brothers and in a
sister
3_16545 F ABCB11:
p.[Y93S(;)V597L(;)R1128]
ABCB11:
p.[V444A]; [=]
ABCB4:
p.[I237=]; [=]
29 5 466 1.2 12 DIC
Neonatal jaundice
Itching
Juvenile
cholelithiasis
ICP
Segregation not
assessed
4_15502 F ABCB11:
p.[A523G]; [A523G]
ABCB11:
p.[V444A];
[V444A]
ATP8B1:
p.[R952Q]; [=]
ABCB4:
p.[I237=]; [=]
20 15 672 11.2 20.9 Familiarity
DIC
Neonatal jaundice
itching
The mutations are
inherited from
healthy parents
4a_15505 F ABCB11:
p.[A523G]; [A523G]
ABCB11:
p.[V444A];
[V444A]
ABCB4:
p.[I237=]; [=]
7 13 1012 3.5 10.1 Familiarity
DIC
Neonatal jaundice
itching
Affected sister of
case 4
The mutations
segregate in both
affected sisters
5_15507 M ABCB11:
p.[E135K]; [S1100Qfs*38]
ABCB11:
p.[V444A];
[V444A]
16 8 467 2.2 11.5 Neonatal jaundice
Itching
The mutations are
inherited from
healthy parents
6_17248 F ABCB4:
p.[K672*]; [=]
ABCB11:
p.[V444A];
[V444A]
ABCB4:
p.[I237=]; [=] TJP2:
p.[T1124=]; [=]
39 363 562 0.4 7.7 Familiarity
DIC
Itching
The mutation was
inherited from the
affected father
J Gastroenterol
123
cholestatic disease characterized by normal GGT and high
BA levels, neonatal jaundice, juvenile cholelithiasis com-
plicated by LPAC, and a history of ICP.
We found two B/LB variants: c.913T[A (p.F305I) andc.2855G[A (p.R952Q). The SNP p.R952Q was found infive cases and in two patients was combined with LP
mutations, one on ABCB11 and one on TJP2, respectively.
AF of p.R952Q was significantly higher in comparison
with AF of East Asian population (10.4 vs. 0.03%:
p\ 0.0001) and comparable with European and worldwidedata (Table 3).
Analysis of variants in the context of sequence and
structural annotations on structures or homology models
revealed that two of them, i.e., p.K203E and p.F305I are
found in spatial proximity within the ATPase catalytic
domain (Fig. 1a), suggesting potential converging effect on
the enzymatic activity, while the p.R952Q is found at the
transmembrane (i.e., phospholipid translocating) domain.
The LP variants, p.P23L and p.D1219H, reside at the N-
and C-terminals of the protein, outside the structured por-
tion (Fig. 1b).
ABCB11 variations
Nine variants were identified in the coding region of
ABCB11 and six resulted in P/LP mutations: three causa-
tive mutations, c.278A[C (p.Y93S), c.1789G[C
(p.V597L) and c.3382C[T (R1128C) were combined in ayoung female (29 years) presenting with a history of
neonatal jaundice, DIC, ICP, juvenile cholelithiasis, itch-
ing, normal GGT levels (case 3). Two sisters (20 and
7 years) were carriers of the LP homozygous mutation
c.1568C[G (p.A523G): both had history of neonataljaundice, itching and DIC, (cases 4–4a). Last two P/LP
mutations, c.3297delC (p.S1100Qfs*38) and c.403G[A(p.E135K), were responsible for a double heterozygosis in
a boy with history of itching and neonatal jaundice, listed
for liver transplantation (case 5); c.3297delC was recently
described [22] while c.403G[A mutation was linked byAnzivino et al. to ICP conditions [23]. The VUS
c.1268A[G (p.H423R) was found in a 43-year-old womanwho presented only high GGT without fibrosis.
