- Email: [email protected]
Genetic determinants of cholangiopathies: Molecular and systems genetics
Genetic determinants of cholangiopathies: molecular and systems genetics Matthias C. Reichert, Rabea A. Hall, Marcin Krawczyk, Frank Lammert PII: DOI: Reference:
S0925-4439(17)30261-2 doi:10.1016/j.bbadis.2017.07.029 BBADIS 64841
To appear in:
BBA - Molecular Basis of Disease
Received date: Revised date: Accepted date:
19 June 2017 24 July 2017 25 July 2017
Please cite this article as: Matthias C. Reichert, Rabea A. Hall, Marcin Krawczyk, Frank Lammert, Genetic determinants of cholangiopathies: molecular and systems genetics, BBA - Molecular Basis of Disease (2017), doi:10.1016/j.bbadis.2017.07.029
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Genetic determinants of cholangiopathies: molecular and systems genetics Matthias C. Reichert1*, Rabea A. Hall1*, Marcin Krawczyk1,2 and Frank Lammert1,3 1
Department of Medicine II, Saarland University Medical Center, Homburg, Germany;
2
PT
Laboratory of Metabolic Liver Diseases, Centre for Preclinical Research, Department of
General, Transplant and Liver Surgery, Medical University of Warsaw, Warsaw, Poland; 3
RI
Chair of Internal Medicine II, Saarland University, Saarbrücken, Germany
SC
*These authors contributed equally.
NU
Corresponding author:
Department of Medicine II Saarland University Medical Center Saarland University
Germany
TE
66424 Homburg
D
Kirrberger Str. 100
MA
Prof. Dr. med. Frank Lammert
+49 6841 1623201
Fax:
+49 6841 1623267
Email:
[email protected]
AC CE P
Phone:
Electric word count: 3,817 Number of tables: 2
DISCLOSURE STATEMENT There are no conflicts of interest.
FINANCIAL SUPPORT
1
ACCEPTED MANUSCRIPT The experimental studies that are referred to in this meeting report were, in part, supported by grants from the Federal Ministry of Education and Research of Germany
RI
PT
(BMBF LiSyM 031L0051 to FL).
ABBREVIATIONS alkaline phosphatase
ASBT
apical sodium bile acid transporter
BRIC
benign recurrent intrahepatic cholestasis
CC
collaborative cross
DILI
drug-induced liver injury
ɣ-GT
ɣ-glutamyl transpeptidase
GRP
genetic reference populations
GWAS
genome-wide association studies
HCC
hepatocellular carcinoma
ICP
intrahepatic cholestasis of pregnancy
LPAC
low phospholipid-associated cholelithiasis
PBC
primary biliary cirrhosis
PBED
partial biliary external diversion
PFIC
progressive familial intrahepatic cholestasis
PSC
primary sclerosing cholangitis
RIL
recombinant inbred lines
UDCA
ursodeoxycholic acid
AC CE P
TE
D
MA
NU
SC
AP
2
ACCEPTED MANUSCRIPT Abstract Familial cholangiopathies are rare but potentially severe diseases. Their spectrum
PT
ranges from fairly benign conditions as, for example, benign recurrent intrahepatic
RI
cholestasis to low-phospholipid associated cholelithiasis and progressive familial intrahepatic cholestasis (PFIC). Many cholangiopathies such as primary biliary
SC
cholangitis (PBC) or primary sclerosing cholangitis (PSC) affect first the bile ducts
NU
("ascending pathophysiology") but others, such as PFIC, start upstream in hepatocytes and cause progressive damage "descending" down the biliary tree and leading to end-
MA
stage liver disease. In recent years our understanding of cholestatic diseases has improved, since we have been able to pinpoint numerous disease-causing mutations
D
that cause familial cholangiopathies. Accordingly, six PFIC subtypes (PFIC type 1 - 6)
TE
have now been defined. Given the availability of genotyping resources, these findings
AC CE P
can be introduced in the diagnostic work-up of patients with peculiar cholestasis. In addition, functional studies have defined the pathophysiological consequences of some of the detected variants. Furthermore, ABCB4 variants do not only cause PFIC type 3 but confer an increased risk for chronic liver disease in general. In the near future these findings will serve to develop new therapeutic strategies for patients with liver diseases. Here we present the latest data on the genetic background of familial cholangiopathies and discuss their application in clinical practice for the differential diagnosis of cholestasis of unknown aetiology. As look in the future we present "system genetics" as a novel experimental tool for the study of cholangiopathies and disease-modifying genes.
3
ACCEPTED MANUSCRIPT Keywords:
Cholestasis,
complex
disease,
genetic
test,
phosphatidylcholine
translocator, quantitative trait locus.
PT
Introduction
RI
Most cholangiopathies are heterogeneous and of unclear aetiology[1, 2]. However, it is known that apart from risk factors such as age, gender and ethnicity, they are influenced
SC
by genetic modifiers[3]. Symptoms, disease progression, comorbidities and the effect of
NU
treatments can vary strongly, since the patients carry a wide spectrum of genetic mutations and variants[4]. Consequently, the diseases often present with unclear
MA
phenotype-to-genotype correlations. This complex structure and the heterogeneity of the diseases hampers the search for effective medical treatments. In addition, the definition
D
of cholangiopathies is often non-comprehensive, because they are accompanied by
TE
several comorbidities and symptoms that might not be recognized as early stages of
AC CE P
disease. Therefore, there is a demand for early markers of disease occurrence, progression and predisposing risk factors. Genetic variants leading to deficiencies in transport proteins of the hepatobiliary system have been associated to cholestatic diseases such as progressive familial intrahepatic cholangitis (PFIC)[2] and gallstone disease (cholelithiasis). For example, three different forms of PFIC (types 1- 3) are caused by mutations in transporters located in the hepatocanalicular membrane (i.e. ATP8B1, ABCB11 and ABCB4, respectively)[2]. Next to the primary effects of these risk genes that define the type of disease, additional signaling pathways may be under genetic control and contribute to the (sub)phenotypes of the disease. All these factors result in the individual disease susceptibility of each patient. Case-control studies and genome-wide association studies (GWAS)[5] in human cohorts aim to elucidate underlying disease mechanisms by finding genetic variants that predispose a patient to 4
ACCEPTED MANUSCRIPT a certain disease. However, the assembly of human cohorts requires the recruitment of a large number of patients as well as the elaborate assimilation of recruitment centers
PT
for disease characterization and accurate documentation. Recent GWAS performed in
RI
PBC[6] and PSC[7] have illustrated these limitations, which were overcome by international collaboration and large consortia as well as more detailed phenotypic and
follow-up.
Notwithstanding
these
SC
characterization
efforts
and
the
new
been implemented in clinical algorithms.
NU
pathophysiological insights, the identified risk loci have low effect sizes and have not
MA
Nevertheless, the knowledge about the genetic causes of cholestatic liver diseases has increased significantly in recent years[4]. Currently available genetic tests
D
can be applied for both distinct groups of genetically caused cholestatic liver diseases:
TE
(i) monogenic diseases, such as PFIC, benign recurrent intrahepatic cholestasis (BRIC),
AC CE P
Dubin-Johnson and Rotor syndromes; (ii) polygenic diseases, such as gallstones or drug-induced liver injury (DILI). Single or multiple variants, whole genes (at least all their exons) or panel of genes can be tested. However, the interpretation of the results may be complex. Below we summarize our knowledge about the genetic background of selected biliary diseases. We discuss how disease-causing-variants contribute to the development of the disease. In addition, we present system genetics in experimental models as novel approach to further improve our knowledge about the genetic background of cholangiopathies.
Gallstones Gallstone disease is one of the most common biliary diseases. In Europe approximately 20% of adults carry gallstones[8, 9]. In adults these are predominantly cholesterol 5
ACCEPTED MANUSCRIPT stones. Formerly environmental and lifestyle factors were regarded to be the major determinants of gallstone formation. Now, it is known that genetic factors may account
PT
for up to 40% of total gallstone risk. Indeed, in addition to the well-known exogenous risk
RI
factors, such as overnutrition and physical inactivity, genetic risk factors have been identified. In mice, gallstone formation can be induced by administration of a lithogenic
SC
diet of 1% cholesterol and 0.5% cholic acid. Here, it was observed that different inbred
NU
mouse strains vary in gallstone susceptibility[10]. The differences were analyzed in experimental crosses by several groups and multiple quantitative trait loci (QTLs) and
MA
genes involved in gallstone formation were identified[11]. Wittenburg et al.[12] identified the locus harboring the Abcg5/g8 genes as one of the major QTL for gallstone formation.
D
In line with these results, the first GWAS study in liver diseases, which was performed
TE
more than 10 years ago and included gallstone patients form Germany and Chile,
AC CE P
identified the amino acid substitution ABCG8 p.D19H as risk factor for the development of gallstones[13]. ABCG8 encodes, together with the adjacent oppositely oriented ABCG5, two hemitransporters that are secreting cholesterol across the apical membrane of hepatocytes into bile canaliculi and into the gut lumen. These results were replicated in additional cohorts, rendering this ABCG8 variant the most common genetic risk factor for cholelithiasis[14]. In the meantime, the Gilbert syndrome variant in the promoter of the UGT1A1 gene was shown to also substantially increase the gallstone risk[15, 16]. Genetic screening is currently not recommended by guidelines for gallstone disease[9]. Sequencing of ABCB4 may be considered in young patients with gallstones to confirm the diagnosis of the Low phospholipid-associated cholelithiasis (LPAC) syndrome. 6
AC CE P
TE
D
MA
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
7
ACCEPTED MANUSCRIPT Primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) PSC is a rare (prevalence 1:10.000) inflammatory disease of the bile ducts. The first and
PT
early lesions are in "downstream" bile ducts, which then cause bile salt-mediated toxic
recurrent
biliary
infections
and
RI
injury of the "upstream" liver parenchyma; this "ascending" disease course may lead to liver
cirrhosis[17].
The
risk
to
develop
SC
cholangiocarcinoma is markedly increased (up to 15%). Currently no medical therapy is
NU
established. On the other hand, siblings have an increased risk to develop PSC, pointing to interacting hereditary and environmental factors. GWAS studies in PSC patients
MA
helped to identify multiple associations with Human Leukocyte Antigen (HLA) genotypes and non-HLA genes modulating T-cell functions, indicating the relevance of systemic
D
immune responses and crosstalk between immune cells and cholangiocytes [18, 19].
TE
Detecting these risk variants has currently no clinical application, and testing is not
AC CE P
recommended in clinical practice guidelines yet. Therefore, it is not surprising that several mouse models were developed that try to mimic PSC[20, 21]. So far these models do not fully resemble the disease-specific features.
A
common
model
is
the
Abcb4
knockout
mouse,
which
shows
histopathological features sclerosing cholangitis and biliary fibrosis and progresses to develop hepatocellular cancer at about 12-16 months of age[22, 23]. However, certain disease features are missing such as cholangiocarcinoma and inflammatory bowel disease. PBC (formerly known as primary biliary cirrhosis) is a chronic cholestatic autoimmune disorder of the small bile ducts[24]. Predominantly women are affected (90%). The progressive extinction of small bile ducts leads to liver fibrosis, and cirrhosis may develop. When started at an early stage, therapy with ursodexoycholic acid (UDCA) 8
ACCEPTED MANUSCRIPT can stop the progression of the disease in most patients. As additional therapy, obeticholic acid - a potent ligand of the central nuclear bile acid receptor FXR (NR1H4) -
PT
has been approved in combination with UDCA in PBC patients with an inadequate
RI
response to UDCA (or as monotherapy in patients unable to tolerate UDCA)[25]. In GWAS the strongest genetic associations in PBC are HLA associations. Most non-HLA
SC
associations in PBC overlap with other autoimmune diseases, with risk loci indicating
NU
altered immunoregulatory pathways, dysregulated mucosal immunity, or aberrant microbial handling[26-28]. Less than 20% of the variability is explained by GWAS,
MA
pointing to the relevance of rare variants and epigenetics. Gene testing for risk variants
D
has no relevance in clinical care of PBC patients yet.
TE
Low phospholipid-associated cholelithiasis (LPAC) syndrome and ABCB4
AC CE P
deficiency
The LPAC syndrome is characterized by low concentrations of phospholipids in bile and an increased risk for gallstones, which may recur after cholecystectomy[29]. LPAC is caused by the reduced transport of phosphatidylcholine into bile due to the dysfunction of the hepatocanalicular phospholipid translocator ABCB4, which can be caused by different ABCB4 gene variants. LPAC is a clinical diagnosis, based on the following three criteria[30]:
biliary symptoms before the age of 40 years;
detection of intrahepatic microlithiasis/sludge by ultrasound (hyperechogenic foci);
recurrence of symptomatic cholelithasis after cholecystectomy.
9
ACCEPTED MANUSCRIPT Most (but not all) patients with LPAC benefit from therapy with UDCA, which should be started early at young age. The diagnosis can be confirmed by microscopic examination
PT
of endoscopically acquired hepatic bile, which contains aggregated cholesterol crystals
RI
or microliths, the measurement of biliary phospholipid concentrations (in frozen bile), or the sequencing of all ABCB4 exons. In more than half of the patients, a pathogenic risk
SC
variant in ABCB4 has been detected[30].
NU
ABCB4 deficiency summarizes several conditions that are caused by gene variants in ABCB4. The clinical spectrum of ABCB4-associated liver phenotypes is
MA
broad. The phenotypes range from benign diseases such as intrahepatic cholestasis of pregnancy (ICP) due to mild and/or heterozygous variants to LPAC and PFIC type 3,
D
resulting in biliary liver cirrhosis, in the setting of severe homozygous ABCB4 mutations.
TE
The detection of genetic variants can be helpful for diagnosis and counseling of patients,
AC CE P
but genotype-phenotype-correlations are imprecise. Recently, a new classification for ABCB4 mutations has been suggested (class I - V), as follows (Table 1): (I) nonsense variations; (II) missense variations that primarily affect maturation; (III) activity; or (IV) stability; and (V) variants without detectable effects[31]. Furthermore, variants in ABCB4 have been found to be associated with gallbladder cancer risk[32]. They might also promote the progression of liver fibrosis in patients with chronic liver diseases in general[33]. Of note, large-scale whole-genome sequencing of the Icelandic population has demonstrated significant associations between ABCB4 variants with liver diseases and liver enzyme activities in the general population[34]: The common ABCB4 variant c.711A>T was identified as general risk factor for elevated aminotransferases and higher impact variants were associated with early-onset gallstone disease, ICP, liver
10
ACCEPTED MANUSCRIPT cirrhosis, and hepatobiliary cancers, thus setting their carriers "on the highway to liver
PT
disease"[35].
RI
Familiar intrahepatic cholestasis
This clinically heterogenic group of diseases is characterized by familial aggregation and
SC
intrahepatic cholestasis and can be further classified into (i) PFIC types 1-6 with mostly
NU
dismal prognosis; and (ii) BRIC with good prognosis. The diseases are caused by mutations in the hepatocanalicular transporters ATP8B1, ABCB11 or ABCB4, or the
MA
TJP2, NR1H4 and MYO5B genes (Table 2).
