University of Groningen
Exome sequencing in patient-parent trios suggests new candidate genes for early-onset
primary sclerosing cholangitis
Haisma, Sjoukje-Marije; Weersma, Rinse K; Joosse, Maria E; de Koning, Barbara Ae; de
Meij, Tim; Koot, Bart Gp; Wolters, Victorien; Norbruis, Obbe; Daly, Mark J; Stevens, Christine
Published in:
Liver International
DOI:
10.1111/liv.14831
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Citation for published version (APA):
Haisma, S-M., Weersma, R. K., Joosse, M. E., de Koning, B. A., de Meij, T., Koot, B. G., Wolters, V.,
Norbruis, O., Daly, M. J., Stevens, C., Xavier, R. J., Koskela, J., Rivas, M. A., Visschedijk, M. C., Verkade,
H. J., Barbieri, R., Jansen, D. B., Festen, E. A., van Rheenen, P. F., & van Diemen, C. C. (2021). Exome
sequencing in patient-parent trios suggests new candidate genes for early-onset primary sclerosing
cholangitis. Liver International. https://doi.org/10.1111/liv.14831
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Liver International. 2021;00:1–14. wileyonlinelibrary.com/journal/liv
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1Received: 8 June 2020
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Revised: 29 January 2021|
Accepted: 7 February 2021DOI: 10.1111/liv.14831
O R I G I N A L A R T I C L E
Exome sequencing in patient- parent trios suggests new
candidate genes for early- onset primary sclerosing cholangitis
Sjoukje- Marije Haisma
1| Rinse K. Weersma
2| Maria E. Joosse
3|
Barbara A. E. de Koning
3| Tim de Meij
4| Bart G. P. Koot
5| Victorien Wolters
6|
Obbe Norbruis
7| Mark J. Daly
8| Christine Stevens
8| Ramnik J. Xavier
9|
Jukka Koskela
9,10,11| Manuel A. Rivas
12| Marijn C. Visschedijk
2|
Henkjan J. Verkade
1| Ruggero Barbieri
2,13| Dianne B. H. Jansen
2|
Eleonora A. M. Festen
2,13| Patrick F. van Rheenen
1| Cleo C. van Diemen
131Department of Paediatric Gastroenterology Hepatology and Nutrition, University of Groningen, University Medical Center Groningen, Groningen,
The Netherlands
2Department of Gastroenterology and Hepatology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands
3Department of Paediatric Gastroenterology, Erasmus University Medical Center, Sophia Children's Hospital, Rotterdam, The Netherlands
4Department of Pediatric Gastroenterology, VU University Medical Center, Amsterdam, The Netherlands
5Pediatric Gastroenterology, Emma Children's Hospital, Amsterdam UMC, University of Amsterdam, Amsterdam, The Netherlands
6Department of Pediatric Gastroenterology, University Medical Center Utrecht – Wilhelmina Children's Hospital, Utrecht, The Netherlands
7Department of Pediatrics, Isala Hospital, Zwolle, The Netherlands
8Broad Institute of Harvard and Massachusetts Institute of Technology, Boston, MA, USA
9Massachusetts General Hospital, Gastroenterology, Boston, MA, USA
10Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland
11Clinic of Gastroenterology Helsinki, Helsinki University and Helsinki University Hospital, Helsinki, Finland
12Stanford University, Stanford, CA, United States
This is an open access article under the terms of the Creative Commons Attribution- NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
© 2021 The Authors. Liver International published by John Wiley & Sons Ltd. Patrick F van Rheenen, Cleo C van Diemen are shared last authors.
Abbreviations: GWAS, genome- wide association studies; HLA, human leukocyte antigen; IBD, inflammatory bowel disease; MHC, major histocompatibility complex; PSC, primary sclerosing cholangitis; WES, whole- exome sequencing.
13Department of Genetics, University of
Groningen, University Medical Center Groningen, Groningen, The Netherlands
*Correspondence
Dr. Cleo C van Diemen, PhD, University Medical Center Groningen, Department of Genetics, Internal Code CB50, PO Box 30001, 9700 RB Groningen, The Netherlands. Email: c.c.van.diemen@umcg.nl
FUNDING INFORMATION
This work was supported by the European Crohn's and Colitis Organization (ECCO) [grant number Grant_2017/ECCO/ PatrickvanRheenen].