Benign mutations detected were among those previously
described [7, 11, 24]: c.2029A[G (p.M677V), detected innine patients, and c.1331T[C (p.V444A), found in 40cases (83.3% of population studied). Of note, SNP
c.1331T[C was reported associated to DIC and ICP inprevious reports [7, 24] and recently to cholestatic phe-
notype in patients without disease-causing mutations in the
respective gene [11]. AF of p.V444A was significantly
more frequent in our cohort of patients without P/LP
mutations in comparison with European and worldwide
populations (81 vs. 60%: p = 0.008; 81 vs. 57%:
p = 0.002). We also observed a higher AF of p.M677V in
Table 2 continued
ID Sex LP and P mutations Additional variants Age
(years)
GGT
(U(L)
PA
(U/l)
Bilirubin
(mg/dl)
FibroScan
(kPa)
Notes
7_15873 M ABCB4:
p.[A364V]; [=]
ABCB4:
p.[I237=]; [=] TJP2
gene:
p.[Q128K(;)M668I]
57 379 606 0.4 35.8 DIC
HCC
No affected relatives.
Segregation not
assessed
8_17362 F TJP2:
p.[T62M]; [=]
ABCB11:
p.[V444A(;)M677V]
ABCB4:
p.[I237=]; [I237=]
51 84 205 0.6 4.4 DIC
ICP
No affected relatives.
Segregation not
assessed
9_17374 M TJP2:
p.[T62M]; [=]
ABCB11:
p.[V444A(;)M677V]
50 130 175 0.6 3.3 No affected relatives.
Segregation not
assessed
10_16643 M TJP2:
p.[I875T]; [=]
ABCB11:
p.[V444A];
[V444A]
ABCB4:
p.[T775M]; [=]
TJP2:
p.[R12H]; [R12H]
37 217 399 0.6 8.1 Familiarity
Both mutations
segregate in an
affected brother
with history of high
GGT
ID 4 and 4a are affected sibling. Sequence variants are reported according to HGVS recommendations (http://varnomen.hgvs.org)
J Gastroenterol
123
http://varnomen.hgvs.org
our cohort with and without P/LP mutations respect to all
other populations (13.5 vs. 1.76% European, 0% East
Asian, 2.7% worldwide: all p\ 0.0001) (Table 3).All nine reported mutations for ABCB11 were mapped
to homolog structures through Mechismo (Fig. 1a). P/LP
variants were found to be located either at ATP-binding
domain, like p.A523G, p.V597L, p.S1100fs and p.R1128C,
or at transmembrane domain, like p.E135K or p.Y93S,
which are also found in spatial proximity (Fig. 1b).
Moreover, the variant p.H423R was found at the interface
with the ATP-binding pocket (Supplementary Table 2).
This overall suggests that deleterious mutations on
ABCB11 might similarly affect the ATP-binding cassette
(ABC) transporter’s functionality by impairing either ABC
catalytic activity, which couples ATP-hydrolysis to
transport, or by perturbing transmembrane region, which
mediates ligands transport to the extracellular side [25].
ABCB4 variations
We identified six variants in the coding region of ABCB4
and two were P/LP mutations. Disease-causing variants
were: c.2014A[T (p.Lys672*), previously undescribed,was found in a 39-year-old woman with familiarity for
cholestatic diseases (mutation was inherited from the
affected father with high GGT), history of DIC and itching
(case 6) while c.1091C[T (p.A364V), described byDegiorgio et al. [26], was present in a 57-year-old cirrhotic
patient with DIC and hepatocellular carcinoma (case 7).