D
Progressive familiar intrahepatic cholestasis (PFIC)
TE
This is a group of rare diseases (incidence 1:50.000). In children it can cause severe
AC CE P
cholestatic liver disease. It commonly manifests in the first year of life (2-9 months) with pruritus and jaundice. The intra- and extrahepatic bile ducts are not dilated. The course of the disease and the clinical phenotypes are heterogeneous, typically with rapid progression to liver cirrhosis at the age of 3-15 years and death if no liver transplantation is performed. Most cases of PFIC are caused by mutations in hepatocanalicular transporter genes with autosomal recessive inheritance. The diagnosis of PFIC is established through genetic testing. In these monogenic cholestatic liver diseases, sequence analysis of the whole gene, comprising at least the coding region, flanking splice sites and possibly regulatory regions, is recommended.
11
ACCEPTED MANUSCRIPT PFIC type 1 Formerly known as Byler syndrome, this cholestatic liver disease typically manifests
PT
within the first year of life with marked pruritus and progression to liver cirrhosis at the
RI
age of 2-7 years. Of note, extrahepatic manifestations (malabsorption, pancreatitis, deafness, pneumonia) can occur. Laboratory analysis reveals raised alkaline
SC
phosphatase (AP) and normal ɣ-glutamyl transpeptidase (ɣ-GT) activities. Liver biopsy
NU
initially shows canalicular cholestasis, later liver cirrhosis develops. PFIC1 is due to mutations of the ATP8B1 (FIC1) gene[36], encoding the transporter responsible for
MA
maintaining the asymmetry of the hepatocanalicular membrane by enrichment of phosphatidylserine in the inner leaflet (flippase). Distorted membrane composition
D
impacts bile acid secretion, which is reflected by low bile acid concentrations in hepatic
TE
bile of PFIC1 patients[2]. As one of the possible mechanisms, downregulation of FXR
AC CE P
could result in repression of the bile acid export pump (ABCB11) in the liver and induction of the apical sodium bile acid transporter (ASBT) in the intestine[17]. The expression of ATP8B1 is higher in the intestine than in the liver and therefore might influence the enterohepatic cycling of bile acids. Many PFIC1 patients need to undergo liver transplantation but in particular this subtype of PFIC might also benefit from early partial biliary external diversion (PBED)[37], for which several different surgical techniques were described[38]. Shah et al. [39] reported data on Atp8b1G308V-knockin mice, which demonstrated showed similar defects in bile acid homeostasis. Still the disease does not progress to a stage resembling advanced human cholestatic disease[39]. Yet, the study by Shah et al.[39] showed that the severity of the ATP8B1 deficiency depends on the genetic background of the strain. 12
ACCEPTED MANUSCRIPT
PFIC type 2
PT
Mutations in ABCB11 (BSEP) cause PFIC2. ABCB11 functions as the bile salt export
RI
pump in the hepatocanalicular membrane, and ABCB11 mutations cause decreased bile acid secretion, accumulation of bile salts in the liver, and hepatocellular injury. PFIC2
SC
manifests typically within the first six months of life with marked pruritus and jaundice.
NU
The clinical course is typically progressive with development of liver cirrhosis within the first two years of life. In later life, the risk for hepatocellular and cholangiocellular cancers
MA
is increased. In laboratory work-up, ALT is higher and AP activities are lower than in PFIC1, with both showing normal ɣ-GT[40]. Histopathologically, initially typically
D
canalicular cholestasis with giant cell hepatitis and lobular fibrosis is found. Functional
TE
ABCB11 gene variants are causative, and up to 60% of European patients harbour one
AC CE P
of the two "hot-spot" mutations p.E297G or p.D482G, but diagnosis is established by gene sequencing in most patients. In the mouse the Abcb11 knockout leads to mild cholestasis only, and the lack of Abcb11 expression seems to be compensated by upregulation of other ATP transporter genes, e.g. Abca1 and Abcb1b. In Abcb11-knockout mice the secretion of hydrophobic bile acids is reduced, while hydrophilic bile acids (β- and Ω-muricholic acid) are secreted by the compensatory transporters. This was confirmed by generating a triple knockout mouse with ABCB11 and ABCA1 (transcripts a and b) deficiency, which presented more severe phenotypes resembling human cholestatic disease, such as inflammatory infiltrates, biliary fibrosis, and deteriorated microvilli in the canaliculi[2, 41]. Nevertheless, disease severity was still generally milder in the mouse model than in humans[2, 41], since mice can detoxify hydrophobic bile salts by hydroxylation. Hence, even though 13
ACCEPTED MANUSCRIPT mouse and humans share most of their genetic information, mouse models need to be evaluated for pathophysiological similarities. As seen in this example, the experimental
RI
PT
setup can often be adjusted to allow further modelling.
PFIC type 3
SC
Patients with PFIC3 carry a defective hepatocanalicular phosphatidylcholine translocator
NU
ABCB4. The formation of mixed micelles in bile depends on the correct composition of bile, which mainly consists of water, bile acids, phosphatiylcholine, and cholesterol. Low
MA
biliary phospholipid concentrations and the resulting imbalance of these three components leads to bile acid toxicity as well as cholesterol supersaturation and
D
crystallization[22, 42, 43]. The severity of the disease varies with the type of mutation
TE
and the remaining function of the transporter. PFIC3 can manifest within the first year of
AC CE P
life (up to 30%) and liver cirrhosis then often develops already in childhood, but ABCB4 deficiency may also present later in life with moderate jaundice. Laboratory tests show increased aminotransferase activities and - in contrast to PFIC types 1 and 2 - elevated ɣ-GT activities. Liver biopsy reveals neoductular proliferations and in further course progression of liver fibrosis. Patients with PFIC3 may benefit from higher doses of UDCA (15 - 20 mg/kg body weight/day) but often liver transplantation is required. The Abcb4-knockout mouse is deficient for the orthologous murine gene and an appropriate PFIC3 model, albeit sponatenous disease severity is less advanced than in humans and older mice develop hepatocellular cancer but not cholangiocarcinoma. Of note, knockout mice present with bile that is supersaturated with cholesterol and develop intra- and extrahepatic gallstones similar to patients with LPAC[44].
14
ACCEPTED MANUSCRIPT PFIC types 4 - 6 Recently a fourth type of PFIC was described in children (Table 2). It is caused by
PT
mutations in the gene encoding the tight junction protein TJP2. This leads to dysfunctional tight junctions and early onset cholestasis. Similar to PFIC1 and PFIC2,
RI
serum ɣ-GT are not increased, in contrast to high bilirubin and bile acid concentrations.
SC
Some patients develop hepatocellular carcinoma (HCC) and most of them require liver
NU
transplantation[45].
Mutations in NR1H4 encoding the nuclear receptor FXR were found in children,
MA
who presented with neonatal cholestasis and rapid progression to end-stage liver disease[46]. This condition has been called PFIC type 5. Lately, mutations in MYO5B, a
D
gene encoding for a large protein that is required for hepatocyte polarization, were
TE
detected. Whereas severe MYO5B mutations underlie systemic microvillus inclusion
AC CE P
disease, mild variants cause persistent, transient or recurrent cholestasis as liverrestricted phenotypes. The disease has been labeled PFIC type 6[47].
Benign recurrent intrahepatic cholestasis (BRIC) This disease was formerly called Summerskill-Walshe-Tygstrup-Syndrome and was first described in 1959. It is a very rare disease with familial clustering. The first flare typically occurs in young patients. BRIC is characterized by prolonged episodes of cholestasis with jaundice, pruritus, and malabsorption[48]. These episodes might be triggered by not well defined exogenous factors (infections, hormones or other drugs). BRIC flares resolve completely after weeks or months without any major damage to the liver. They might recur after an asymptomatic period of months to years. Liver biopsy shows bland intrahepatic cholestasis. During remission, liver morphology and function are normal. As 15
ACCEPTED MANUSCRIPT per definition, no progression to liver cirrhosis occurs in contrast to PFIC. Genetically different mutations in the ATP8B1 (BRIC type 1) or ABCB11 genes (BRIC type 2) can be
PT
detected[49, 50]. 45% of BRIC1 patients carry the amino acid substitution p.I661T.
RI
Sequencing of both genes confirms the diagnosis. Although larger studies are missing,
NU
nasobiliary drainage, or plasmapheresis[51, 52].
SC
case reports highlight the successful treatment of BRIC with UDCA, rifampicin,
Association studies and systems genetics in experimental models
MA
Trying to tackle complex diseases like cholangiopathies has shown us how important it is to understand complex biological systems. Systems genetics approaches encompass
populations,
which
TE
of
D
multiple levels of (patho)biological data. Systems genetics is defined as systems biology are
inter-breeding
individuals
AC CE P
in a specific environment[53]. Animal models may fill the gap where human studies fail to succeed and can help to achieve a better understanding of critical pathways that are under genetic control[54-56]. Mice and humans share about 95% of their genome and many disease genes were also identified in mice. In contrast to human studies genetically different mice can be analyzed under controlled environmental conditions. In addition the mouse genome can be changed to analyze potential modifier genes in detail. Inbred mouse strains, which are homozygous at all loci, often display different disease susceptibility due to their distinct genetic backgrounds[57]. Genetic mapping in mouse strains enhances the power of detecting modifier genes and identifying complex genetic interactions. Genomewide quantitative trait locus (QTL) analysis, as described in more detail below, represents a promising approach to detect genetic variants that are associated with specific phenotypes and interact with each other. 16
ACCEPTED MANUSCRIPT In experimental crosses of two (inbred) strains the first generation (F1) of offsprings is genetically heterozygous but equal. Then in the next generation (F2) the
PT
strain-specific genetic information is distributed across the genomes of their progeny and
RI
each offspring is genetically unique. Accordingly, F2 mice represent a population of mice displaying highly variable phenotypes. They are a promising tool to identify new disease
SC
modifiers in association studies. QTL analysis detect genomic regions that harbor genes
NU
associated with disease susceptibility[58, 59]. More than 200 mice are typically required for the analysis of an F2 cross. Each mouse is genotyped for genetic markers covering
MA
the whole genome, but it is unique, which limits the phenotypic characterization of multiple traits. Nevertheless, an F2 cross can be employed for the analysis of genetic
D
background effects when using genetically modified mice. In a recent QTL study we
TE
transferred the Abcb4 knockout from the fibrosis resistant FVB/NJ strain to the fibrosis
AC CE P
susceptible BALB/cJ strain by repeated backcrossing. These two congenic knockout strains were then crossed to generate F2 progeny, all of which are ABCB4 deficient. At the age of 16 weeks the F2 mice developed biliary fibrosis of variable severity. Each mouse was genotyped for single nucleotide variants across all chromosomes. Phenotypic differences were quantified by hepatic collagen contents and other subphenotypes, and then the genetic regions linked to these phenotypes were mapped as described above. Similar to genetic studies in human cohorts, the identified risk loci showed small effect sizes. However, in this model system the analysis of gene-gene interactions and epistatic effects is possible, which allowed us to identify genetic regions that are significantly linked to the phenotypes. These regions contain genetic modifiers dependent on ABCB4 deficiency. From this point it is possible to go back to patient cohorts to study the orthologous modifier genes in humans. The mouse studies enable 17
ACCEPTED MANUSCRIPT the identification of new modifiers that remain masked in human populations or affect phenotypes that cannot be quantified non-invasively in patients.
PT
An alternative to the F2 cross are experimental crosses that are used to establish
RI
genetic reference populations (GRP). Here, the F2 generation is inbred by continuous brothersister matings for more than 20 generations to generate recombinant inbred
SC
lines (RIL). This results in homozygous but heterogenic RILs, where each line consists
NU
of mice that resemble genetic clones. The genetic identical mice allow the analysis of different models and experimental conditions. One of the best studied genetic reference
MA
populations are the BXD recombinant inbred lines[60]. They were generated starting with the two inbred strains C57BL/6J and DBA/2J, and now more than 100 RILs exist
TE
D
that have been screened for several phenotypes, including liver diseases. The advantage of these lines is that they were also genotyped and the genetic data was
AC CE P
uploaded into GeneNetwork (www.genenetwork.org)[61], where it is freely available for every scientist working with this GRP together with all phenotypic data from other projects or groups. GeneNetwork enables QTL and multiscale correlation analyses. This approach saves resources and at the same time contributes to the dissection of disease networks at multiple biological levels. It represents a new step towards a systems biology approach and a better understanding of complex diseases[62-65]. The collaborative cross (CC) represents the "next generation" of GRPs and the future tool for systems genetics in mouse models. The CC lines are generated in a collaboration project by the Complex Trait Consortium (www.complextrait.org)[66], and as to date about 300 lines have been generated. By introducing eight strains in an eightway cross, the genetic diversity of the offsprings was increased aiming for finer mapping resolution than in F2 crosses or the BXD RILs. The improved resolution reduces the 18
ACCEPTED MANUSCRIPT number of candidate genes in the identified QTL regions. Nevertheless, the groups generating the CC lines still have to overcome several challenges, e.g. difficult breeding
PT
the lines to achieve fully inbreds and high complexity of the analysis based on eight
RI
instead of two alleles[67]. Yet, the first disease studies were published recently and demonstrated the power of CC lines for the genetic dissection of complex diseases[68,
SC
69]. These technical advances in mice allow a better understanding of the complex
NU
genetics of cholangiopathies. The increasing complexity of the experimental systems provides new insights that can hopefully be translated to human populations in the near
MA
future.
D
Key points
TE
• Genetics lead to the dissection of familial and multifactorial cholangiopathies.
AC CE P
• With the identification of underlying genetic contributors cholestatic diseases are being reclassified.
• Systems genetics reveals the full complexity of genotype-phenotype relationships in liver.
• In the future the genetic findings might open new avenues for treating patients with cholangiopathies.
19
ACCEPTED MANUSCRIPT References [1] K.N. Lazaridis, N.F. LaRusso, Primary Sclerosing Cholangitis, N Engl J Med, 375
PT
(2016) 1161-1170.
RI
[2] E. Jacquemin, Progressive familial intrahepatic cholestasis, Clin Res Hepatol
SC
Gastroenterol, 36 Suppl 1 (2012) S26-35.
[3] G.M. Hirschfield, R.W. Chapman, T.H. Karlsen, F. Lammert, K.N. Lazaridis, A.L.
NU
Mason, The genetics of complex cholestatic disorders, Gastroenterology, 144 (2013)
MA
1357-1374.
[4] T.H. Karlsen, F. Lammert, R.J. Thompson, Genetics of liver disease: From
D
pathophysiology to clinical practice, J Hepatol, 62 (2015) S6-S14.
TE
[5] M. Krawczyk, R. Mullenbach, S.N. Weber, V. Zimmer, F. Lammert, Genome-wide association studies and genetic risk assessment of liver diseases, Nat Rev
AC CE P
Gastroenterol Hepatol, 7 (2010) 669-681. [6] A.F. Gulamhusein, B.D. Juran, K.N. Lazaridis, Genome-Wide Association Studies in Primary Biliary Cirrhosis, Semin Liver Dis, 35 (2015) 392-401. [7] A. Paziewska, A. Habior, A. Rogowska, W. Zych, K. Goryca, J. Karczmarski, M. Dabrowska, F. Ambrozkiewicz, B. Walewska-Zielecka, M. Krawczyk, H. Cichoz-Lach, P. Milkiewicz, A. Kowalik, K. Mucha, J. Raczynska, J. Musialik, G. Boryczka, M. Wasilewicz, I. Ciecko-Michalska, M. Ferenc, M. Janiak, A. Kanikowska, R. Stankiewicz, M. Hartleb, T. Mach, M. Grzymislawski, J. Raszeja-Wyszomirska, E. Wunsch, T. Bobinski, M. Mikula, J. Ostrowski, A novel approach to genome-wide association analysis identifies genetic associations with primary biliary cholangitis and primary sclerosing cholangitis in Polish patients, BMC Med Genomics, 10 (2017) 2.