Handling Editor: Ana Lleol
Abstract
BACKGROUND & AIMS: Primary sclerosing cholangitis (PSC) is a rare bile duct disease strongly associated with inflammatory bowel disease (IBD). Whole- exome sequencing (WES) has contributed to understanding the molecular basis of very early- onset IBD, but rare protein- altering genetic variants have not been identified for early- onset PSC. We performed WES in patients diagnosed with PSC ≤ 12 years to investigate the contribution of rare genetic variants to early- onset PSC.
METHODS: In this multicentre study, WES was performed on 87 DNA samples from 29 patient- parent trios with early- onset PSC. We selected rare (minor allele frequency < 2%) coding and splice- site variants that matched recessive (homozygous and compound het-erozygous variants) and dominant (de novo) inheritance in the index patients. Variant
1 | INTRODUCTION
Primary sclerosing cholangitis (PSC) is a rare chronic cholestatic dis-ease characterized by progressive inflammation and obliterative fi-brosis of the intra- and extrahepatic bile ducts.1 Disease progression
is inevitable in the majority of PSC patients, with the development of biliary cirrhosis and portal hypertension requiring repeated endo-scopic procedures. Liver transplantation is the only curative treat-ment option, but the disease recurs in 20%- 25% of transplanted patients.2 Cholangiocarcinoma and colorectal cancer are feared
complications in PSC and the most common causes of death.3
There is a strong relation between PSC and inflammatory bowel disease (IBD). Patients who initially present with isolated PSC may go on to develop IBD years later.4,5 In adult- onset disease,
approx-imately two thirds of patients with PSC have concurrent IBD.1 The
co- occurrence of PSC and IBD is higher in children than in adults, varying from 76% to 97%.2,6,7 The pathogenesis of PSC is largely
unknown, but there is a strong genetic component. Genome- wide association studies (GWAS) in adult- onset PSC, carried out by the International PSC Study Group, identified 23 risk loci and 9 sugges-tive findings.8 Not surprisingly, considering the large clinical overlap,
the human leukocyte antigen (HLA) locus is by far the strongest sig-nal in GWAS in both PSC and IBD.9,10
Next to the common variants found by GWAS that so far explain <10% of PSC susceptibility, rare variants with large monogenic effect size may play a role in the onset of PSC. These variants are so rare in
allele frequency (many of them private variants) that their genetic sig-nals are hard to detect by GWAS. In contrast, whole- exome sequenc-ing (WES) in patients with extreme phenotypes, such as early- onset IBD, has led to the identification of a growing number of rare mono-genic disorders presenting with IBD- like intestinal inflammation.11,12
Additionally, a rare monogenic variant (a loss of function mutation in doublecortin domain containing protein 2 (DCDC2) has recently been identified in a PSC- like disorder called neonatal sclerosing cholangitis.13
Although neonatal sclerosing cholangitis is a different entity than PSC, histological similarities of the cholangiocytes of these patients lacking primary cilia suggest that some of the underlying pathogenic mecha-nisms could be shared between the two diseases. Likewise, we expect that a monogenic or oligogenic inheritance pattern may play a role in a subset of patients with early- onset PSC as well. We therefore per-formed WES in a Dutch cohort of patients with early- onset PSC and their parents to identify rare, but possibly causative genetic variants.
2 | METHODS
2.1 | Study design, participants and setting
In this multicentre parent- offspring study, we collected DNA from PSC patients with disease- onset prior to their 13th birthday and from their biological parents. PSC diagnosis was confirmed by cholangiography (presence of multifocal strictures, focal dilatation or beading of the pathogenicity was predicted by an in- house developed algorithm (GAVIN), and PSC- relevant variants were selected using gene expression data and gene function.
RESULTS: In 22 of 29 trios we identified at least 1 possibly pathogenic variant. We prioritized 36 genes, harbouring a total of 54 variants with predicted pathogenic ef-fects. In 18 genes, we identified 36 compound heterozygous variants, whereas in the other 18 genes we identified 18 de novo variants. Twelve of 36 candidate risk genes are known to play a role in transmembrane transport, adaptive and innate immunity, and epithelial barrier function.
CONCLUSIONS: The 36 candidate genes for early- onset PSC need further verifica-tion in other patient cohorts and evaluaverifica-tion of gene funcverifica-tion before a causal role can be attributed to its variants.