Two VUS, p.L73V (c.217C[G) and c.2324C[T
Table 3 Allele frequencies of detected SNPs
Gene Mutation Protein dbSNPs AF full
cohort
(a) (%)
AF Pts w/o
P/LP
mutations
(b) (%)
AF
EU
(1)
(%)
AF East
Asia (2)
(%)
AF
world
(3) (%)
p values f
ATP8B1 c.2855G[A p.R952Q rs12968116 10.4 10.8 12 0.03 8.3 a-1 = 0.9151 b-1 = 0.8297a-2 < 0.0001 b-2 < 0.0001
a-3 = 0.5888 b-3 = 0.5744
ABCB11 c.1331T[C p.V444A rs2287622 83 81 60 73 57 a-1 < 0.0001 b-1 = 0.008a-2 = 0.1329 b-2 = 0.3318
a-3 =
E135K Y93S
A523GH423RV444A V597L
M677V R1128C
S1100Qfs
Pholip_ATPase
ATP8B1 ABCB4ABCB11
cytosol
extracellular
ABC_tran
PDZ
PDZ
PDZ
SH3_2Guanylate_kin
TJP2
ATP8B1P23
LN45
TK20
3EF30
5IR95
2Q D1219H
PhoLip_ATPase E1-E2 ATPase Cation_ATPase Phospholipid-translocating P-type ATPase C-terminal
ABCB11Y93
SE13
5KH42
3R V444A
A523G
V597L
M677V
S1100Q
fs*38R11
28C
ABC_membrane ABC_tran ABC_membrane ABC_tran
0 93 135 371 423 523 586 677 755 858 1029 1100 12141247 1321
ABCB4L73
V A36
4V R652G L67
2*
T775M
ABC_membrane ABC_tran ABC_membrane ABC_tran
0 73 121 237 364 424 524 561 624 672 711 775 985 1052 1210 1286
TJP2
V3LR12H
T62M Q12
8KR32
2W
M668I
I875T
PDZ PDZ PDZ SH3_2 Guanylate_kin
3 33 62 91 319 668 875 1190
2338 66 145 172 203 305 413 440 476 532 632 658667 779789 902924952 992 11731203 12170
R4E
(L)B or VUS(L)PPhosphorylation siteAcetylation site
R952Q
K203E
F305IE1_E2_ATPase
(P23L)(N45T)
(D1219H)
ABC_membraneL73V
A364V
R652G
T775M
(K672*)
T62M
Q128K
R322W M668I
I875T
A256V
(V3L)(R4E)(R12H)
(A256V)
(a)
(b)
J Gastroenterol
123
(p.T775M), were identified, previously associated to dif-
ferent PFIC3 phenotypes [10, 26].
The missense variant p.T775M was detected together
with LP c.2717T[C (p.I906T) in exon 18 of TJP2 in a37-year-old male presenting a familiarity for cholestasis
(case 3), while the variant p.L73V was detected in a
23-year-old female with high GGT levels, without evi-
dence of significant liver fibrosis at FibroScan�. The
known LB c.711A[T (p.I237=) [11, 27] and c.1954A[G(p.R652G) [11, 28] mutations were found in 16 and four
cases, respectively. The SNP p.I237= was associated in
previous reports with elevated GGT-cholestasis, ICP, and
gallstones [11].
AF of p.I237= was significantly more common in our
cohort of patients with disease-causing mutations than in
European and worldwide population (33 vs. 18%:
p = 0.004; 33 vs. 21%: p = 0.033; Table 3).
AF of p.R652G was similar between our cohort and both
European and worldwide population while it appeared to
be more common in East Asian subgroup (10.8 vs. 27%;
p = 0.00106).
Similarly to ABCB11, ABCB4 non-synonymous muta-
tions were mapped to homolog structures with exception of
the stop-gain p.L672*. Mutations were essentially located
in transmembrane region (Fig. 1a), suggesting a perturba-
tion of ligand transport mechanisms. The stop-gain
p.L672*, located in the middle of the gene (Fig. 1b), would
result in a halved transcript and protein sequence, lacking
one ATP-binding cassette and one transmembrane domain,
further stressing its high severity.
TJP2 variations
Variants identified in the coding region of TJP2 were ten:
three LP mutations, two VUS, and five B/lB variants.