20
ACCEPTED MANUSCRIPT [8] R. Aerts, F. Penninckx, The burden of gallstone disease in Europe, Aliment Pharmacol Ther, 18 Suppl 3 (2003) 49-53.
PT
[9] L. European Association for the Study of the, EASL Clinical Practice Guidelines on
RI
the prevention, diagnosis and treatment of gallstones, J Hepatol, 65 (2016) 146-181. [10] M. Alexander, O.W. Portman, Different susceptibilities to the formation of
SC
cholesterol gallstones in mice, Hepatology, 7 (1987) 257-265.
NU
[11] D.Q. Wang, N.H. Afdhal, Genetic analysis of cholesterol gallstone formation: searching for Lith (gallstone) genes, Curr Gastroenterol Rep, 6 (2004) 140-150.
MA
[12] H. Wittenburg, M.A. Lyons, R. Li, G.A. Churchill, M.C. Carey, B. Paigen, FXR and ABCG5/ABCG8 as determinants of cholesterol gallstone formation from quantitative trait
D
locus mapping in mice, Gastroenterology, 125 (2003) 868-881.
TE
[13] S. Buch, C. Schafmayer, H. Volzke, C. Becker, A. Franke, H. von Eller-Eberstein, C.
AC CE P
Kluck, I. Bassmann, M. Brosch, F. Lammert, J.F. Miquel, F. Nervi, M. Wittig, D. Rosskopf, B. Timm, C. Holl, M. Seeger, A. ElSharawy, T. Lu, J. Egberts, F. Fandrich, U.R. Folsch, M. Krawczak, S. Schreiber, P. Nurnberg, J. Tepel, J. Hampe, A genomewide association scan identifies the hepatic cholesterol transporter ABCG8 as a susceptibility factor for human gallstone disease, Nat Genet, 39 (2007) 995-999. [14] M. Krawczyk, D.Q. Wang, P. Portincasa, F. Lammert, Dissecting the genetic heterogeneity of gallbladder stone formation, Semin Liver Dis, 31 (2011) 157-172. [15] S. Stender, R. Frikke-Schmidt, B.G. Nordestgaard, A. Tybjaerg-Hansen, Extreme bilirubin levels as a causal risk factor for symptomatic gallstone disease, JAMA Intern Med, 173 (2013) 1222-1228.
21
ACCEPTED MANUSCRIPT [16] H.U. Marschall, M. Krawczyk, F. Grunhage, D. Katsika, C. Einarsson, F. Lammert, Gallstone disease in Swedish twins is associated with the Gilbert variant of UGT1A1,
PT
Liver Int, 33 (2013) 904-908.
RI
[17] P.L. Jansen, A. Ghallab, N. Vartak, R. Reif, F.G. Schaap, J. Hampe, J.G. Hengstler, The ascending pathophysiology of cholestatic liver disease, Hepatology, 65 (2017) 722-
SC
738.
NU
[18] D. Ellinghaus, L. Jostins, S.L. Spain, A. Cortes, J. Bethune, B. Han, Y.R. Park, S. Raychaudhuri, J.G. Pouget, M. Hubenthal, T. Folseraas, Y. Wang, T. Esko, A. Metspalu,
MA
H.J. Westra, L. Franke, T.H. Pers, R.K. Weersma, V. Collij, M. D'Amato, J. Halfvarson, A.B. Jensen, W. Lieb, F. Degenhardt, A.J. Forstner, A. Hofmann, I.B.D.G.C. C.
International
Genetics
D
International,
of
Ankylosing
Spondylitis,
P.S.C.S.G.
TE
International, C. Genetic Analysis of Psoriasis, E. Psoriasis Association Genetics, S.
AC CE P
Schreiber, U. Mrowietz, B.D. Juran, K.N. Lazaridis, S. Brunak, A.M. Dale, R.C. Trembath, S. Weidinger, M. Weichenthal, E. Ellinghaus, J.T. Elder, J.N. Barker, O.A. Andreassen, D.P. McGovern, T.H. Karlsen, J.C. Barrett, M. Parkes, M.A. Brown, A. Franke, Analysis of five chronic inflammatory diseases identifies 27 new associations and highlights disease-specific patterns at shared loci, Nat Genet, 48 (2016) 510-518. [19] J.Z. Liu, J.R. Hov, T. Folseraas, E. Ellinghaus, S.M. Rushbrook, N.T. Doncheva, O.A. Andreassen, R.K. Weersma, T.J. Weismuller, B. Eksteen, P. Invernizzi, G.M. Hirschfield, D.N. Gotthardt, A. Pares, D. Ellinghaus, T. Shah, B.D. Juran, P. Milkiewicz, C. Rust, C. Schramm, T. Muller, B. Srivastava, G. Dalekos, M.M. Nothen, S. Herms, J. Winkelmann, M. Mitrovic, F. Braun, C.Y. Ponsioen, P.J. Croucher, M. Sterneck, A. Teufel, A.L. Mason, J. Saarela, V. Leppa, R. Dorfman, D. Alvaro, A. Floreani, S. Onengut-Gumuscu, S.S. Rich, W.K. Thompson, A.J. Schork, S. Naess, I. Thomsen, G. 22
ACCEPTED MANUSCRIPT Mayr, I.R. Konig, K. Hveem, I. Cleynen, J. Gutierrez-Achury, I. Ricano-Ponce, D. van Heel, E. Bjornsson, R.N. Sandford, P.R. Durie, E. Melum, M.H. Vatn, M.S. Silverberg,
PT
R.H. Duerr, L. Padyukov, S. Brand, M. Sans, V. Annese, J.P. Achkar, K.M. Boberg, H.U.
RI
Marschall, O. Chazouilleres, C.L. Bowlus, C. Wijmenga, E. Schrumpf, S. Vermeire, M. Albrecht, U.-P. Consortium, J.D. Rioux, G. Alexander, A. Bergquist, J. Cho, S.
SC
Schreiber, M.P. Manns, M. Farkkila, A.M. Dale, R.W. Chapman, K.N. Lazaridis,
NU
P.S.C.S.G. International, A. Franke, C.A. Anderson, T.H. Karlsen, I.B.D.G.C. International, Dense genotyping of immune-related disease regions identifies nine new
MA
risk loci for primary sclerosing cholangitis, Nat Genet, 45 (2013) 670-675. [20] P. Fickert, M.J. Pollheimer, U. Beuers, C. Lackner, G. Hirschfield, C. Housset, V.
D
Keitel, C. Schramm, H.U. Marschall, T.H. Karlsen, E. Melum, A. Kaser, B. Eksteen, M.
TE
Strazzabosco, M. Manns, M. Trauner, Characterization of animal models for primary
AC CE P
sclerosing cholangitis (PSC), J Hepatol, 60 (2014) 1290-1303. [21] J.M. Vierling, Animal models for primary sclerosing cholangitis, Best Pract Res Clin Gastroenterol, 15 (2001) 591-610. [22] J.J. Smit, A.H. Schinkel, R.P. Oude Elferink, A.K. Groen, E. Wagenaar, L. van Deemter, C.A. Mol, R. Ottenhoff, N.M. van der Lugt, M.A. van Roon, et al., Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease, Cell, 75 (1993) 451-462. [23] M. Katzenellenbogen, L. Mizrahi, O. Pappo, N. Klopstock, D. Olam, J. Jacob-Hirsch, N. Amariglio, G. Rechavi, E. Domany, E. Galun, D. Goldenberg, Molecular mechanisms of liver carcinogenesis in the mdr2-knockout mice, Mol Cancer Res, 5 (2007) 11591170.
23
ACCEPTED MANUSCRIPT [24] e.e.e. European Association for the Study of the Liver. Electronic address, G.M. Hirschfield, U. Beuers, C. Corpechot, P. Invernizzi, D. Jones, M. Marzioni, C. Schramm,
PT
EASL Clinical Practice Guidelines: The diagnosis and management of patients with
RI
primary biliary cholangitis, J Hepatol, (2017).
[25] F. Nevens, P. Andreone, G. Mazzella, S.I. Strasser, C. Bowlus, P. Invernizzi, J.P.
SC
Drenth, P.J. Pockros, J. Regula, U. Beuers, M. Trauner, D.E. Jones, A. Floreani, S.
NU
Hohenester, V. Luketic, M. Shiffman, K.J. van Erpecum, V. Vargas, C. Vincent, G.M. Hirschfield, H. Shah, B. Hansen, K.D. Lindor, H.U. Marschall, K.V. Kowdley, R.
MA
Hooshmand-Rad, T. Marmon, S. Sheeron, R. Pencek, L. MacConell, M. Pruzanski, D. Shapiro, P.S. Group, A Placebo-Controlled Trial of Obeticholic Acid in Primary Biliary
D
Cholangitis, N Engl J Med, 375 (2016) 631-643.
TE
[26] H.J. Cordell, Y. Han, G.F. Mells, Y. Li, G.M. Hirschfield, C.S. Greene, G. Xie, B.D.
AC CE P
Juran, D. Zhu, D.C. Qian, J.A. Floyd, K.I. Morley, D. Prati, A. Lleo, D. Cusi, U.S.P.B.C.C. Canadian, P.B.C.G.S.G. Italian, U.-P. Consortium, M.E. Gershwin, C.A. Anderson, K.N. Lazaridis, P. Invernizzi, M.F. Seldin, R.N. Sandford, C.I. Amos, K.A. Siminovitch, International genome-wide meta-analysis identifies new primary biliary cirrhosis risk loci and targetable pathogenic pathways, Nat Commun, 6 (2015) 8019. [27] G.F. Mells, J.A. Floyd, K.I. Morley, H.J. Cordell, C.S. Franklin, S.Y. Shin, M.A. Heneghan, J.M. Neuberger, P.T. Donaldson, D.B. Day, S.J. Ducker, A.W. Muriithi, E.F. Wheater, C.J. Hammond, M.F. Dawwas, U.P. Consortium, C. Wellcome Trust Case Control, D.E. Jones, L. Peltonen, G.J. Alexander, R.N. Sandford, C.A. Anderson, Genome-wide association study identifies 12 new susceptibility loci for primary biliary cirrhosis, Nat Genet, 43 (2011) 329-332.
24
ACCEPTED MANUSCRIPT [28] P.J. Trivedi, G.M. Hirschfield, The Immunogenetics of Autoimmune Cholestasis, Clin Liver Dis, 20 (2016) 15-31.
PT
[29] O. Rosmorduc, B. Hermelin, P.Y. Boelle, R. Parc, J. Taboury, R. Poupon, ABCB4
RI
gene mutation-associated cholelithiasis in adults, Gastroenterology, 125 (2003) 452459.
SC
[30] R. Poupon, O. Rosmorduc, P.Y. Boelle, Y. Chretien, C. Corpechot, O.
NU
Chazouilleres, C. Housset, V. Barbu, Genotype-phenotype relationships in the low-
Hepatology, 58 (2013) 1105-1110.
MA
phospholipid-associated cholelithiasis syndrome: a study of 156 consecutive patients,
[31] J.L. Delaunay, A.M. Durand-Schneider, C. Dossier, T. Falguieres, J. Gautherot, A.
D
Davit-Spraul, T. Ait-Slimane, C. Housset, E. Jacquemin, M. Maurice, A functional
TE
classification of ABCB4 variations causing progressive familial intrahepatic cholestasis
AC CE P
type 3, Hepatology, 63 (2016) 1620-1631. [32] S. Mhatre, Z. Wang, R. Nagrani, R. Badwe, S. Chiplunkar, B. Mittal, S. Yadav, H. Zhang, C.C. Chung, P. Patil, S. Chanock, R. Dikshit, N. Chatterjee, P. Rajaraman, Common genetic variation and risk of gallbladder cancer in India: a case-control genome-wide association study, Lancet Oncol, 18 (2017) 535-544. [33] M. Krawczyk, M. Rau, F. Grunhage, J.M. Schattenberg, H. Bantel, A. Pathil, M. Demir, J. Kluwe, T. Boettler, A. Geier, F. Lammert, N. Clinical Study Group, The ABCB4 p.T175A variant as potential modulator of hepatic fibrosis in patients with chronic liver diseases: Looking beyond the cholestatic realm, Hepatology, (2017). [34] D.F. Gudbjartsson, H. Helgason, S.A. Gudjonsson, F. Zink, A. Oddson, A. Gylfason, S. Besenbacher, G. Magnusson, B.V. Halldorsson, E. Hjartarson, G.T. Sigurdsson, S.N. Stacey, M.L. Frigge, H. Holm, J. Saemundsdottir, H.T. Helgadottir, H. Johannsdottir, G. 25
ACCEPTED MANUSCRIPT Sigfusson, G. Thorgeirsson, J.T. Sverrisson, S. Gretarsdottir, G.B. Walters, T. Rafnar, B. Thjodleifsson, E.S. Bjornsson, S. Olafsson, H. Thorarinsdottir, T. Steingrimsdottir, T.S.
PT
Gudmundsdottir, A. Theodors, J.G. Jonasson, A. Sigurdsson, G. Bjornsdottir, J.J.
RI
Jonsson, O. Thorarensen, P. Ludvigsson, H. Gudbjartsson, G.I. Eyjolfsson, O. Sigurdardottir, I. Olafsson, D.O. Arnar, O.T. Magnusson, A. Kong, G. Masson, U.
SC
Thorsteinsdottir, A. Helgason, P. Sulem, K. Stefansson, Large-scale whole-genome
NU
sequencing of the Icelandic population, Nat Genet, 47 (2015) 435-444. [35] F. Lammert, K. Hochrath, A letter on ABCB4 from Iceland: On the highway to liver
MA
disease, Clin Res Hepatol Gastroenterol, 39 (2015) 655-658. [36] L.W. Klomp, J.C. Vargas, S.W. van Mil, L. Pawlikowska, S.S. Strautnieks, M.J. van
D
Eijk, J.A. Juijn, C. Pabon-Pena, L.B. Smith, J.A. DeYoung, J.A. Byrne, J. Gombert, G.
TE
van der Brugge, R. Berger, I. Jankowska, J. Pawlowska, E. Villa, A.S. Knisely, R.J.
AC CE P
Thompson, N.B. Freimer, R.H. Houwen, L.N. Bull, Characterization of mutations in ATP8B1 associated with hereditary cholestasis, Hepatology, 40 (2004) 27-38. [37] I. Jankowska, P. Czubkowski, A. Wierzbicka, J. Pawlowska, P. Kalicinski, P. Socha, Influence of Partial External Biliary Diversion on the Lipid Profile in Children With Progressive Familial Intrahepatic Cholestasis, J Pediatr Gastroenterol Nutr, 63 (2016) 598-602. [38] M. Gunaydin, B. Tander, D. Demirel, G. Caltepe, A.G. Kalayci, E. Eren, U. Bicakci, R. Rizalar, E. Ariturk, F. Bernay, Different techniques for biliary diversion in progressive familial intrahepatic cholestasis, J Pediatr Surg, 51 (2016) 386-389. [39] S. Shah, U.R. Sanford, J.C. Vargas, H. Xu, A. Groen, C.C. Paulusma, J.P. Grenert, L. Pawlikowska, S. Sen, R.P. Elferink, L.N. Bull, Strain background modifies phenotypes in the ATP8B1-deficient mouse, PLoS One, 5 (2010) e8984. 26
ACCEPTED MANUSCRIPT [40] L. Pawlikowska, S. Strautnieks, I. Jankowska, P. Czubkowski, K. Emerick, A. Antoniou, C. Wanty, B. Fischler, E. Jacquemin, S. Wali, S. Blanchard, I.M. Nielsen, B.