K E Y W O R D S
genetic, inflammatory bowel disease, sclerosing cholangitis Key points
It is rare to diagnose a child with primary sclerosing cholangitis (PSC) before its 13th birthday. We screened the portion of the DNA that codes for proteins in 29 young PSC children and their biological parents and identified 54 rare genetic variants in 36 genes with an assumed deleteri-ous effect on protein function. Whether these variants play a part in the aetiology of PSC will require further verification.
biliary tree) or liver histology (presence of bile duct damage, onion- skinned peri- ductal fibrosis, inflammation, portal oedema or fibrosis, ductopenia, ductular proliferation or cholestasis), or both. Patients with sclerosing cholangitis as a result of secondary causes such as sur-gery, trauma, cancer or infection were excluded from participation.
Patients were recruited in five tertiary care hospitals in the Netherlands— University Medical Center Groningen (UMCG, a re-ferral paediatric liver transplant centre), Erasmus University Medical Center– Sophia Children's Hospital, VU University Medical Center, Amsterdam University Medical Center– Emma Children's Hospital, University Medical Center Utrecht– Wilhelmina Children's Hospital— and one large general teaching hospital, the Isala Hospital. Eligible patients were those regularly attending the (paediatric) gastroenter-ology and hepatgastroenter-ology clinics as part of standard care. After informed consent was given, the following information was obtained from the local patient records and entered in an online clinical registry using Castor Electronic Data Capture (Amsterdam, the Netherlands): age at PSC diagnosis, findings on cholangiography and/or histology, and follow- up data on medication use and appearance of biliary cirrhosis, portal hypertension or liver transplantation. Between January 2017 and July 2017, blood was collected from patients and volunteering parents for genomic DNA extraction according to standard protocols.
2.2 | Ethical considerations
The Medical Ethical Committee of the UMCG approved the study protocol (METC 2016/289), and secondary approval was obtained from all participating centres. All participating parents and teenagers 12- 19 years old gave informed consent prior study inclusion.
2.3 | Whole- exome sequencing
Libraries were prepared using the Illumina Nextera prep kit and hy-brid capture (Illumina Rapid Capture Enrichment – 37 Mb target), and sequencing was performed using the Illumina HiSeq 2500 at the Broad Institute of MIT and Harvard. All raw data underwent qual-ity control steps (https://hub.docker.com/r/broad insti tute/gatk/) without any noticeable negative features to achieve 86.06 million high- quality reads per sample with 98.85% of reads aligned, on aver-age, resulting in a coverage of 81% of the target region with a read depth of >30X. Sequence reads were aligned to the human refer-ence genome using Novoalign (http://www.novoc raft.com). Next, the Genome Analysis Toolkit of the Broad Institute14 was used for
calling single- nucleotide variant and insertions/deletions.
2.3.1 | Variant annotation
Variants were annotated with SNPEff15 using publicly available data
from Ensembl and Refseq, and with GAVIN, an annotation tool with an algorithm that scores the likely pathogenicity of the variants.16
Additional annotations at the variant, exon and gene level were ob-tained from the 1000 Genomes Project (http://www.1000g enomes. org); National Heart, Lung and Blood Institute GO Exome Sequencing Project Exome Variant Server (http://evs.gs.washi ngton.edu/EVS); PolyPhen217 and the Exome Aggregation Consortium (ExAC, http://
exac.broad insti tute.org). To facilitate further discoveries in this fairly narrow field of research, the VCF files of our study are now publicly available in The Groningen Data Catalogue ( catalogus.helpdesk@ umcg.nl ) at (https://groni ngend ataca talog us.nl/menu/groni ngend ataca talog ue/datae xplor er/detai ls/umcg_colle ction s/aaaac 526ld ngr6q wh2xc 53aaae).
2.3.2 | Variant filtering
We used variants with a sequence coverage of ten or greater. We used a Genomics Data Management System (Alissa Interpret – Agilent technologies) to create a filtering tree specifically designed for this study (see Figure 1).