The LP mutations were: c.278C[T (p.T62M) in twocases (cases 8 and 9), one who experienced DIC and ICP,
c.1057C[T (p.R322W), combined to LP heterozygousp.D1219H on ATP8B1 in a 71-year-old man presenting
only high GGT (case 1), and c.2717T[C (p.I875T) asso-ciated with VUS p.T775M in ABCB4 in one patient with
familiarity for cholestatic diseases (case 10).
Differently from what has been reported in a previous
report [6], all three patients presented high GGT. Two VUS
were c.43G[C (p.V3L) and c.46A[G (p.R4E) while B/LBmutations were c.71G[A (p.R12H), c.475C[A (p.Q128K),c.860C[T (p.A256V), c.2097G[A (p.M668I), andc.3465G[AT (p.T1124=).
VUS p.R4E was present in a 31-year-old female asso-
ciated to benign C1954A[G and C711A[T mutations inABCB4: the patient had a history of ICP, juvenile
cholelithiasis and recurrent DIC to different drugs used for
multiple sclerosis. Finally, a 31-year-old man with high
GGT and BA levels presented another VUS p.V3L.
SNP p.M668I was significantly more frequent in our
cohort of patients with disease-causing mutation compared
to European, East Asian, and worldwide population (14.6
vs. 6.3%: p = 0.0385, 0.03%: p\ 0.001, and 5.3%:p = 0.0043) (Table 3).
Most deleterious TJP2 mutations were found at the level
of PDZ, SH3, and guanylate kinase domains (Fig. 1a, b).
This suggests a potential perturbation of TJP2’s function,
i.e., organizing tight and adherent junctions by binding to
the cytoplasmic C termini of junctional transmembrane
proteins and linking them to the actin cytoskeleton [29].
Indeed, one variant (p.R322W) is predicted to affect the
homo- and hetero-dimerization interface with TJP1
(Fig. 2).
Summary mutation profile for FIC patients
Excluding the common SNP V444A on ABCB11, (present
in 83.3% of cases) and I237= on ABCB4 (33%), 26/48
patients (58.3%), have at least one mutation in one or more
genes: eight patients had only one mutated allele while 18
patients had two or more mutated alleles. ABCB11 and
TJP2 resulted most affected genes and they had mutual
exclusive but non-significant tendency. In particular,
ABCB11/M677V, TJP2/M668I, TJP2/Q128K, and TJP2/
R12H had a tendency to affect patients in a mutual
exclusive way between each other (but in combination,
within the same patient, with various mutations). Figure 3
summarizes mutations discovered contemporary, showing
allele variants in the four genes, according to pathogenicity
prediction (P/LP vs. B, LB, and VUS mutations), zygosity,
and the clinical data (age, FibroScan�, GGT, neonatal
jaundice, itching history, bile acids and cholestasis at
histology).
It is possible that patients with multiple alleles mutated
have more severe phenotypes by a synergistic effect at
different bile transporters’ sites as predicted by Mechismo.
However, patients having C 2 non-synonymous mutations
(with at least one predicted P/LP), tend to have higher liver
stiffness comparing to patients with\ 2 non-synonymousmutations (kPa, 7.9 [3.3–35.8] vs. 4.9 [3.1–29.9],
bFig. 1 a Protein sequence annotations of FIC-selected non-synony-mous mutations displayed as pink and red lollipops to indicate
respectively B/LB or VUS and P/LP predicted consequences. Circle
diameters are proportional to the number of patients affected. Blue
and green lollipops indicate phosphorylation and acetylation sites.
Protein sequence regions corresponding to conserved domains are
highlighted by colored boxes and are indicated by their respective
Pfam names; b non-synonymous mutation annotations on available3D structures as assessed through Mechismo, are displayed following
the same coloring scheme as in a. Labels in parenthesis indicatemutations with no structural information
J Gastroenterol
123
p = 0.028), indicating a potential role of these variants as
disease modifiers.