PT
Bourke, S. McQuaid, F. Lacaille, J.A. Byrne, A.M. van Eerde, K.L. Kolho, L. Klomp, R.
RI
Houwen, P. Bacchetti, S. Lobritto, V. Hupertz, P. McClean, G. Mieli-Vergani, B. Shneider, A. Nemeth, E. Sokal, N.B. Freimer, A.S. Knisely, P. Rosenthal, P.F.
SC
Whitington, J. Pawlowska, R.J. Thompson, L.N. Bull, Differences in presentation and
NU
progression between severe FIC1 and BSEP deficiencies, J Hepatol, 53 (2010) 170178.
MA
[41] R. Wang, H.L. Chen, L. Liu, J.A. Sheps, M.J. Phillips, V. Ling, Compensatory role of P-glycoproteins in knockout mice lacking the bile salt export pump, Hepatology, 50
D
(2009) 948-956.
TE
[42] P. Fickert, A. Fuchsbichler, M. Wagner, G. Zollner, A. Kaser, H. Tilg, R. Krause, F.
AC CE P
Lammert, C. Langner, K. Zatloukal, H.U. Marschall, H. Denk, M. Trauner, Regurgitation of bile acids from leaky bile ducts causes sclerosing cholangitis in Mdr2 (Abcb4) knockout mice, Gastroenterology, 127 (2004) 261-274. [43] J. Eckstein, N. Berndt, H.G. Holzhutter, Computer simulations suggest a key role of membranous nanodomains in biliary lipid secretion, PLoS Comput Biol, 11 (2015) e1004033. [44] F. Lammert, D.Q. Wang, S. Hillebrandt, A. Geier, P. Fickert, M. Trauner, S. Matern, B. Paigen, M.C. Carey, Spontaneous cholecysto- and hepatolithiasis in Mdr2-/- mice: a model for low phospholipid-associated cholelithiasis, Hepatology, 39 (2004) 117-128. [45] M. Sambrotta, S. Strautnieks, E. Papouli, P. Rushton, B.E. Clark, D.A. Parry, C.V. Logan, L.J. Newbury, B.M. Kamath, S. Ling, T. Grammatikopoulos, B.E. Wagner, J.C. Magee, R.J. Sokol, G. Mieli-Vergani, G. University of Washington Center for Mendelian, 27
ACCEPTED MANUSCRIPT J.D. Smith, C.A. Johnson, P. McClean, M.A. Simpson, A.S. Knisely, L.N. Bull, R.J. Thompson, Mutations in TJP2 cause progressive cholestatic liver disease, Nat Genet,
PT
46 (2014) 326-328.
RI
[46] N. Gomez-Ospina, C.J. Potter, R. Xiao, K. Manickam, M.S. Kim, K.H. Kim, B.L. Shneider, J.L. Picarsic, T.A. Jacobson, J. Zhang, W. He, P. Liu, A.S. Knisely, M.J.
SC
Finegold, D.M. Muzny, E. Boerwinkle, J.R. Lupski, S.E. Plon, R.A. Gibbs, C.M. Eng, Y.
NU
Yang, G.C. Washington, M.H. Porteus, W.E. Berquist, N. Kambham, R.J. Singh, F. Xia, G.M. Enns, D.D. Moore, Mutations in the nuclear bile acid receptor FXR cause
MA
progressive familial intrahepatic cholestasis, Nat Commun, 7 (2016) 10713. [47] E. Gonzales, S.A. Taylor, A. Davit-Spraul, A. Thebaut, N. Thomassin, C. Guettier,
D
P.F. Whitington, E. Jacquemin, MYO5B mutations cause cholestasis with normal serum
TE
gamma-glutamyl transferase activity in children without microvillous inclusion disease,
AC CE P
Hepatology, 65 (2017) 164-173.
[48] W.L. van der Woerd, S.W. van Mil, J.M. Stapelbroek, L.W. Klomp, S.F. van de Graaf, R.H. Houwen, Familial cholestasis: progressive familial intrahepatic cholestasis, benign recurrent intrahepatic cholestasis and intrahepatic cholestasis of pregnancy, Best Pract Res Clin Gastroenterol, 24 (2010) 541-553. [49] S.W. van Mil, W.L. van der Woerd, G. van der Brugge, E. Sturm, P.L. Jansen, L.N. Bull, I.E. van den Berg, R. Berger, R.H. Houwen, L.W. Klomp, Benign recurrent intrahepatic cholestasis type 2 is caused by mutations in ABCB11, Gastroenterology, 127 (2004) 379-384. [50] J. Stindt, P. Ellinger, K. Weissenberger, C. Droge, D. Herebian, E. Mayatepek, B. Homey, S. Braun, J. Schulte am Esch, M. Horacek, A. Canbay, L. Schmitt, D. Haussinger, R. Kubitz, A novel mutation within a transmembrane helix of the bile salt 28
ACCEPTED MANUSCRIPT export pump (BSEP, ABCB11) with delayed development of cirrhosis, Liver Int, 33 (2013) 1527-1535.
PT
[51] V.S. Hegade, M. Krawczyk, A.E. Kremer, J. Kuczka, F. Gaouar, E.M. Kuiper, H.R.
RI
van Buuren, F. Lammert, C. Corpechot, D.E. Jones, The safety and efficacy of nasobiliary drainage in the treatment of refractory cholestatic pruritus: a multicentre
SC
European study, Aliment Pharmacol Ther, 43 (2016) 294-302.
NU
[52] G. Folvik, O. Hilde, G.O. Helge, Benign recurrent intrahepatic cholestasis: review and long-term follow-up of five cases, Scand J Gastroenterol, 47 (2012) 482-488.
MA
[53] R.A. Hall, F. Lammert, Systems Genetics of Liver Fibrosis, Methods Mol Biol, 1488 (2017) 455-466.
D
[54] L. Hood, J.R. Heath, M.E. Phelps, B. Lin, Systems biology and new technologies
TE
enable predictive and preventative medicine, Science, 306 (2004) 640-643.
AC CE P
[55] R.C. Jansen, Studying complex biological systems using multifactorial perturbation, Nat Rev Genet, 4 (2003) 145-151. [56] M. Civelek, A.J. Lusis, Systems genetics approaches to understand complex traits, Nat Rev Genet, 15 (2014) 34-48. [57] S. Hillebrandt, C. Goos, S. Matern, F. Lammert, Genome-wide analysis of hepatic fibrosis in inbred mice identifies the susceptibility locus Hfib1 on chromosome 15, Gastroenterology, 123 (2002) 2041-2051. [58] E.S. Lander, D. Botstein, Mapping mendelian factors underlying quantitative traits using RFLP linkage maps, Genetics, 121 (1989) 185-199. [59] K.W. Broman, Review of statistical methods for QTL mapping in experimental crosses, Lab Anim (NY), 30 (2001) 44-52.
29
ACCEPTED MANUSCRIPT [60] J.L. Peirce, L. Lu, J. Gu, L.M. Silver, R.W. Williams, A new set of BXD recombinant inbred lines from advanced intercross populations in mice, BMC Genet, 5 (2004) 7.
PT
[61] J. Wang, R.W. Williams, K.F. Manly, WebQTL: web-based complex trait analysis,
RI
Neuroinformatics, 1 (2003) 299-308.
[62] R.A. Hall, R. Liebe, K. Hochrath, A. Kazakov, R. Alberts, U. Laufs, M. Bohm, H.P.
SC
Fischer, R.W. Williams, K. Schughart, S.N. Weber, F. Lammert, Systems genetics of
NU
liver fibrosis: identification of fibrogenic and expression quantitative trait loci in the BXD murine reference population, PLoS One, 9 (2014) e89279.
MA
[63] R. Liebe, R.A. Hall, R.W. Williams, S. Dooley, F. Lammert, Systems genetics of hepatocellular damage in vivo and in vitro: identification of a critical network on
D
chromosome 11 in mouse, Physiol Genomics, 45 (2013) 931-939.
TE
[64] D. Gatti, A. Maki, E.J. Chesler, R. Kirova, O. Kosyk, L. Lu, K.F. Manly, R.W.
AC CE P
Williams, A. Perkins, M.A. Langston, D.W. Threadgill, I. Rusyn, Genome-level analysis of genetic regulation of liver gene expression networks, Hepatology, 46 (2007) 548-557. [65] P.A. Andreux, E.G. Williams, H. Koutnikova, R.H. Houtkooper, M.F. Champy, H. Henry, K. Schoonjans, R.W. Williams, J. Auwerx, Systems Genetics of Metabolism: The Use of the BXD Murine Reference Panel for Multiscalar Integration of Traits, Cell, 150 (2012) 1287-1299. [66] G.A. Churchill, D.C. Airey, H. Allayee, J.M. Angel, A.D. Attie, J. Beatty, W.D. Beavis, J.K. Belknap, B. Bennett, W. Berrettini, A. Bleich, M. Bogue, K.W. Broman, K.J. Buck, E. Buckler, M. Burmeister, E.J. Chesler, J.M. Cheverud, S. Clapcote, M.N. Cook, R.D. Cox, J.C. Crabbe, W.E. Crusio, A. Darvasi, C.F. Deschepper, R.W. Doerge, C.R. Farber, J. Forejt, D. Gaile, S.J. Garlow, H. Geiger, H. Gershenfeld, T. Gordon, J. Gu, W. Gu, G. de Haan, N.L. Hayes, C. Heller, H. Himmelbauer, R. Hitzemann, K. Hunter, H.C. Hsu, F.A. 30
ACCEPTED MANUSCRIPT Iraqi, B. Ivandic, H.J. Jacob, R.C. Jansen, K.J. Jepsen, D.K. Johnson, T.E. Johnson, G. Kempermann, C. Kendziorski, M. Kotb, R.F. Kooy, B. Llamas, F. Lammert, J.M.
PT
Lassalle, P.R. Lowenstein, L. Lu, A. Lusis, K.F. Manly, R. Marcucio, D. Matthews, J.F.
RI
Medrano, D.R. Miller, G. Mittleman, B.A. Mock, J.S. Mogil, X. Montagutelli, G. Morahan, D.G. Morris, R. Mott, J.H. Nadeau, H. Nagase, R.S. Nowakowski, B.F. O'Hara, A.V.
SC
Osadchuk, G.P. Page, B. Paigen, K. Paigen, A.A. Palmer, H.J. Pan, L. Peltonen-Palotie,
NU
J. Peirce, D. Pomp, M. Pravenec, D.R. Prows, Z. Qi, R.H. Reeves, J. Roder, G.D. Rosen, E.E. Schadt, L.C. Schalkwyk, Z. Seltzer, K. Shimomura, S. Shou, M.J. Sillanpaa,
MA
L.D. Siracusa, H.W. Snoeck, J.L. Spearow, K. Svenson, L.M. Tarantino, D. Threadgill, L.A. Toth, W. Valdar, F.P. de Villena, C. Warden, S. Whatley, R.W. Williams, T.
D
Wiltshire, N. Yi, D. Zhang, M. Zhang, F. Zou, The Collaborative Cross, a community
TE
resource for the genetic analysis of complex traits, Nat Genet, 36 (2004) 1133-1137.
AC CE P
[67] D.W. Threadgill, G.A. Churchill, Ten years of the Collaborative Cross, Genetics, 190 (2012) 291-294.
[68] H.J. Atamni, M. Botzman, R. Mott, I. Gat-Viks, F.A. Iraqi, Mapping liver fat femaledependent quantitative trait loci in collaborative cross mice, Mamm Genome, 27 (2016) 565-573.
[69] H.J. Atamni, R. Mott, M. Soller, F.A. Iraqi, High-fat-diet induced development of increased fasting glucose levels and impaired response to intraperitoneal glucose challenge in the collaborative cross mouse genetic reference population, BMC Genet, 17 (2016) 10.
31
ACCEPTED MANUSCRIPT Table 1
Activity
p.I541F
Frameshift
p.L556R
Deletions
p.Q855L
MA
Nonsense
therapy (likely to be tested in association with UDCA)
TE
Pharmacological
Class V
Stability
No detectable defect
p.S346I
p.T424A
p.R652G
p.F357L
p.N510S
p.T175A
p.P726L p.T775M G954S Nuclear
Nuclear
chaperones (e.g.
receptor
receptor
ciclosporin)
agonists
agonists
(e.g.
(e.g.
fibrates,
fibrates,
obeticholic
obeticholic
acid),
acid),
statins
statins
AC CE P
Potential
Class IV
RI
Maturation
D
Variants
Class III
SC
Defect
Class II
NU
Class I
PT
Classification of ABCB4 variants associated with PFIC3
32
ACCEPTED MANUSCRIPT
PT
Table 2
4 TJP2
Smit et al. Cell 1993
Sambrotta et al. Nat Genet (2014)
NU
3 ABCB4 (MDR3)
MA
1 (Byler syndrome) 2 ATP8B1 (FIC1) ABCB11 (BSEP) Reference Klomp et al. Pawlikowska Hepatology 2004 et al. J Hepatol 2010 Transport Phosphatidylserine Bile acid flippase export Phenotypes Biliary cirrhosis Neonatal giant cell BRIC type 1 hepatitis
Labs
Low ɣ-GT
Biliary cirrhosis
AC
Extrahepatic manifestations: Malabsoprtion, pancreatitis, deafness, pneumonia
CE
PT ED
Type Gene
SC
RI
Phenotypic characteristics of PFIC types 1-6
BRIC type 2 ICP Gallstones DILI HCC Low ɣ-GT
Phosphatidylcholine translocator Biliary cirrhosis with neoductuli
Tight junction protein 2 Early onset chronic cholestasis
Gallstones (LPAC) HCC ICP
5 NR1H4 (FXR) GomezOspina et al. Nat Comm (2016) Nuclear bile acid receptor Neonatal cholestasis with rapid progression to end-stage liver disease
6 MYO5B Gonzalez et al. Hepatology (2017) Myosin 5b Giant-cell hepatocytes and persistent intralobular cholestasis
Respiratory/CNS Cholangiocarcinoma symptoms Vitamin KTransient or HCC independent recurrent coagulopathy cholestasis
High ɣ-GT
Low ɣ-GT
Low ɣ-GT AFP increased
Microvillus inclusion disease Low ɣ-GT
33
ACCEPTED MANUSCRIPT
UDCA OLT
OLT
OLT
UDCA OLT
PT ED
MA
NU
SC
RI
PT
OLT
CE
OLT PBED
AC
Therapy
34
ACCEPTED MANUSCRIPT
Highlights Genetics lead to the dissection of familial and multifactorial cholangiopathies.
•
With the identification of underlying genetic contributors of cholestatic diseases are reclassified.
•
Systems genetics reveals the full complexity of genotype-phenotype relationships in liver.
•
In the future the genetic findings might open new avenues for patients with cholangiopathies.