We performed patient- parent trio analyses. On the variant- level, we selected variants matching recessive (homozygous and com-pound heterozygous variants) and dominant (de novo) inheritance in the index patients. HLA variants were excluded from this analysis as it is unfeasible to distinguish functional polymorphism from random variation in a patient cohort of modest size. Minor allele frequency (MAF) cut- offs from the GnomAD database (http://gnomad.broad insti tute.org/) were <2% for recessive variants and <0.01% for dom-inant (de novo) variants. Variants were selected when they fulfilled the following two criteria: (a) deemed to be coding (missense- and nonsense mutations, frameshift insertions and deletions) or to have
F I G U R E 1 Variant selection. * Population databases used:
ExAC, GnoMAD, 1000 Genomes project. Abbreviation: MAF, Minor allele frequency
an effect on splicing; and (b) predicted to be (likely) pathogenic ac-cording to GAVIN.16
2.3.3 | Variant categorization
We then searched in Genecards (www.genec ards.com), Reactome (www.react ome.org) and OMIM (Online Mendelian Inheritance in Man; (www.omim.org) databases for information about gene func-tion and their involvement in diseases. We prioritized the genes into three categories (Box 1).
2.3.4 | Variant verification and validation
De novo variants were manually checked for coverage and allelic bal-ance in the BAM files. If there was doubt about the validity of the variant, confirmatory Sanger sequencing was performed.
3 | RESULTS
3.1 | Patient and disease characteristics
A total of 29 patient- parent trios were enrolled in this study (see Figure 1). Table 1 shows the characteristics of the affected persons, who were diagnosed with PSC at a median age of 10 years (range: 2- 12) and were predominantly male (72%). Twenty- two of 29 patients (76%) had concurrent IBD, with ulcerative colitis significantly more prevalent than Crohn's disease (73% vs 27%). Other autoimmune dis-orders included celiac disease (n = 1), idiopathic thrombocytopenic purpura (n = 1) and vitiligo (n = 1). None of the parents was known to have liver disease, but three had IBD. The median time (range) be-tween PSC diagnosis and study enrolment was 4 (0- 30) years. Biliary complications, including cholangitis or bile duct obstruction, had oc-curred in one patient (3%), and cirrhosis with portal hypertension had developed in nine (31%). Two patients underwent a liver trans-plantation after a disease duration of 10 and 11 years, respectively,
and two other patients are currently listed for liver transplantation. One of the cirrhotic patients had experienced bleeding of oesopha-geal varices and required a transjugular intrahepatic portosystemic shunt procedure.
3.2 | Patient- parent trio analyses of WES data
Figure 1 provides an overview of WES variant selection and prioriti-zation. In 22 of 29 trios, we identified at least 1 candidate pathogenic variant. We identified a total of 54 candidate variants with predicted pathogenic effect, annotated to 36 genes (see Table 2). In 15 trios we identified 36 unique compound heterozygous variants, and in 12 trios we identified 18 unique de novo variants. Twelve of 36 can-didate risk genes were assigned to prioritization category 1, 11 to category 2 and 13 to category 3.
The category 1 genes are depicted in Figure 2. The genes SLC9B1 and ABCB6 encode for membrane transporter proteins and relate to the ‘Transport of glucose and other sugars, bile salts and organic acids,
metal ions and amine compounds’ pathway (www.pathc ards.genec
ards.org). The gene NOTCH2NLA regulates the ‘Notch Signaling’ pathway (www.pathc ards.genec ards.org) and NOTCH2 variants are related to syndromes with cholestatic phenotypes (www.omim.org/ entry/ 600275). The genes MARCH1, PTX4, TIRAP, ADAM32, DDX47,
USP17L2, EHD1 and EDC4 are associated with defects of the
im-mune system. The CDHR2 gene is involved in the epithelial barrier function.
4 | DISCUSSION
4.1 | Key results
In this study we examined the exomes of patients with early- onset PSC and their biological parents. With this trio- analysis approach we identified 54 rare variants with predicted large effects on protein function in 36 genes. We prioritized 12 candidate risk genes that are most likely to contribute to the development of PSC in patients with early- onset disease based on their presumed role in (auto) immunity pathways, membrane transport (including bile salt homeostasis) and epithelial barrier functioning.
4.2 | Prioritized genes and their possible role in PSC
pathogenesis
Previous GWAS studies on the genetics of PSC suggested the au-toimmune origin of the disease, with strong associations with the genes encoding for the HLA complex on chromosome 6, along with several susceptibility genes that are critically involved in T- cell func-tion.1,8 Liver tissue from biopsied patients with PSC showed mainly
T cells and, to a lesser degree, macrophages and neutrophils in the infiltrates.1 We found a de novo stop- gain variant located at the
BOX 1 Prioritization of genes
Category 1 Well- known gene function connected with PSC or a similar phenotype (i.e., immunological, inflammatory or bile salt homeostasis).