Subanalysis of patients with P and LP mutations vs.
remaining cholestatic population
Significant differences between subjects with and without
P/LP mutations were not observed in sex, age at symptoms
presentation, and the following risk factors for PFIC: DIC
history, family or personal history of ICP, juvenile
cholelithiasis and family history for cholestatic diseases
(Table 4). However, at least one of these risk factors was
present in 9/11 patients with P/LP mutations (Table 2).
Patients with P/LP mutations had more frequently neonatal
jaundice (45.5 vs. 13.5%, p = 0.036) and itching (54.5 vs.
18.9%, p = 0.029).
Regarding laboratory tests, significant differences were
observed between the two subgroups only in terms of BA
concentration (23.8 [4.4–403] vs. 8.8 [2.3–114],
p = 0.003).
Liver stiffness was greater in subjects with P/LP muta-
tions in comparison with the others (8.1 [3.3–35.8] vs. 4.8
[3.1–29.9], p = 0.009). All subjects of the first group
showed histological features of intrahepatic cholestasis
while 72% of patients without P/LP mutations presented it
(p = 0.018). The univariate analysis for predictors of P/LP
mutations indicated that AP (odds ratio [OR] 0.995, 95%
CI 0.990–0.999, p = 0.019), liver stiffness (OR 0.921,
95% CI 0.841–1.010, p = 0.081), itching (OR 5.143, 95%
CI 1.214–21.795, p = 0.026), neonatal jaundice (OR
5.330, 95% CI (1.172–24.277, p = 0.030) showed a
p\ 0.1 (Supplementary Table 3). Multivariate analysis(Supplementary Table 3) showed that only itching was an
independent predictor of P/LP mutations in patients with
cryptogenic cholestasis (OR 5.801, 95% CI 1.244–27.060,
p = 0.025).
Fig. 2 a Prediction of the functional consequences of TJP2’sp.R322W variant through Mechismo. Green and orange arrows,
respectively, indicate an enabling and mixed (i.e., both enabling and
disabling) effect of the p.R322W mutation towards interactors.
b Structural details of the TJP2 dimerization interface predicted to beaffected by the p.R322W mutation
*(L)P(L)B or VUSHM
Age at testFibroscan
High GGT Yes No
7 71
3.1 35.8
Patient ID
NA NA NA
Bile acidsNeonatal jaundice
Itching
Cholestasis at histology
2.3 403
Yes No
Yes No
Yes No
Fig. 3 Bi-dimensional representation showing genes affected by non-synonymous mutations (row) in each patient (column). Mutated genes
are sorted according to their mutation rate and each mutation is
colored according to pathogenicity prediction (P/LP in red, B/LB and
VUS in pink). Homozygous mutations are indicated by an asterisk.
For each patient, the following clinical data are also displayed: age of
patient, FibroScan� values, GGT levels, neonatal jaundice, itching
history, bile acids and cholestasis at histology
J Gastroenterol
123
Discussion
ABCB11 and ABCB4 are ATP-binding cassette proteins
and members of MDR/TAP subfamily. They are membrane
proteins, with a long intracellular domain, which makes
them partly similar to ATP8B1, though it shares no close
homology with either of them. These three genes are pre-
dicted, but equivalents in non-human species, to interact
both physically and functionally [30]. Specifically,
ATP8B1 homolog in Drosophila MRP has been seen to
interact with ABCB11/B4 homolog CG31729 and yeast
equivalents YOR1 and DNF3 are genetic interactors. The
majority of mutations described in these three genes lie in
or near the intracellular domains in these three proteins.
TJP2, while lacking trans-membrane domains is never-
theless a peripheral membrane protein attached (when not
in the nucleus) via lipid head groups to the cytoplasmic
side of the membrane. Functionally, the formation of tight-
junctions is thought to be related to separation of bile from
plasma (which in itself is intricately linked to their trans-
port) [31]. It is possible that some or all of these proteins
form a complex at some point during the production or
transport of liver metabolites.