AC
CE
PT ED
MA
NU
SC
RI
PT
•
35
S0925-4439(17)30261-2 doi:10.1016/j.bbadis.2017.07.029 BBADIS 64841
To appear in:
BBA - Molecular Basis of Disease
Received date: Revised date: Accepted date:
19 June 2017 24 July 2017 25 July 2017
Please cite this article as: Matthias C. Reichert, Rabea A. Hall, Marcin Krawczyk, Frank Lammert, Genetic determinants of cholangiopathies: molecular and systems genetics, BBA - Molecular Basis of Disease (2017), doi:10.1016/j.bbadis.2017.07.029
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Genetic determinants of cholangiopathies: molecular and systems genetics Matthias C. Reichert1*, Rabea A. Hall1*, Marcin Krawczyk1,2 and Frank Lammert1,3 1
Department of Medicine II, Saarland University Medical Center, Homburg, Germany;
2
PT
Laboratory of Metabolic Liver Diseases, Centre for Preclinical Research, Department of
General, Transplant and Liver Surgery, Medical University of Warsaw, Warsaw, Poland; 3
RI
Chair of Internal Medicine II, Saarland University, Saarbrücken, Germany
SC
*These authors contributed equally.
NU
Corresponding author:
Department of Medicine II Saarland University Medical Center Saarland University
Germany
TE
66424 Homburg
D
Kirrberger Str. 100
MA
Prof. Dr. med. Frank Lammert
+49 6841 1623201
Fax:
+49 6841 1623267
Email:
[email protected]
AC CE P
Phone:
Electric word count: 3,817 Number of tables: 2
DISCLOSURE STATEMENT There are no conflicts of interest.
FINANCIAL SUPPORT
1
ACCEPTED MANUSCRIPT The experimental studies that are referred to in this meeting report were, in part, supported by grants from the Federal Ministry of Education and Research of Germany
RI
PT
(BMBF LiSyM 031L0051 to FL).
ABBREVIATIONS alkaline phosphatase
ASBT
apical sodium bile acid transporter
BRIC
benign recurrent intrahepatic cholestasis
CC
collaborative cross
DILI
drug-induced liver injury
ɣ-GT
ɣ-glutamyl transpeptidase
GRP
genetic reference populations
GWAS
genome-wide association studies
HCC
hepatocellular carcinoma
ICP
intrahepatic cholestasis of pregnancy
LPAC
low phospholipid-associated cholelithiasis
PBC
primary biliary cirrhosis
PBED
partial biliary external diversion
PFIC
progressive familial intrahepatic cholestasis
PSC
primary sclerosing cholangitis
RIL
recombinant inbred lines
UDCA
ursodeoxycholic acid
AC CE P
TE
D
MA
NU
SC
AP
2
ACCEPTED MANUSCRIPT Abstract Familial cholangiopathies are rare but potentially severe diseases. Their spectrum
PT
ranges from fairly benign conditions as, for example, benign recurrent intrahepatic
RI
cholestasis to low-phospholipid associated cholelithiasis and progressive familial intrahepatic cholestasis (PFIC). Many cholangiopathies such as primary biliary
SC
cholangitis (PBC) or primary sclerosing cholangitis (PSC) affect first the bile ducts
NU
("ascending pathophysiology") but others, such as PFIC, start upstream in hepatocytes and cause progressive damage "descending" down the biliary tree and leading to end-
MA
stage liver disease. In recent years our understanding of cholestatic diseases has improved, since we have been able to pinpoint numerous disease-causing mutations
D
that cause familial cholangiopathies. Accordingly, six PFIC subtypes (PFIC type 1 - 6)
TE
have now been defined. Given the availability of genotyping resources, these findings
AC CE P
can be introduced in the diagnostic work-up of patients with peculiar cholestasis. In addition, functional studies have defined the pathophysiological consequences of some of the detected variants. Furthermore, ABCB4 variants do not only cause PFIC type 3 but confer an increased risk for chronic liver disease in general. In the near future these findings will serve to develop new therapeutic strategies for patients with liver diseases. Here we present the latest data on the genetic background of familial cholangiopathies and discuss their application in clinical practice for the differential diagnosis of cholestasis of unknown aetiology. As look in the future we present "system genetics" as a novel experimental tool for the study of cholangiopathies and disease-modifying genes.
3
ACCEPTED MANUSCRIPT Keywords:
Cholestasis,
complex
disease,
genetic
test,
phosphatidylcholine
translocator, quantitative trait locus.
PT
Introduction
RI
Most cholangiopathies are heterogeneous and of unclear aetiology[1, 2]. However, it is known that apart from risk factors such as age, gender and ethnicity, they are influenced
SC
by genetic modifiers[3]. Symptoms, disease progression, comorbidities and the effect of
NU
treatments can vary strongly, since the patients carry a wide spectrum of genetic mutations and variants[4]. Consequently, the diseases often present with unclear
MA
phenotype-to-genotype correlations. This complex structure and the heterogeneity of the diseases hampers the search for effective medical treatments. In addition, the definition
D
of cholangiopathies is often non-comprehensive, because they are accompanied by
TE
several comorbidities and symptoms that might not be recognized as early stages of
AC CE P
disease. Therefore, there is a demand for early markers of disease occurrence, progression and predisposing risk factors. Genetic variants leading to deficiencies in transport proteins of the hepatobiliary system have been associated to cholestatic diseases such as progressive familial intrahepatic cholangitis (PFIC)[2] and gallstone disease (cholelithiasis). For example, three different forms of PFIC (types 1- 3) are caused by mutations in transporters located in the hepatocanalicular membrane (i.e. ATP8B1, ABCB11 and ABCB4, respectively)[2]. Next to the primary effects of these risk genes that define the type of disease, additional signaling pathways may be under genetic control and contribute to the (sub)phenotypes of the disease. All these factors result in the individual disease susceptibility of each patient. Case-control studies and genome-wide association studies (GWAS)[5] in human cohorts aim to elucidate underlying disease mechanisms by finding genetic variants that predispose a patient to 4
ACCEPTED MANUSCRIPT a certain disease. However, the assembly of human cohorts requires the recruitment of a large number of patients as well as the elaborate assimilation of recruitment centers
PT
for disease characterization and accurate documentation. Recent GWAS performed in
RI
PBC[6] and PSC[7] have illustrated these limitations, which were overcome by international collaboration and large consortia as well as more detailed phenotypic and
follow-up.
Notwithstanding
these
SC
characterization
efforts
and
the
new
been implemented in clinical algorithms.
NU
pathophysiological insights, the identified risk loci have low effect sizes and have not
MA
Nevertheless, the knowledge about the genetic causes of cholestatic liver diseases has increased significantly in recent years[4]. Currently available genetic tests
D
can be applied for both distinct groups of genetically caused cholestatic liver diseases:
TE
(i) monogenic diseases, such as PFIC, benign recurrent intrahepatic cholestasis (BRIC),
AC CE P
Dubin-Johnson and Rotor syndromes; (ii) polygenic diseases, such as gallstones or drug-induced liver injury (DILI). Single or multiple variants, whole genes (at least all their exons) or panel of genes can be tested. However, the interpretation of the results may be complex. Below we summarize our knowledge about the genetic background of selected biliary diseases. We discuss how disease-causing-variants contribute to the development of the disease. In addition, we present system genetics in experimental models as novel approach to further improve our knowledge about the genetic background of cholangiopathies.
Gallstones Gallstone disease is one of the most common biliary diseases. In Europe approximately 20% of adults carry gallstones[8, 9]. In adults these are predominantly cholesterol 5
ACCEPTED MANUSCRIPT stones. Formerly environmental and lifestyle factors were regarded to be the major determinants of gallstone formation. Now, it is known that genetic factors may account
PT
for up to 40% of total gallstone risk. Indeed, in addition to the well-known exogenous risk
RI
factors, such as overnutrition and physical inactivity, genetic risk factors have been identified. In mice, gallstone formation can be induced by administration of a lithogenic
SC
diet of 1% cholesterol and 0.5% cholic acid. Here, it was observed that different inbred
NU
mouse strains vary in gallstone susceptibility[10]. The differences were analyzed in experimental crosses by several groups and multiple quantitative trait loci (QTLs) and
MA
genes involved in gallstone formation were identified[11]. Wittenburg et al.[12] identified the locus harboring the Abcg5/g8 genes as one of the major QTL for gallstone formation.
D
In line with these results, the first GWAS study in liver diseases, which was performed
TE
more than 10 years ago and included gallstone patients form Germany and Chile,
AC CE P
identified the amino acid substitution ABCG8 p.D19H as risk factor for the development of gallstones[13]. ABCG8 encodes, together with the adjacent oppositely oriented ABCG5, two hemitransporters that are secreting cholesterol across the apical membrane of hepatocytes into bile canaliculi and into the gut lumen. These results were replicated in additional cohorts, rendering this ABCG8 variant the most common genetic risk factor for cholelithiasis[14]. In the meantime, the Gilbert syndrome variant in the promoter of the UGT1A1 gene was shown to also substantially increase the gallstone risk[15, 16]. Genetic screening is currently not recommended by guidelines for gallstone disease[9]. Sequencing of ABCB4 may be considered in young patients with gallstones to confirm the diagnosis of the Low phospholipid-associated cholelithiasis (LPAC) syndrome. 6
AC CE P
TE
D
MA
NU
SC
RI
PT
ACCEPTED MANUSCRIPT
7
ACCEPTED MANUSCRIPT Primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) PSC is a rare (prevalence 1:10.000) inflammatory disease of the bile ducts. The first and
PT
early lesions are in "downstream" bile ducts, which then cause bile salt-mediated toxic
recurrent
biliary
infections
and
RI
injury of the "upstream" liver parenchyma; this "ascending" disease course may lead to liver
cirrhosis[17].
The
risk
to
develop
SC
cholangiocarcinoma is markedly increased (up to 15%). Currently no medical therapy is
NU
established. On the other hand, siblings have an increased risk to develop PSC, pointing to interacting hereditary and environmental factors. GWAS studies in PSC patients
MA
helped to identify multiple associations with Human Leukocyte Antigen (HLA) genotypes and non-HLA genes modulating T-cell functions, indicating the relevance of systemic
D
immune responses and crosstalk between immune cells and cholangiocytes [18, 19].
TE
Detecting these risk variants has currently no clinical application, and testing is not
AC CE P
recommended in clinical practice guidelines yet. Therefore, it is not surprising that several mouse models were developed that try to mimic PSC[20, 21]. So far these models do not fully resemble the disease-specific features.
A
common
model
is
the
Abcb4
knockout
mouse,
which
shows
histopathological features sclerosing cholangitis and biliary fibrosis and progresses to develop hepatocellular cancer at about 12-16 months of age[22, 23]. However, certain disease features are missing such as cholangiocarcinoma and inflammatory bowel disease. PBC (formerly known as primary biliary cirrhosis) is a chronic cholestatic autoimmune disorder of the small bile ducts[24]. Predominantly women are affected (90%). The progressive extinction of small bile ducts leads to liver fibrosis, and cirrhosis may develop. When started at an early stage, therapy with ursodexoycholic acid (UDCA) 8
ACCEPTED MANUSCRIPT can stop the progression of the disease in most patients. As additional therapy, obeticholic acid - a potent ligand of the central nuclear bile acid receptor FXR (NR1H4) -
PT
has been approved in combination with UDCA in PBC patients with an inadequate
RI
response to UDCA (or as monotherapy in patients unable to tolerate UDCA)[25]. In GWAS the strongest genetic associations in PBC are HLA associations. Most non-HLA
SC
associations in PBC overlap with other autoimmune diseases, with risk loci indicating
NU
altered immunoregulatory pathways, dysregulated mucosal immunity, or aberrant microbial handling[26-28]. Less than 20% of the variability is explained by GWAS,
MA
pointing to the relevance of rare variants and epigenetics. Gene testing for risk variants
D
has no relevance in clinical care of PBC patients yet.
TE
Low phospholipid-associated cholelithiasis (LPAC) syndrome and ABCB4
AC CE P
deficiency
The LPAC syndrome is characterized by low concentrations of phospholipids in bile and an increased risk for gallstones, which may recur after cholecystectomy[29]. LPAC is caused by the reduced transport of phosphatidylcholine into bile due to the dysfunction of the hepatocanalicular phospholipid translocator ABCB4, which can be caused by different ABCB4 gene variants. LPAC is a clinical diagnosis, based on the following three criteria[30]:
biliary symptoms before the age of 40 years;
detection of intrahepatic microlithiasis/sludge by ultrasound (hyperechogenic foci);
recurrence of symptomatic cholelithasis after cholecystectomy.
9
ACCEPTED MANUSCRIPT Most (but not all) patients with LPAC benefit from therapy with UDCA, which should be started early at young age. The diagnosis can be confirmed by microscopic examination
PT
of endoscopically acquired hepatic bile, which contains aggregated cholesterol crystals
RI
or microliths, the measurement of biliary phospholipid concentrations (in frozen bile), or the sequencing of all ABCB4 exons. In more than half of the patients, a pathogenic risk
SC
variant in ABCB4 has been detected[30].
NU
ABCB4 deficiency summarizes several conditions that are caused by gene variants in ABCB4. The clinical spectrum of ABCB4-associated liver phenotypes is
MA
broad. The phenotypes range from benign diseases such as intrahepatic cholestasis of pregnancy (ICP) due to mild and/or heterozygous variants to LPAC and PFIC type 3,
D
resulting in biliary liver cirrhosis, in the setting of severe homozygous ABCB4 mutations.
TE
The detection of genetic variants can be helpful for diagnosis and counseling of patients,
AC CE P
but genotype-phenotype-correlations are imprecise. Recently, a new classification for ABCB4 mutations has been suggested (class I - V), as follows (Table 1): (I) nonsense variations; (II) missense variations that primarily affect maturation; (III) activity; or (IV) stability; and (V) variants without detectable effects[31]. Furthermore, variants in ABCB4 have been found to be associated with gallbladder cancer risk[32]. They might also promote the progression of liver fibrosis in patients with chronic liver diseases in general[33]. Of note, large-scale whole-genome sequencing of the Icelandic population has demonstrated significant associations between ABCB4 variants with liver diseases and liver enzyme activities in the general population[34]: The common ABCB4 variant c.711A>T was identified as general risk factor for elevated aminotransferases and higher impact variants were associated with early-onset gallstone disease, ICP, liver
10
ACCEPTED MANUSCRIPT cirrhosis, and hepatobiliary cancers, thus setting their carriers "on the highway to liver
PT
disease"[35].
RI
Familiar intrahepatic cholestasis
This clinically heterogenic group of diseases is characterized by familial aggregation and
SC
intrahepatic cholestasis and can be further classified into (i) PFIC types 1-6 with mostly
NU
dismal prognosis; and (ii) BRIC with good prognosis. The diseases are caused by mutations in the hepatocanalicular transporters ATP8B1, ABCB11 or ABCB4, or the
MA
TJP2, NR1H4 and MYO5B genes (Table 2).