Category 2 Unknown gene function and not (or rarely) reported in literature, and therefore cannot be excluded from having a potential role in the disease pathogenesis,
Category 3 Well- known gene function not directly associated with the disease phenotype.
T A B LE 1 C ha ra ct er is tic s o f p at ie nt s w ith e ar ly - o ns et P SC ( n = 2 9) Pa tie nt id en tif ic at io n num be r A ge a t P SC d ia gn os is (in y ea rs ) PS C c on fir m ed b y IB D p re se nt ? 1s t d eg re e r el at iv e w ith I B D o r P SC ? Co m pl ic at ed d is ea se c ou rs e? C an di da te g en e iden tif ied [P rio rit iz ed g ene s pr in te d i n b ol d] M RC P ERC P Li ver h is tolo gy [N o, U C or C D ] B ili ar y c om pl ic at io ns [ B]; C irr ho si s w ith p or ta l hy pe rt en si on [ C ]; l iv er tr an sp la nt at ion [L T] 1 11 CD No - - 2 10 U C IB D ( fa th er ) - - 3 7 U C No - AB CB6 4 8 No No - AD AM 32 , M AR CH 1, PT X4 , PL XD C1 5 11 U C IB D ( m ot he r) - - 6 12 No No - ED C4 7 8 No No C , L T PH C2 , T N RC 18 8 11 No No - - 9 12 No No - H M CN2 10 10 CD No C CC N 4 11 7 No No C - 12 9 CD No - D N AH6 , E H D1 , L AM A2 13 7 No No C D N AH7 14 12 U C No - KR TA P5 - 1 , M CM 8 15 12 CD No - CA SK IN2 16 10 U C No - PP FI A4 17 5 U C No - CD H R2 , D D X4 7 18 2 CD No - CA LC RL , U SP 17 L2 19 11 U C No - ANK RD 36 , D N AH 11 , N O TC H 2N LA 20 10 U C No - - 21 11 U C No C JM JD 1C , D AC T1 22 11 U C No C , L T TS PY L5 23 10 U C No - SL C9 B1 24 5 U C No C , L T TR DN 25 8 U C No - FA M 23 4B 26 7 U C No B SM CH D1 , TI RA P 27 12 U C IB D ( fa th er ) - - 28 12 CD No C CH ST 11 29 6 U C No C KIF 4A , Z NF 30
T A B LE 2 C om pl et e l is t o f 3 6 c an di da te r is k g en es f or e ar ly - o ns et P SC f ro m 2 2 p at ie nt - p ar en t t rio s Tr io C hr : p os iti on :al le le s rs n um be r C an di da te r is k ge ne In he rit anc e mo de (p ar en tal al le le ) G nom A D al le le co un t P op ul at io n fr eq uen cy A m in o A ci d cha ng e C A D D - sco re Li te ra tu re ca te go ry Pr ot ei n func tio n 3 2: 220 07 55 21 :C /T rs 14 82 11 04 2 AB CB6 Com pou nd he te ro zy gou s (m ot he r) 217 = 0.0 00 77 p. R72 3Q 35 .0 1 B in ds h em e a nd p or ph yr in s a nd f un ct io ns in t he ir A TP - d ep en de nt u pt ak e i nt o t he m itoc ho nd ria . 20 ,21 M ut at io ns i n t hi s g en e un de rli e f am ili al p se ud oh yp er ka le m ia ( O M IM 60 9 15 3) a nd d ys ch ro m at os is u ni ve rs al is he re di ta ria n ( O M IM 6 15 4 02 ). 2: 220 07 80 06 :C /T rs 14 55 269 96 AB CB6 Com pou nd he te ro zy gou s (fa th er ) 12 36 = 0.0 04 3 p.G 58 8S 32 .0 4 8: 39 0444 52 :G /A rs 74 59 52 92 7 AD AM 32 Com pou nd he te ro zy gou s (fa th er ) 30 = 0 p. A 314 T 12 .9 3 1 Th is g en e e nc od es a m em be r o f t he d is in te gr in fa m ily o f m em br an an ch or ed p ro te in s t ha t pl ay a r ol e i n d iv er se b io lo gi ca l p ro ce ss es s uc h as b ra in d ev el op m en t, f er til iz at io n, t um ou r de velo pm en t a nd in fla mm at io n. 28 8: 39 103 683 :A /G rs 15 01 14 293 