PFIC are considered pediatric diseases related to liver
failure. Mutations in ATP8B1, ABCB11, ABCB4 and TJP2
have been associated to a plethora of cholestatic disorders;
hepatocellular carcinoma and cholangiocarcinoma (to
ABCB11 and TJP2), ICP (linked to ATP8B1, ABCB11 and
ABCB4), LPAC (to ABCB4), DIC (to ABCB11 and ABCB4)
and BRIC (to ATP8B1 and ABCB11), are related and may
coexist in the same patient. Furthermore, all four genes may
be responsible for progressive forms of cholestasis, tradi-
tionally considered exclusive of childhood [5, 6, 32, 33].
Of interest, we found a mean age of 37 years in subjects
with disease-causing variants to confirm that P/LP muta-
tions are not present only in children. Few studies linked
mutations in PFIC genes with non-progressive diseases,
especially in heterozygous subjects. In our cohort of
patients with at least one disease-causing mutation, only
four patients were homozygous or compound heterozygous
for disease-causing variants in the same gene.
Colombo et al. [10] described pathogenic mutations in
ABCB4 in about a quarter of asymptomatic children where
cholestatic disease was incidentally discovered via liver
enzyme abnormalities and, not surprisingly, some of these
patients carried a single heterozygous mutation. Gordo-
Table 4 Main features of patients according to presence of P/LP mutations
Pts with P/LP mutations N = 11 Pts without P/LP mutations N = 37 p value
Male N/Tot N (%) 5/11 (45.5) 23/37 (62.2) 0.260
Age: (years, mean ± SD)
At time of genetic test 37 ± 19 44 ± 14 0.244
At symptoms presentation 27 ± 12 33 ± 15 0.308
Risk factor of cholestasis N/Tot N (%)
DIC history 6/11 (54.4) 11/37 (29.7) 0.126
Neonatal jaundice 5/11 (45.5) 5/37 (13.5) 0.036
Itching history 6/11 (54.5) 7/37 (18.9) 0.029
ICP history 2/11 (18.2) 4/37 (10.8) 0.420
Juvenile cholelithiasis 3/11 (27.3) 5/37 (13.5) 0.259
Family history 5/11 (45.5) 12/37 (32.4) 0.327
Laboratory, median (range)
GammaGT (UI/l) 121 (5–379) 148 (18–597) 0.425
AP (UI/l) 467 (138–1012) 280 (122–610) 0.061
ALT (UI/l) 41 (9–387) 46 (9–358) 0.608
Bilirubin (mg/dl) 1.2 (0.4–11.2) 0.7 (0.4–6.2) 0.484
Bile acids (lmol/l) 23.8 (4.4–403) 8.8 (2.3–114) 0.034
Cholesterol (mg/dl) 182 (132–303) 215 (87–328) 0.484
Albumin (g/dl) 4.3 (2.9–5.2) 4 (3.6–4.7) 4.3 (2.9–5.2) 0.126
Platelets (103/lL) 223 (128–412) 233 (61–417) 0.873
FibroScan (kPa), median (range) 8.1 (3.3–35.8) 4.8 (3.1–29.9) 0.009
Histologic features of cholestasis N/Tot N (%) 10/10 (100) 21/34 (61.8) 0.018
N number, Tot N total number, DIC drug-induced cholestasis, SD standard deviation, P/LP mutations pathogenic and likely mutations, ICP
intrahepatic cholestasis of pregnancy, AP alkaline phosphatase, ALT alanine transaminases, kPa kiloPascal
J Gastroenterol
123
Gilart et al. [9] reported similar results in another cohort of
pediatric patients in whom defects in a single allele of
ABCB4 were identified in 9/67 subjects. Some authors
suggested that cryptogenic cholestasis in adults should be
added to the spectrum of conditions associated with
ABCB4 mutations [34–36]. Furthermore, adults with PSC
had early onset of disease if MDR3 deficiency was found
[34].