D
Progressive familiar intrahepatic cholestasis (PFIC)
TE
This is a group of rare diseases (incidence 1:50.000). In children it can cause severe
AC CE P
cholestatic liver disease. It commonly manifests in the first year of life (2-9 months) with pruritus and jaundice. The intra- and extrahepatic bile ducts are not dilated. The course of the disease and the clinical phenotypes are heterogeneous, typically with rapid progression to liver cirrhosis at the age of 3-15 years and death if no liver transplantation is performed. Most cases of PFIC are caused by mutations in hepatocanalicular transporter genes with autosomal recessive inheritance. The diagnosis of PFIC is established through genetic testing. In these monogenic cholestatic liver diseases, sequence analysis of the whole gene, comprising at least the coding region, flanking splice sites and possibly regulatory regions, is recommended.
11
ACCEPTED MANUSCRIPT PFIC type 1 Formerly known as Byler syndrome, this cholestatic liver disease typically manifests
PT
within the first year of life with marked pruritus and progression to liver cirrhosis at the
RI
age of 2-7 years. Of note, extrahepatic manifestations (malabsorption, pancreatitis, deafness, pneumonia) can occur. Laboratory analysis reveals raised alkaline
SC
phosphatase (AP) and normal ɣ-glutamyl transpeptidase (ɣ-GT) activities. Liver biopsy
NU
initially shows canalicular cholestasis, later liver cirrhosis develops. PFIC1 is due to mutations of the ATP8B1 (FIC1) gene[36], encoding the transporter responsible for
MA
maintaining the asymmetry of the hepatocanalicular membrane by enrichment of phosphatidylserine in the inner leaflet (flippase). Distorted membrane composition
D
impacts bile acid secretion, which is reflected by low bile acid concentrations in hepatic
TE
bile of PFIC1 patients[2]. As one of the possible mechanisms, downregulation of FXR
AC CE P
could result in repression of the bile acid export pump (ABCB11) in the liver and induction of the apical sodium bile acid transporter (ASBT) in the intestine[17]. The expression of ATP8B1 is higher in the intestine than in the liver and therefore might influence the enterohepatic cycling of bile acids. Many PFIC1 patients need to undergo liver transplantation but in particular this subtype of PFIC might also benefit from early partial biliary external diversion (PBED)[37], for which several different surgical techniques were described[38]. Shah et al. [39] reported data on Atp8b1G308V-knockin mice, which demonstrated showed similar defects in bile acid homeostasis. Still the disease does not progress to a stage resembling advanced human cholestatic disease[39]. Yet, the study by Shah et al.[39] showed that the severity of the ATP8B1 deficiency depends on the genetic background of the strain. 12
ACCEPTED MANUSCRIPT
PFIC type 2
PT
Mutations in ABCB11 (BSEP) cause PFIC2. ABCB11 functions as the bile salt export
RI
pump in the hepatocanalicular membrane, and ABCB11 mutations cause decreased bile acid secretion, accumulation of bile salts in the liver, and hepatocellular injury. PFIC2
SC
manifests typically within the first six months of life with marked pruritus and jaundice.
NU
The clinical course is typically progressive with development of liver cirrhosis within the first two years of life. In later life, the risk for hepatocellular and cholangiocellular cancers
MA
is increased. In laboratory work-up, ALT is higher and AP activities are lower than in PFIC1, with both showing normal ɣ-GT[40]. Histopathologically, initially typically
D
canalicular cholestasis with giant cell hepatitis and lobular fibrosis is found. Functional
TE
ABCB11 gene variants are causative, and up to 60% of European patients harbour one
AC CE P
of the two "hot-spot" mutations p.E297G or p.D482G, but diagnosis is established by gene sequencing in most patients. In the mouse the Abcb11 knockout leads to mild cholestasis only, and the lack of Abcb11 expression seems to be compensated by upregulation of other ATP transporter genes, e.g. Abca1 and Abcb1b. In Abcb11-knockout mice the secretion of hydrophobic bile acids is reduced, while hydrophilic bile acids (β- and Ω-muricholic acid) are secreted by the compensatory transporters. This was confirmed by generating a triple knockout mouse with ABCB11 and ABCA1 (transcripts a and b) deficiency, which presented more severe phenotypes resembling human cholestatic disease, such as inflammatory infiltrates, biliary fibrosis, and deteriorated microvilli in the canaliculi[2, 41]. Nevertheless, disease severity was still generally milder in the mouse model than in humans[2, 41], since mice can detoxify hydrophobic bile salts by hydroxylation. Hence, even though 13
ACCEPTED MANUSCRIPT mouse and humans share most of their genetic information, mouse models need to be evaluated for pathophysiological similarities. As seen in this example, the experimental
RI
PT
setup can often be adjusted to allow further modelling.
PFIC type 3
SC
Patients with PFIC3 carry a defective hepatocanalicular phosphatidylcholine translocator
NU
ABCB4. The formation of mixed micelles in bile depends on the correct composition of bile, which mainly consists of water, bile acids, phosphatiylcholine, and cholesterol. Low
MA
biliary phospholipid concentrations and the resulting imbalance of these three components leads to bile acid toxicity as well as cholesterol supersaturation and
D
crystallization[22, 42, 43]. The severity of the disease varies with the type of mutation
TE
and the remaining function of the transporter. PFIC3 can manifest within the first year of
AC CE P
life (up to 30%) and liver cirrhosis then often develops already in childhood, but ABCB4 deficiency may also present later in life with moderate jaundice. Laboratory tests show increased aminotransferase activities and - in contrast to PFIC types 1 and 2 - elevated ɣ-GT activities. Liver biopsy reveals neoductular proliferations and in further course progression of liver fibrosis. Patients with PFIC3 may benefit from higher doses of UDCA (15 - 20 mg/kg body weight/day) but often liver transplantation is required. The Abcb4-knockout mouse is deficient for the orthologous murine gene and an appropriate PFIC3 model, albeit sponatenous disease severity is less advanced than in humans and older mice develop hepatocellular cancer but not cholangiocarcinoma. Of note, knockout mice present with bile that is supersaturated with cholesterol and develop intra- and extrahepatic gallstones similar to patients with LPAC[44].
14
ACCEPTED MANUSCRIPT PFIC types 4 - 6 Recently a fourth type of PFIC was described in children (Table 2). It is caused by
PT
mutations in the gene encoding the tight junction protein TJP2. This leads to dysfunctional tight junctions and early onset cholestasis. Similar to PFIC1 and PFIC2,
RI
serum ɣ-GT are not increased, in contrast to high bilirubin and bile acid concentrations.
SC
Some patients develop hepatocellular carcinoma (HCC) and most of them require liver
NU
transplantation[45].
Mutations in NR1H4 encoding the nuclear receptor FXR were found in children,
MA
who presented with neonatal cholestasis and rapid progression to end-stage liver disease[46]. This condition has been called PFIC type 5. Lately, mutations in MYO5B, a
D
gene encoding for a large protein that is required for hepatocyte polarization, were
TE
detected. Whereas severe MYO5B mutations underlie systemic microvillus inclusion
AC CE P
disease, mild variants cause persistent, transient or recurrent cholestasis as liverrestricted phenotypes. The disease has been labeled PFIC type 6[47].
Benign recurrent intrahepatic cholestasis (BRIC) This disease was formerly called Summerskill-Walshe-Tygstrup-Syndrome and was first described in 1959. It is a very rare disease with familial clustering. The first flare typically occurs in young patients. BRIC is characterized by prolonged episodes of cholestasis with jaundice, pruritus, and malabsorption[48]. These episodes might be triggered by not well defined exogenous factors (infections, hormones or other drugs). BRIC flares resolve completely after weeks or months without any major damage to the liver. They might recur after an asymptomatic period of months to years. Liver biopsy shows bland intrahepatic cholestasis. During remission, liver morphology and function are normal. As 15
ACCEPTED MANUSCRIPT per definition, no progression to liver cirrhosis occurs in contrast to PFIC. Genetically different mutations in the ATP8B1 (BRIC type 1) or ABCB11 genes (BRIC type 2) can be
PT
detected[49, 50]. 45% of BRIC1 patients carry the amino acid substitution p.I661T.
RI
Sequencing of both genes confirms the diagnosis. Although larger studies are missing,
NU
nasobiliary drainage, or plasmapheresis[51, 52].
SC
case reports highlight the successful treatment of BRIC with UDCA, rifampicin,
Association studies and systems genetics in experimental models
MA
Trying to tackle complex diseases like cholangiopathies has shown us how important it is to understand complex biological systems. Systems genetics approaches encompass
populations,
which
TE
of
D
multiple levels of (patho)biological data. Systems genetics is defined as systems biology are
inter-breeding
individuals
AC CE P
in a specific environment[53]. Animal models may fill the gap where human studies fail to succeed and can help to achieve a better understanding of critical pathways that are under genetic control[54-56]. Mice and humans share about 95% of their genome and many disease genes were also identified in mice. In contrast to human studies genetically different mice can be analyzed under controlled environmental conditions. In addition the mouse genome can be changed to analyze potential modifier genes in detail. Inbred mouse strains, which are homozygous at all loci, often display different disease susceptibility due to their distinct genetic backgrounds[57]. Genetic mapping in mouse strains enhances the power of detecting modifier genes and identifying complex genetic interactions. Genomewide quantitative trait locus (QTL) analysis, as described in more detail below, represents a promising approach to detect genetic variants that are associated with specific phenotypes and interact with each other. 16
ACCEPTED MANUSCRIPT In experimental crosses of two (inbred) strains the first generation (F1) of offsprings is genetically heterozygous but equal. Then in the next generation (F2) the
PT
strain-specific genetic information is distributed across the genomes of their progeny and
RI
each offspring is genetically unique. Accordingly, F2 mice represent a population of mice displaying highly variable phenotypes. They are a promising tool to identify new disease
SC
modifiers in association studies. QTL analysis detect genomic regions that harbor genes
NU
associated with disease susceptibility[58, 59]. More than 200 mice are typically required for the analysis of an F2 cross. Each mouse is genotyped for genetic markers covering
MA
the whole genome, but it is unique, which limits the phenotypic characterization of multiple traits. Nevertheless, an F2 cross can be employed for the analysis of genetic
D
background effects when using genetically modified mice. In a recent QTL study we
TE
transferred the Abcb4 knockout from the fibrosis resistant FVB/NJ strain to the fibrosis
AC CE P
susceptible BALB/cJ strain by repeated backcrossing. These two congenic knockout strains were then crossed to generate F2 progeny, all of which are ABCB4 deficient. At the age of 16 weeks the F2 mice developed biliary fibrosis of variable severity. Each mouse was genotyped for single nucleotide variants across all chromosomes. Phenotypic differences were quantified by hepatic collagen contents and other subphenotypes, and then the genetic regions linked to these phenotypes were mapped as described above. Similar to genetic studies in human cohorts, the identified risk loci showed small effect sizes. However, in this model system the analysis of gene-gene interactions and epistatic effects is possible, which allowed us to identify genetic regions that are significantly linked to the phenotypes. These regions contain genetic modifiers dependent on ABCB4 deficiency. From this point it is possible to go back to patient cohorts to study the orthologous modifier genes in humans. The mouse studies enable 17
ACCEPTED MANUSCRIPT the identification of new modifiers that remain masked in human populations or affect phenotypes that cannot be quantified non-invasively in patients.
PT
An alternative to the F2 cross are experimental crosses that are used to establish
RI
genetic reference populations (GRP). Here, the F2 generation is inbred by continuous brothersister matings for more than 20 generations to generate recombinant inbred
SC
lines (RIL). This results in homozygous but heterogenic RILs, where each line consists
NU
of mice that resemble genetic clones. The genetic identical mice allow the analysis of different models and experimental conditions. One of the best studied genetic reference
MA
populations are the BXD recombinant inbred lines[60]. They were generated starting with the two inbred strains C57BL/6J and DBA/2J, and now more than 100 RILs exist
TE
D
that have been screened for several phenotypes, including liver diseases. The advantage of these lines is that they were also genotyped and the genetic data was
AC CE P
uploaded into GeneNetwork (www.genenetwork.org)[61], where it is freely available for every scientist working with this GRP together with all phenotypic data from other projects or groups. GeneNetwork enables QTL and multiscale correlation analyses. This approach saves resources and at the same time contributes to the dissection of disease networks at multiple biological levels. It represents a new step towards a systems biology approach and a better understanding of complex diseases[62-65]. The collaborative cross (CC) represents the "next generation" of GRPs and the future tool for systems genetics in mouse models. The CC lines are generated in a collaboration project by the Complex Trait Consortium (www.complextrait.org)[66], and as to date about 300 lines have been generated. By introducing eight strains in an eightway cross, the genetic diversity of the offsprings was increased aiming for finer mapping resolution than in F2 crosses or the BXD RILs. The improved resolution reduces the 18
ACCEPTED MANUSCRIPT number of candidate genes in the identified QTL regions. Nevertheless, the groups generating the CC lines still have to overcome several challenges, e.g. difficult breeding
PT
the lines to achieve fully inbreds and high complexity of the analysis based on eight
RI
instead of two alleles[67]. Yet, the first disease studies were published recently and demonstrated the power of CC lines for the genetic dissection of complex diseases[68,
SC
69]. These technical advances in mice allow a better understanding of the complex
NU
genetics of cholangiopathies. The increasing complexity of the experimental systems provides new insights that can hopefully be translated to human populations in the near
MA
future.
D
Key points
TE
• Genetics lead to the dissection of familial and multifactorial cholangiopathies.
AC CE P
• With the identification of underlying genetic contributors cholestatic diseases are being reclassified.
• Systems genetics reveals the full complexity of genotype-phenotype relationships in liver.
• In the future the genetic findings might open new avenues for treating patients with cholangiopathies.
19
ACCEPTED MANUSCRIPT References [1] K.N. Lazaridis, N.F. LaRusso, Primary Sclerosing Cholangitis, N Engl J Med, 375
PT
(2016) 1161-1170.
RI
[2] E. Jacquemin, Progressive familial intrahepatic cholestasis, Clin Res Hepatol
SC
Gastroenterol, 36 Suppl 1 (2012) S26-35.
[3] G.M. Hirschfield, R.W. Chapman, T.H. Karlsen, F. Lammert, K.N. Lazaridis, A.L.
NU
Mason, The genetics of complex cholestatic disorders, Gastroenterology, 144 (2013)
MA
1357-1374.
[4] T.H. Karlsen, F. Lammert, R.J. Thompson, Genetics of liver disease: From
D
pathophysiology to clinical practice, J Hepatol, 62 (2015) S6-S14.
TE
[5] M. Krawczyk, R. Mullenbach, S.N. Weber, V. Zimmer, F. Lammert, Genome-wide association studies and genetic risk assessment of liver diseases, Nat Rev
AC CE P
Gastroenterol Hepatol, 7 (2010) 669-681. [6] A.F. Gulamhusein, B.D. Juran, K.N. Lazaridis, Genome-Wide Association Studies in Primary Biliary Cirrhosis, Semin Liver Dis, 35 (2015) 392-401. [7] A. Paziewska, A. Habior, A. Rogowska, W. Zych, K. Goryca, J. Karczmarski, M. Dabrowska, F. Ambrozkiewicz, B. Walewska-Zielecka, M. Krawczyk, H. Cichoz-Lach, P. Milkiewicz, A. Kowalik, K. Mucha, J. Raczynska, J. Musialik, G. Boryczka, M. Wasilewicz, I. Ciecko-Michalska, M. Ferenc, M. Janiak, A. Kanikowska, R. Stankiewicz, M. Hartleb, T. Mach, M. Grzymislawski, J. Raszeja-Wyszomirska, E. Wunsch, T. Bobinski, M. Mikula, J. Ostrowski, A novel approach to genome-wide association analysis identifies genetic associations with primary biliary cholangitis and primary sclerosing cholangitis in Polish patients, BMC Med Genomics, 10 (2017) 2.