AD AM 32 Com pou nd he te ro zy gou s (m ot he r) 19 2 = 0.0 01 p.G 63 4R 29 .8 4: 16 47 75 27 2: C /T U nk no wn M AR CH1 D e n ovo U no bse rv ed p.W 4* 38 .0 1 D ow nr eg ul at es s ur fa ce e xp re ss io n o f m aj or hi st oc om pa tib ili ty c om pl ex ( M H C ) c la ss II m ol ec ul es a nd o th er g ly co pr ot ei ns b y di re ct in g t he m t o t he l at e e nd os om al / ly so so m al c ompa rt m en t. 18 ,19 16 :1 53 79 11 :C /T rs7 75 40 71 57 PT X4 D e n ovo 3 = 0 .0000 12 p. V6 3 M 12 .5 1 Pe nt ra xi ns a re p ar t o f t he h um or al a rm o f in na te i m m un ity a nd b eh av e a s f un ct io na l an ce st or s o f a nt ib od ie s b y m ed ia tin g ag gl ut in at io n, c om pl emen t ac tiv at io n an d op so ni za tio n. P TX 4 i s a n ew u ni qu e m em be r of t he p en tr ax in s up er fa m ily , c on se rv ed i n ev ol ut io n. F ur th er s tu di es a re n ee de d t o de fin e i ts f un ct io n. 29 17 :3 72 34 30 0: G /A U nk no wn PL XD C1 D e n ovo U no bse rv ed p. A 351 V 23 .8 2 Pl ay s a c rit ic al r ol e i n e nd ot he lia l c el l c ap ill ar y m or ph og en es is . 30 6 16 :67 91 167 7: G /A rs 111 23 16 28 ED C4 Com pou nd he te ro zy gou s (m ot he r) 447 = 0.0 02 p. S2 75 G 10 .9 3 1 En ha nc er O f M RN A D ec ap pi ng . 31 D ise ase s as so ci at ed w ith E D C 4 i nc lu de H um an G ra nu lo cy tic A na pl as m os is a nd A nt er os ep ta l M yo ca rd ia l I nf arc tio n. 16 :6 79 16 92 0: C /T rs 56 31 49 57 7 ED C4 Com pou nd he te ro zy gou s (fa th er ) 21 = 0 p. A 123 0V 27. 7 7 1: 33 79 46 34: G/ C rs 376 86 94 90 PH C2 Com pou nd he te ro zy gou s (fa th er ) 41 = 0 p. I7 53 M 23 .2 2 C om po ne nt o f a P ol yc om b g ro up ( Pc G ) m ul tip ro te in P RC lik e c om pl ex , a c om pl ex cl as s r eq ui re d t o m ai nt ai n t he t ra ns cr ip tio na lly re pr es si ve s ta te o f m an y g en es , i nc lu di ng H ox ge ne s, th rou gh ou t d ev elo pm en t. 32 1: 33 82 01 46 :T /C rs 14 2759 75 0 PH C2 Com pou nd he te ro zy gou s (m ot he r) 40 = 0 p. D 47 1N 18 .51 (Co nti nue s)
Tr io C hr : p os iti on :al le le s rs n um be r C an di da te r is k ge ne In he rit anc e mo de (p ar en tal al le le ) G nom A D al le le co un t P op ul at io n fr eq uen cy A m in o A ci d cha ng e C A D D - sco re Li te ra tu re ca te go ry Pr ot ei n func tio n 7: 54 27 97 1: C /A U nk no wn TN RC 18 D e n ovo 0 p. G 49 5V 14 .8 8 2 Pr ot ei n C od in g g en e. D is ea se s a ss oc ia te d w ith T N RC 18 i nc lu de A tr ia l S ep ta l D ef ec t a nd S ec ke l S yn dr om e. 33 L ea d C pG s at T N RC 18 m ap t o a ct iv e e nh an ce rs i n ki dn ey c or te x a nd a re a ss oc ia te d w ith r en al fib ro si s. . 34 9 9:1 33 04 75 87 :C /G U nk no wn H M CN2 Com pou nd he te ro zy gou s (fa th er ) 8 = 0 p. Q 161 E 22 .8 2 Pr ot ei n C od in g g en e. D is ea se s a ss oc ia te d w ith H M C N 2 i nc lu de P os te rio r M yo ca rd ia l In fa rc tio n. 35 9:1 33 30 58 95 :A /G rs 55 93 74 16 1 H M CN2 Com pou nd he te ro zy gou s (m ot he r) 63 = 0 p. G 42 93 E 3. 