In a recent study analyzing 427 cholestatic patients, 149
subjects presented at least one disease-causing variant on
ATP8B1, ABCB11 and ABCB4. Surprisingly, 25 patients
with only one heterozygous variant experienced symptoms
before first year of life, suggesting the presence of muta-
tions in other genes, epigenetic changes or environmental
factors responsible of cholestatic phenotype severity [11].
However, adult population data lack clinical significance
of heterozygosity in ABCB11, ATP8B1, and TJP2, though
the first two genes have been correlated to BRIC when only
one heterozygous variant was present. In some instances,
these diseases may evolve towards progressive forms (e.g.,
PFIC), thus representing a clinical continuum [22, 37].
Our study represents the first report of simultaneous
sequencing of ATP8B1, ABCB11, ABCB4 and TJP2 in
patients with cryptogenic cholestasis.
In our cohort, 21% of patients had at least one P/LP
mutation and 17% with a disease-causing mutation pre-
sented a liver disorder in adulthood ([ 18 years), sug-gesting once again that variants in PFIC genes are not
exclusive to cholestatic diseases in childhood.
Patients with P/LP mutations had higher liver stiffness
and BA levels than patients without disease-causing vari-
ants and all exhibited histological evidence of lobular
cholestasis, confirming a more aggressive phenotype in
subjects with causative mutations.
Many patients had multiple variants in these four genes.
It is thus tempting to hypothesize a synergistic effect in
determining different cholestasis phenotypes and/or speci-
fic interactions with environmental factors, in particular
certain drugs. A reduction of bile flow represents a
pathophysiological state and can be critical for metabolism
of many drugs. This is supported by MDR3 and BSEP
deficiencies being known to be associated to different
levels of DIC [7, 38]. Examples are SNPs V444A/M677V
in ABCB11 and I237= in ABCB4. We found V444A in
83.3% of patients (38% in homozygous state): this benign
variant was reported with AF in the general population of
56.9 and of 65.9% in cholestatic patients with no BSEP
disease-causing mutations [11]. It is associated with DIC,
ICP and reduced expression of BSEP levels in previous
studies [24, 39–41]. Specifically in DIC, patients carrying
V444A were at increased risk of drug-induced liver injury,
when taking drugs containing a carbocyclic system with
aromatic rings [42].
SNP M677V occurred in 13.5% of our cohort of cho-
lestatic patients without P/LP mutations, more significantly
frequent than in the other zones (AF in word 2.73%).
In our cohort, SNP p.I237= has an allele frequency of
25%, while Dröge et al. [11] reported 15.4% in an assumed
genetic cholestatic population. Our AFs were significantly
increased respect to AFs reported in worldwide and
European population. This ABCB4 polymorphism increa-
ses the risk of development of cholestatic disease, ICP and
LPAC in previous reports [11, 43].
Common SNPs V444A/M677V/I237= were detected in
a very high number of cases, with or without disease-
causing mutations, suggesting that these common variants
might contribute to cholestasis development or worsening.
The integration of standard variant prediction and clas-
sification tools with more advanced bioinformatic analyses,
taking into account biological pathways and/or biomolec-
ular structures, is becoming increasingly used to get a
deeper understanding on the role of genetic variation from
large NGS datasets, as we previously showed for genetic
diseases [44] and cancer [45]. This is also witnessed by
recent work by Dröge et al. [11] for a larger panel of
cholestatic liver disease patients. For example, we showed
that the common SNP p.V444A of ABCB11 is found at the
catalytic domain of the ABC transporter (Fig. 1) in close
proximity but not in direct physical contact with the ATP-
binding cassette. We thus speculate that while it is unlikely
that this variant directly perturbs the enzymatic activity of
the transporter, it might still induce subtle structural rear-
rangements on the catalytic domain leading to a slightly
perturbed, but still functional enzyme. Such a genotype
would give rise to a disease phenotype when in co-presence
with other genetic or environmental factors.