20
ACCEPTED MANUSCRIPT [8] R. Aerts, F. Penninckx, The burden of gallstone disease in Europe, Aliment Pharmacol Ther, 18 Suppl 3 (2003) 49-53.
PT
[9] L. European Association for the Study of the, EASL Clinical Practice Guidelines on
RI
the prevention, diagnosis and treatment of gallstones, J Hepatol, 65 (2016) 146-181. [10] M. Alexander, O.W. Portman, Different susceptibilities to the formation of
SC
cholesterol gallstones in mice, Hepatology, 7 (1987) 257-265.
NU
[11] D.Q. Wang, N.H. Afdhal, Genetic analysis of cholesterol gallstone formation: searching for Lith (gallstone) genes, Curr Gastroenterol Rep, 6 (2004) 140-150.
MA
[12] H. Wittenburg, M.A. Lyons, R. Li, G.A. Churchill, M.C. Carey, B. Paigen, FXR and ABCG5/ABCG8 as determinants of cholesterol gallstone formation from quantitative trait
D
locus mapping in mice, Gastroenterology, 125 (2003) 868-881.
TE
[13] S. Buch, C. Schafmayer, H. Volzke, C. Becker, A. Franke, H. von Eller-Eberstein, C.
AC CE P
Kluck, I. Bassmann, M. Brosch, F. Lammert, J.F. Miquel, F. Nervi, M. Wittig, D. Rosskopf, B. Timm, C. Holl, M. Seeger, A. ElSharawy, T. Lu, J. Egberts, F. Fandrich, U.R. Folsch, M. Krawczak, S. Schreiber, P. Nurnberg, J. Tepel, J. Hampe, A genomewide association scan identifies the hepatic cholesterol transporter ABCG8 as a susceptibility factor for human gallstone disease, Nat Genet, 39 (2007) 995-999. [14] M. Krawczyk, D.Q. Wang, P. Portincasa, F. Lammert, Dissecting the genetic heterogeneity of gallbladder stone formation, Semin Liver Dis, 31 (2011) 157-172. [15] S. Stender, R. Frikke-Schmidt, B.G. Nordestgaard, A. Tybjaerg-Hansen, Extreme bilirubin levels as a causal risk factor for symptomatic gallstone disease, JAMA Intern Med, 173 (2013) 1222-1228.
21
ACCEPTED MANUSCRIPT [16] H.U. Marschall, M. Krawczyk, F. Grunhage, D. Katsika, C. Einarsson, F. Lammert, Gallstone disease in Swedish twins is associated with the Gilbert variant of UGT1A1,
PT
Liver Int, 33 (2013) 904-908.
RI
[17] P.L. Jansen, A. Ghallab, N. Vartak, R. Reif, F.G. Schaap, J. Hampe, J.G. Hengstler, The ascending pathophysiology of cholestatic liver disease, Hepatology, 65 (2017) 722-
SC
738.
NU
[18] D. Ellinghaus, L. Jostins, S.L. Spain, A. Cortes, J. Bethune, B. Han, Y.R. Park, S. Raychaudhuri, J.G. Pouget, M. Hubenthal, T. Folseraas, Y. Wang, T. Esko, A. Metspalu,
MA
H.J. Westra, L. Franke, T.H. Pers, R.K. Weersma, V. Collij, M. D'Amato, J. Halfvarson, A.B. Jensen, W. Lieb, F. Degenhardt, A.J. Forstner, A. Hofmann, I.B.D.G.C. C.
International
Genetics
D
International,
of
Ankylosing
Spondylitis,
P.S.C.S.G.
TE
International, C. Genetic Analysis of Psoriasis, E. Psoriasis Association Genetics, S.
AC CE P
Schreiber, U. Mrowietz, B.D. Juran, K.N. Lazaridis, S. Brunak, A.M. Dale, R.C. Trembath, S. Weidinger, M. Weichenthal, E. Ellinghaus, J.T. Elder, J.N. Barker, O.A. Andreassen, D.P. McGovern, T.H. Karlsen, J.C. Barrett, M. Parkes, M.A. Brown, A. Franke, Analysis of five chronic inflammatory diseases identifies 27 new associations and highlights disease-specific patterns at shared loci, Nat Genet, 48 (2016) 510-518. [19] J.Z. Liu, J.R. Hov, T. Folseraas, E. Ellinghaus, S.M. Rushbrook, N.T. Doncheva, O.A. Andreassen, R.K. Weersma, T.J. Weismuller, B. Eksteen, P. Invernizzi, G.M. Hirschfield, D.N. Gotthardt, A. Pares, D. Ellinghaus, T. Shah, B.D. Juran, P. Milkiewicz, C. Rust, C. Schramm, T. Muller, B. Srivastava, G. Dalekos, M.M. Nothen, S. Herms, J. Winkelmann, M. Mitrovic, F. Braun, C.Y. Ponsioen, P.J. Croucher, M. Sterneck, A. Teufel, A.L. Mason, J. Saarela, V. Leppa, R. Dorfman, D. Alvaro, A. Floreani, S. Onengut-Gumuscu, S.S. Rich, W.K. Thompson, A.J. Schork, S. Naess, I. Thomsen, G. 22
ACCEPTED MANUSCRIPT Mayr, I.R. Konig, K. Hveem, I. Cleynen, J. Gutierrez-Achury, I. Ricano-Ponce, D. van Heel, E. Bjornsson, R.N. Sandford, P.R. Durie, E. Melum, M.H. Vatn, M.S. Silverberg,
PT
R.H. Duerr, L. Padyukov, S. Brand, M. Sans, V. Annese, J.P. Achkar, K.M. Boberg, H.U.
RI
Marschall, O. Chazouilleres, C.L. Bowlus, C. Wijmenga, E. Schrumpf, S. Vermeire, M. Albrecht, U.-P. Consortium, J.D. Rioux, G. Alexander, A. Bergquist, J. Cho, S.
SC
Schreiber, M.P. Manns, M. Farkkila, A.M. Dale, R.W. Chapman, K.N. Lazaridis,
NU
P.S.C.S.G. International, A. Franke, C.A. Anderson, T.H. Karlsen, I.B.D.G.C. International, Dense genotyping of immune-related disease regions identifies nine new
MA
risk loci for primary sclerosing cholangitis, Nat Genet, 45 (2013) 670-675. [20] P. Fickert, M.J. Pollheimer, U. Beuers, C. Lackner, G. Hirschfield, C. Housset, V.
D
Keitel, C. Schramm, H.U. Marschall, T.H. Karlsen, E. Melum, A. Kaser, B. Eksteen, M.
TE
Strazzabosco, M. Manns, M. Trauner, Characterization of animal models for primary
AC CE P
sclerosing cholangitis (PSC), J Hepatol, 60 (2014) 1290-1303. [21] J.M. Vierling, Animal models for primary sclerosing cholangitis, Best Pract Res Clin Gastroenterol, 15 (2001) 591-610. [22] J.J. Smit, A.H. Schinkel, R.P. Oude Elferink, A.K. Groen, E. Wagenaar, L. van Deemter, C.A. Mol, R. Ottenhoff, N.M. van der Lugt, M.A. van Roon, et al., Homozygous disruption of the murine mdr2 P-glycoprotein gene leads to a complete absence of phospholipid from bile and to liver disease, Cell, 75 (1993) 451-462. [23] M. Katzenellenbogen, L. Mizrahi, O. Pappo, N. Klopstock, D. Olam, J. Jacob-Hirsch, N. Amariglio, G. Rechavi, E. Domany, E. Galun, D. Goldenberg, Molecular mechanisms of liver carcinogenesis in the mdr2-knockout mice, Mol Cancer Res, 5 (2007) 11591170.
23
ACCEPTED MANUSCRIPT [24] e.e.e. European Association for the Study of the Liver. Electronic address, G.M. Hirschfield, U. Beuers, C. Corpechot, P. Invernizzi, D. Jones, M. Marzioni, C. Schramm,
PT
EASL Clinical Practice Guidelines: The diagnosis and management of patients with
RI
primary biliary cholangitis, J Hepatol, (2017).
[25] F. Nevens, P. Andreone, G. Mazzella, S.I. Strasser, C. Bowlus, P. Invernizzi, J.P.
SC
Drenth, P.J. Pockros, J. Regula, U. Beuers, M. Trauner, D.E. Jones, A. Floreani, S.
NU
Hohenester, V. Luketic, M. Shiffman, K.J. van Erpecum, V. Vargas, C. Vincent, G.M. Hirschfield, H. Shah, B. Hansen, K.D. Lindor, H.U. Marschall, K.V. Kowdley, R.
MA
Hooshmand-Rad, T. Marmon, S. Sheeron, R. Pencek, L. MacConell, M. Pruzanski, D. Shapiro, P.S. Group, A Placebo-Controlled Trial of Obeticholic Acid in Primary Biliary
D
Cholangitis, N Engl J Med, 375 (2016) 631-643.
TE
[26] H.J. Cordell, Y. Han, G.F. Mells, Y. Li, G.M. Hirschfield, C.S. Greene, G. Xie, B.D.
AC CE P
Juran, D. Zhu, D.C. Qian, J.A. Floyd, K.I. Morley, D. Prati, A. Lleo, D. Cusi, U.S.P.B.C.C. Canadian, P.B.C.G.S.G. Italian, U.-P. Consortium, M.E. Gershwin, C.A. Anderson, K.N. Lazaridis, P. Invernizzi, M.F. Seldin, R.N. Sandford, C.I. Amos, K.A. Siminovitch, International genome-wide meta-analysis identifies new primary biliary cirrhosis risk loci and targetable pathogenic pathways, Nat Commun, 6 (2015) 8019. [27] G.F. Mells, J.A. Floyd, K.I. Morley, H.J. Cordell, C.S. Franklin, S.Y. Shin, M.A. Heneghan, J.M. Neuberger, P.T. Donaldson, D.B. Day, S.J. Ducker, A.W. Muriithi, E.F. Wheater, C.J. Hammond, M.F. Dawwas, U.P. Consortium, C. Wellcome Trust Case Control, D.E. Jones, L. Peltonen, G.J. Alexander, R.N. Sandford, C.A. Anderson, Genome-wide association study identifies 12 new susceptibility loci for primary biliary cirrhosis, Nat Genet, 43 (2011) 329-332.
24
ACCEPTED MANUSCRIPT [28] P.J. Trivedi, G.M. Hirschfield, The Immunogenetics of Autoimmune Cholestasis, Clin Liver Dis, 20 (2016) 15-31.
PT
[29] O. Rosmorduc, B. Hermelin, P.Y. Boelle, R. Parc, J. Taboury, R. Poupon, ABCB4
RI
gene mutation-associated cholelithiasis in adults, Gastroenterology, 125 (2003) 452459.
SC
[30] R. Poupon, O. Rosmorduc, P.Y. Boelle, Y. Chretien, C. Corpechot, O.
NU
Chazouilleres, C. Housset, V. Barbu, Genotype-phenotype relationships in the low-
Hepatology, 58 (2013) 1105-1110.
MA
phospholipid-associated cholelithiasis syndrome: a study of 156 consecutive patients,
[31] J.L. Delaunay, A.M. Durand-Schneider, C. Dossier, T. Falguieres, J. Gautherot, A.
D
Davit-Spraul, T. Ait-Slimane, C. Housset, E. Jacquemin, M. Maurice, A functional
TE
classification of ABCB4 variations causing progressive familial intrahepatic cholestasis
AC CE P
type 3, Hepatology, 63 (2016) 1620-1631. [32] S. Mhatre, Z. Wang, R. Nagrani, R. Badwe, S. Chiplunkar, B. Mittal, S. Yadav, H. Zhang, C.C. Chung, P. Patil, S. Chanock, R. Dikshit, N. Chatterjee, P. Rajaraman, Common genetic variation and risk of gallbladder cancer in India: a case-control genome-wide association study, Lancet Oncol, 18 (2017) 535-544. [33] M. Krawczyk, M. Rau, F. Grunhage, J.M. Schattenberg, H. Bantel, A. Pathil, M. Demir, J. Kluwe, T. Boettler, A. Geier, F. Lammert, N. Clinical Study Group, The ABCB4 p.T175A variant as potential modulator of hepatic fibrosis in patients with chronic liver diseases: Looking beyond the cholestatic realm, Hepatology, (2017). [34] D.F. Gudbjartsson, H. Helgason, S.A. Gudjonsson, F. Zink, A. Oddson, A. Gylfason, S. Besenbacher, G. Magnusson, B.V. Halldorsson, E. Hjartarson, G.T. Sigurdsson, S.N. Stacey, M.L. Frigge, H. Holm, J. Saemundsdottir, H.T. Helgadottir, H. Johannsdottir, G. 25
ACCEPTED MANUSCRIPT Sigfusson, G. Thorgeirsson, J.T. Sverrisson, S. Gretarsdottir, G.B. Walters, T. Rafnar, B. Thjodleifsson, E.S. Bjornsson, S. Olafsson, H. Thorarinsdottir, T. Steingrimsdottir, T.S.
PT
Gudmundsdottir, A. Theodors, J.G. Jonasson, A. Sigurdsson, G. Bjornsdottir, J.J.
RI
Jonsson, O. Thorarensen, P. Ludvigsson, H. Gudbjartsson, G.I. Eyjolfsson, O. Sigurdardottir, I. Olafsson, D.O. Arnar, O.T. Magnusson, A. Kong, G. Masson, U.
SC
Thorsteinsdottir, A. Helgason, P. Sulem, K. Stefansson, Large-scale whole-genome
NU
sequencing of the Icelandic population, Nat Genet, 47 (2015) 435-444. [35] F. Lammert, K. Hochrath, A letter on ABCB4 from Iceland: On the highway to liver
MA
disease, Clin Res Hepatol Gastroenterol, 39 (2015) 655-658. [36] L.W. Klomp, J.C. Vargas, S.W. van Mil, L. Pawlikowska, S.S. Strautnieks, M.J. van
D
Eijk, J.A. Juijn, C. Pabon-Pena, L.B. Smith, J.A. DeYoung, J.A. Byrne, J. Gombert, G.
TE
van der Brugge, R. Berger, I. Jankowska, J. Pawlowska, E. Villa, A.S. Knisely, R.J.
AC CE P
Thompson, N.B. Freimer, R.H. Houwen, L.N. Bull, Characterization of mutations in ATP8B1 associated with hereditary cholestasis, Hepatology, 40 (2004) 27-38. [37] I. Jankowska, P. Czubkowski, A. Wierzbicka, J. Pawlowska, P. Kalicinski, P. Socha, Influence of Partial External Biliary Diversion on the Lipid Profile in Children With Progressive Familial Intrahepatic Cholestasis, J Pediatr Gastroenterol Nutr, 63 (2016) 598-602. [38] M. Gunaydin, B. Tander, D. Demirel, G. Caltepe, A.G. Kalayci, E. Eren, U. Bicakci, R. Rizalar, E. Ariturk, F. Bernay, Different techniques for biliary diversion in progressive familial intrahepatic cholestasis, J Pediatr Surg, 51 (2016) 386-389. [39] S. Shah, U.R. Sanford, J.C. Vargas, H. Xu, A. Groen, C.C. Paulusma, J.P. Grenert, L. Pawlikowska, S. Sen, R.P. Elferink, L.N. Bull, Strain background modifies phenotypes in the ATP8B1-deficient mouse, PLoS One, 5 (2010) e8984. 26
ACCEPTED MANUSCRIPT [40] L. Pawlikowska, S. Strautnieks, I. Jankowska, P. Czubkowski, K. Emerick, A. Antoniou, C. Wanty, B. Fischler, E. Jacquemin, S. Wali, S. Blanchard, I.M. Nielsen, B.