Disease-causing mutations in TJP2 are also well estab-
lished. Sambrotta et al. [6] associated them with progres-
sive forms of normal GGT cholestasis in pediatric patients,
while our four patients had heterozygous status, different
ages of onset, and high GGT levels. Our results highlight
the variability of clinical presentation in patients having
mutations in ATP8B1, ABCB11, ABCB4, and TJP2, espe-
cially when only one allele was involved. Our structural
analysis, while corroborating pathogenicity predictions by
standard bioinformatic tools, also provided novel mecha-
nistic insights on the functional consequences of these
variants. For example, the TJP2 p.R322W mutation, pre-
dicted to be pathogenic, was shown by Mechismo to affect
interfaces mediating homo- and hetero-dimers (Fig. 2),
thus suggesting to likely impact TJP2’s physiological
function.
Similarly to other studies, we found it difficult to assess
the causative role of many missense variants. We classified
the variants detected in our cohort according to ACMG
criteria, and some missense resulted as VUS. The patients
J Gastroenterol
123
showing only VUS were not included in our analyses of
mutated patients (Table 4 and Supplementary Table 1) but
some of these variants might be re-classified as likely
pathogenic when related to the clinical phenotype.
However, in our cohort of patients, the etiology of the
cholestatic disease remains elusive, with only 21%
attributable to mutations in the known causative genes.
Mutational combinations at different loci as well as the
several VUS detected in one or more FIC-genes and their
potential synergistic effect could also contribute to the
disease onset.
The main limit of our study is that we have considered
four genes responsible for intrahepatic cholestasis, which
does not exclude that other genes could potentially be
associated with this condition. Other studies proposed
multi-gene panels able to facilitate genetic diagnosis, but
only in children with intrahepatic cholestasis [13, 46, 47].
For instance, the recent discovery of new genes
responsible of FIC: myosin-5B gene, linked to microvillus
inclusion disease and NR1H4 gene, encoding farnesoid
X-receptor, a bile acid-activated nuclear hormone receptor
that regulates bile acid metabolism and suppression of bile
acid production. Both genes are able to lead to progressive
cholestatic liver disease [48–50].
Nevertheless, the highest rates of BA levels, liver
fibrosis, itching, and bile histologic alterations detected in
the patients with P/lP mutations make it possible to classify
the pathogenicity of variants in accordance with the
ACMG standards [16].
In conclusion, the remarkable number of cases with
causative mutations found in adults with cryptogenic
cholestasis confirms the usefulness of mutational screening
in ATP8B1, ABCB11, ABCB4, and TJP2, especially in
those cases with itching history, regardless of age of
cholestasis onset. Remains to be evaluated the synergistic
role of mutations at different loci and of VUS in these four
genes. Further observational studies are needed to under-
stand the long-term clinical significance of mutations in
genes responsible for FIC. Finally, the high proportion of
unsolved cases suggests novel genetic etiologies that
remain to be elucidated.
Author contributions GV and PA designed the study and collecteddata. AM, VM, and MS performed the DNA sequencing and applied
prediction tools; AD supervised the histological evaluations, FR and
RBR performed protein modeling by Mechismo; SG, AM, VM, GV,
RV, and PA analyzed the patients’ data. GV wrote the manuscript; all
authors critically revised the manuscript.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict ofinterest.
Financial support No grants and other financial support werereceived.
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Cryptogenic cholestasis in young and adults: ATP8B1, ABCB11, ABCB4, and TJP2 gene variants analysis by high-throughput sequencingAbstractBackgroundAimMethodsResultsConclusions
IntroductionMaterials and methodsPatientsNGS analysisPrediction of functional consequences of variants and classificationComparison with an international databaseStatistical analysis
ResultsPatient characteristicsNGS results and variantsATP8B1 variantsABCB11 variationsABCB4 variationsTJP2 variationsSummary mutation profile for FIC patientsSubanalysis of patients with P and LP mutations vs. remaining cholestatic population
DiscussionAuthor contributionsReferences
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