PT
Bourke, S. McQuaid, F. Lacaille, J.A. Byrne, A.M. van Eerde, K.L. Kolho, L. Klomp, R.
RI
Houwen, P. Bacchetti, S. Lobritto, V. Hupertz, P. McClean, G. Mieli-Vergani, B. Shneider, A. Nemeth, E. Sokal, N.B. Freimer, A.S. Knisely, P. Rosenthal, P.F.
SC
Whitington, J. Pawlowska, R.J. Thompson, L.N. Bull, Differences in presentation and
NU
progression between severe FIC1 and BSEP deficiencies, J Hepatol, 53 (2010) 170178.
MA
[41] R. Wang, H.L. Chen, L. Liu, J.A. Sheps, M.J. Phillips, V. Ling, Compensatory role of P-glycoproteins in knockout mice lacking the bile salt export pump, Hepatology, 50
D
(2009) 948-956.
TE
[42] P. Fickert, A. Fuchsbichler, M. Wagner, G. Zollner, A. Kaser, H. Tilg, R. Krause, F.
AC CE P
Lammert, C. Langner, K. Zatloukal, H.U. Marschall, H. Denk, M. Trauner, Regurgitation of bile acids from leaky bile ducts causes sclerosing cholangitis in Mdr2 (Abcb4) knockout mice, Gastroenterology, 127 (2004) 261-274. [43] J. Eckstein, N. Berndt, H.G. Holzhutter, Computer simulations suggest a key role of membranous nanodomains in biliary lipid secretion, PLoS Comput Biol, 11 (2015) e1004033. [44] F. Lammert, D.Q. Wang, S. Hillebrandt, A. Geier, P. Fickert, M. Trauner, S. Matern, B. Paigen, M.C. Carey, Spontaneous cholecysto- and hepatolithiasis in Mdr2-/- mice: a model for low phospholipid-associated cholelithiasis, Hepatology, 39 (2004) 117-128. [45] M. Sambrotta, S. Strautnieks, E. Papouli, P. Rushton, B.E. Clark, D.A. Parry, C.V. Logan, L.J. Newbury, B.M. Kamath, S. Ling, T. Grammatikopoulos, B.E. Wagner, J.C. Magee, R.J. Sokol, G. Mieli-Vergani, G. University of Washington Center for Mendelian, 27
ACCEPTED MANUSCRIPT J.D. Smith, C.A. Johnson, P. McClean, M.A. Simpson, A.S. Knisely, L.N. Bull, R.J. Thompson, Mutations in TJP2 cause progressive cholestatic liver disease, Nat Genet,
PT
46 (2014) 326-328.
RI
[46] N. Gomez-Ospina, C.J. Potter, R. Xiao, K. Manickam, M.S. Kim, K.H. Kim, B.L. Shneider, J.L. Picarsic, T.A. Jacobson, J. Zhang, W. He, P. Liu, A.S. Knisely, M.J.
SC
Finegold, D.M. Muzny, E. Boerwinkle, J.R. Lupski, S.E. Plon, R.A. Gibbs, C.M. Eng, Y.
NU
Yang, G.C. Washington, M.H. Porteus, W.E. Berquist, N. Kambham, R.J. Singh, F. Xia, G.M. Enns, D.D. Moore, Mutations in the nuclear bile acid receptor FXR cause
MA
progressive familial intrahepatic cholestasis, Nat Commun, 7 (2016) 10713. [47] E. Gonzales, S.A. Taylor, A. Davit-Spraul, A. Thebaut, N. Thomassin, C. Guettier,
D
P.F. Whitington, E. Jacquemin, MYO5B mutations cause cholestasis with normal serum
TE
gamma-glutamyl transferase activity in children without microvillous inclusion disease,
AC CE P
Hepatology, 65 (2017) 164-173.
[48] W.L. van der Woerd, S.W. van Mil, J.M. Stapelbroek, L.W. Klomp, S.F. van de Graaf, R.H. Houwen, Familial cholestasis: progressive familial intrahepatic cholestasis, benign recurrent intrahepatic cholestasis and intrahepatic cholestasis of pregnancy, Best Pract Res Clin Gastroenterol, 24 (2010) 541-553. [49] S.W. van Mil, W.L. van der Woerd, G. van der Brugge, E. Sturm, P.L. Jansen, L.N. Bull, I.E. van den Berg, R. Berger, R.H. Houwen, L.W. Klomp, Benign recurrent intrahepatic cholestasis type 2 is caused by mutations in ABCB11, Gastroenterology, 127 (2004) 379-384. [50] J. Stindt, P. Ellinger, K. Weissenberger, C. Droge, D. Herebian, E. Mayatepek, B. Homey, S. Braun, J. Schulte am Esch, M. Horacek, A. Canbay, L. Schmitt, D. Haussinger, R. Kubitz, A novel mutation within a transmembrane helix of the bile salt 28
ACCEPTED MANUSCRIPT export pump (BSEP, ABCB11) with delayed development of cirrhosis, Liver Int, 33 (2013) 1527-1535.
PT
[51] V.S. Hegade, M. Krawczyk, A.E. Kremer, J. Kuczka, F. Gaouar, E.M. Kuiper, H.R.
RI
van Buuren, F. Lammert, C. Corpechot, D.E. Jones, The safety and efficacy of nasobiliary drainage in the treatment of refractory cholestatic pruritus: a multicentre
SC
European study, Aliment Pharmacol Ther, 43 (2016) 294-302.
NU
[52] G. Folvik, O. Hilde, G.O. Helge, Benign recurrent intrahepatic cholestasis: review and long-term follow-up of five cases, Scand J Gastroenterol, 47 (2012) 482-488.
MA
[53] R.A. Hall, F. Lammert, Systems Genetics of Liver Fibrosis, Methods Mol Biol, 1488 (2017) 455-466.
D
[54] L. Hood, J.R. Heath, M.E. Phelps, B. Lin, Systems biology and new technologies
TE
enable predictive and preventative medicine, Science, 306 (2004) 640-643.
AC CE P
[55] R.C. Jansen, Studying complex biological systems using multifactorial perturbation, Nat Rev Genet, 4 (2003) 145-151. [56] M. Civelek, A.J. Lusis, Systems genetics approaches to understand complex traits, Nat Rev Genet, 15 (2014) 34-48. [57] S. Hillebrandt, C. Goos, S. Matern, F. Lammert, Genome-wide analysis of hepatic fibrosis in inbred mice identifies the susceptibility locus Hfib1 on chromosome 15, Gastroenterology, 123 (2002) 2041-2051. [58] E.S. Lander, D. Botstein, Mapping mendelian factors underlying quantitative traits using RFLP linkage maps, Genetics, 121 (1989) 185-199. [59] K.W. Broman, Review of statistical methods for QTL mapping in experimental crosses, Lab Anim (NY), 30 (2001) 44-52.
29
ACCEPTED MANUSCRIPT [60] J.L. Peirce, L. Lu, J. Gu, L.M. Silver, R.W. Williams, A new set of BXD recombinant inbred lines from advanced intercross populations in mice, BMC Genet, 5 (2004) 7.
PT
[61] J. Wang, R.W. Williams, K.F. Manly, WebQTL: web-based complex trait analysis,
RI
Neuroinformatics, 1 (2003) 299-308.
[62] R.A. Hall, R. Liebe, K. Hochrath, A. Kazakov, R. Alberts, U. Laufs, M. Bohm, H.P.
SC
Fischer, R.W. Williams, K. Schughart, S.N. Weber, F. Lammert, Systems genetics of
NU
liver fibrosis: identification of fibrogenic and expression quantitative trait loci in the BXD murine reference population, PLoS One, 9 (2014) e89279.
MA
[63] R. Liebe, R.A. Hall, R.W. Williams, S. Dooley, F. Lammert, Systems genetics of hepatocellular damage in vivo and in vitro: identification of a critical network on
D
chromosome 11 in mouse, Physiol Genomics, 45 (2013) 931-939.
TE
[64] D. Gatti, A. Maki, E.J. Chesler, R. Kirova, O. Kosyk, L. Lu, K.F. Manly, R.W.
AC CE P
Williams, A. Perkins, M.A. Langston, D.W. Threadgill, I. Rusyn, Genome-level analysis of genetic regulation of liver gene expression networks, Hepatology, 46 (2007) 548-557. [65] P.A. Andreux, E.G. Williams, H. Koutnikova, R.H. Houtkooper, M.F. Champy, H. Henry, K. Schoonjans, R.W. Williams, J. Auwerx, Systems Genetics of Metabolism: The Use of the BXD Murine Reference Panel for Multiscalar Integration of Traits, Cell, 150 (2012) 1287-1299. [66] G.A. Churchill, D.C. Airey, H. Allayee, J.M. Angel, A.D. Attie, J. Beatty, W.D. Beavis, J.K. Belknap, B. Bennett, W. Berrettini, A. Bleich, M. Bogue, K.W. Broman, K.J. Buck, E. Buckler, M. Burmeister, E.J. Chesler, J.M. Cheverud, S. Clapcote, M.N. Cook, R.D. Cox, J.C. Crabbe, W.E. Crusio, A. Darvasi, C.F. Deschepper, R.W. Doerge, C.R. Farber, J. Forejt, D. Gaile, S.J. Garlow, H. Geiger, H. Gershenfeld, T. Gordon, J. Gu, W. Gu, G. de Haan, N.L. Hayes, C. Heller, H. Himmelbauer, R. Hitzemann, K. Hunter, H.C. Hsu, F.A. 30
ACCEPTED MANUSCRIPT Iraqi, B. Ivandic, H.J. Jacob, R.C. Jansen, K.J. Jepsen, D.K. Johnson, T.E. Johnson, G. Kempermann, C. Kendziorski, M. Kotb, R.F. Kooy, B. Llamas, F. Lammert, J.M.
PT
Lassalle, P.R. Lowenstein, L. Lu, A. Lusis, K.F. Manly, R. Marcucio, D. Matthews, J.F.
RI
Medrano, D.R. Miller, G. Mittleman, B.A. Mock, J.S. Mogil, X. Montagutelli, G. Morahan, D.G. Morris, R. Mott, J.H. Nadeau, H. Nagase, R.S. Nowakowski, B.F. O'Hara, A.V.
SC
Osadchuk, G.P. Page, B. Paigen, K. Paigen, A.A. Palmer, H.J. Pan, L. Peltonen-Palotie,
NU
J. Peirce, D. Pomp, M. Pravenec, D.R. Prows, Z. Qi, R.H. Reeves, J. Roder, G.D. Rosen, E.E. Schadt, L.C. Schalkwyk, Z. Seltzer, K. Shimomura, S. Shou, M.J. Sillanpaa,
MA
L.D. Siracusa, H.W. Snoeck, J.L. Spearow, K. Svenson, L.M. Tarantino, D. Threadgill, L.A. Toth, W. Valdar, F.P. de Villena, C. Warden, S. Whatley, R.W. Williams, T.
D
Wiltshire, N. Yi, D. Zhang, M. Zhang, F. Zou, The Collaborative Cross, a community
TE
resource for the genetic analysis of complex traits, Nat Genet, 36 (2004) 1133-1137.
AC CE P
[67] D.W. Threadgill, G.A. Churchill, Ten years of the Collaborative Cross, Genetics, 190 (2012) 291-294.
[68] H.J. Atamni, M. Botzman, R. Mott, I. Gat-Viks, F.A. Iraqi, Mapping liver fat femaledependent quantitative trait loci in collaborative cross mice, Mamm Genome, 27 (2016) 565-573.
[69] H.J. Atamni, R. Mott, M. Soller, F.A. Iraqi, High-fat-diet induced development of increased fasting glucose levels and impaired response to intraperitoneal glucose challenge in the collaborative cross mouse genetic reference population, BMC Genet, 17 (2016) 10.
31
ACCEPTED MANUSCRIPT Table 1
Activity
p.I541F
Frameshift
p.L556R
Deletions
p.Q855L
MA
Nonsense
therapy (likely to be tested in association with UDCA)
TE
Pharmacological
Class V
Stability
No detectable defect
p.S346I
p.T424A
p.R652G
p.F357L
p.N510S
p.T175A
p.P726L p.T775M G954S Nuclear
Nuclear
chaperones (e.g.
receptor
receptor
ciclosporin)
agonists
agonists
(e.g.
(e.g.
fibrates,
fibrates,
obeticholic
obeticholic
acid),
acid),
statins
statins
AC CE P
Potential
Class IV
RI
Maturation
D
Variants
Class III
SC
Defect
Class II
NU
Class I
PT
Classification of ABCB4 variants associated with PFIC3
32
ACCEPTED MANUSCRIPT
PT
Table 2
4 TJP2
Smit et al. Cell 1993
Sambrotta et al. Nat Genet (2014)
NU
3 ABCB4 (MDR3)
MA
1 (Byler syndrome) 2 ATP8B1 (FIC1) ABCB11 (BSEP) Reference Klomp et al. Pawlikowska Hepatology 2004 et al. J Hepatol 2010 Transport Phosphatidylserine Bile acid flippase export Phenotypes Biliary cirrhosis Neonatal giant cell BRIC type 1 hepatitis
Labs
Low ɣ-GT
Biliary cirrhosis
AC
Extrahepatic manifestations: Malabsoprtion, pancreatitis, deafness, pneumonia
CE
PT ED
Type Gene
SC
RI
Phenotypic characteristics of PFIC types 1-6
BRIC type 2 ICP Gallstones DILI HCC Low ɣ-GT
Phosphatidylcholine translocator Biliary cirrhosis with neoductuli
Tight junction protein 2 Early onset chronic cholestasis
Gallstones (LPAC) HCC ICP
5 NR1H4 (FXR) GomezOspina et al. Nat Comm (2016) Nuclear bile acid receptor Neonatal cholestasis with rapid progression to end-stage liver disease
6 MYO5B Gonzalez et al. Hepatology (2017) Myosin 5b Giant-cell hepatocytes and persistent intralobular cholestasis
Respiratory/CNS Cholangiocarcinoma symptoms Vitamin KTransient or HCC independent recurrent coagulopathy cholestasis
High ɣ-GT
Low ɣ-GT
Low ɣ-GT AFP increased
Microvillus inclusion disease Low ɣ-GT
33
ACCEPTED MANUSCRIPT
UDCA OLT
OLT
OLT
UDCA OLT
PT ED
MA
NU
SC
RI
PT
OLT
CE
OLT PBED
AC
Therapy
34
ACCEPTED MANUSCRIPT
Highlights Genetics lead to the dissection of familial and multifactorial cholangiopathies.
•
With the identification of underlying genetic contributors of cholestatic diseases are reclassified.
•
Systems genetics reveals the full complexity of genotype-phenotype relationships in liver.
•
In the future the genetic findings might open new avenues for patients with cholangiopathies.
AC
CE
PT ED
MA
NU
SC
RI
PT
•
35