17 1 10 8: 13 42 25 27 3: G /A U nk no wn CC N 4 D e n ovo U no bse rv ed p.C 79Y 27. 2 3 M ed ia te s d iv er se d ev el op m en ta l p ro ce ss es , su ch a s c on tr ol o f c el l p ro lif er at io n, a dh es io n, ce ll p ol ar ity a nd e st ab lis hm en t o f c el l f at es . 36 12 2: 84 81 60 32: T/ A U nk no wn D N AH6 D e n ovo U no bse rv ed p. L8 55H 22 .8 3 Fo rc e g en er at in g p ro te in o f r es pi ra to ry c ili a. Pr od uc es f or ce t ow ar ds t he m in us e nd s o f m ic ro tu bu le s. D is ea se s a ss oc ia te d i nc lu de Pr im ar y C ili ar y D ys ki ne si a a nd A no m al ou s Le ft C or on ar y A rt er y F ro m T he P ul m on ar y A rt er y. 37: 11 :64 64 564 8: G /A rs 74 72 58 453 EH D1 Com pou nd he te ro zy gou s (m ot he r) U no bse rv ed p. F97 L 27. 8 1 Im po rt an t m ot if i n p ro te in s i nv ol ve d i n p ro te in - pr ot ei n i nt er ac tio ns a nd i n i nt ra ce llu la r so rt in g. T he p ro te in e nc od ed b y t hi s g en e i s th ou gh t t o p la y a r ol e i n t he e nd oc yt os is o f IG F1 r ec ep to rs . 38 11 :64 64 58 41 :C /T U nk no wn EH D1 Com pou nd he te ro zy gou s (fa th er ) 2 = 0 p. = 15 .6 8 6: 12 962 19 52 :A /T U nk no wn LAM A2 Com pou nd he te ro zy gou s (fa th er ) U no bse rv ed p. I10 37 F 18.8 5 3 It i s t ho ug ht t o m ed ia te t he a tt ac hm en t, m ig ra tio n a nd o rg an iz at io n o f c el ls i nt o tis su es d ur in g e m br yo ni c d ev el op m en t b y in te ra ct in g w ith o th er e xt ra ce llu la r m at rix co m po ne nt s. D is ea se s a ss oc ia te d w ith LA M A 2 i nc lu de M us cu la r D ys tr op hy . 39 6: 12 98 24 34 5: T/ C rs 151 33 47 75 LAM A2 Com pou nd he te ro zy gou s (m ot he r) 22 = 0 p. P2 82 3S 25 .3 13 2: 19 67 20 58 9: C /A rs 7497 76 50 4 D N AH7 Com pou nd he te ro zy gou s (fa th er ) 6 = 0 p. R 28 47S 12 .0 6 3 Fo rc e g en er at in g p ro te in o f r es pi ra to ry ci lia . P ro du ce s f or ce t ow ar ds t he min us e nds o f micr ot ub ul es . 40D ise ase s as so ci at ed w ith D N A H 7 i nc lu de S itu s In ve rs us a nd D ex tr oc ar di a W ith S itu s Inv er su s. 2:1 96 88 91 60 :A /G rs1 82 08 63 16 D N AH7 Com pou nd he te ro zy gou s (m ot he r) 43 9 = 0.0 02 p. R 24 6C 29 .2 T A B LE 2 (Co nti nue d) (Co nti nue s)
Tr io C hr : p os iti on :al le le s rs n um be r C an di da te r is k ge ne In he rit anc e mo de (p ar en tal al le le ) G nom A D al le le co un t P op ul at io n fr eq uen cy A m in o A ci d cha ng e C A D D - sco re Li te ra tu re ca te go ry Pr ot ei n func tio n 14 11 :1 60 61 44 : AC A AG AG C C AC AG CCCCCC TT G G /. rs 76 11 47 27 1 KR TA P5 - 1 D e n ovo U no bse rv ed p. S1 05 W fs *1 15 - 3 In t he h ai r c or te x, h ai r k er at in i nt er m ed ia te fil amen ts a re em be dde d in a n in ter fil amen to us m at rix , c on si st in g o f h ai r k er at in - a ss oc ia te d pr ot ei n ( K RT A P) , w hi ch a re e ss en tia l f or t he fo rm at io n o f a r ig id a nd r es is ta nt h ai r s ha ft th ro ug h t he ir e xt en si ve d is ul ph id e b on d c ro ss - lin ki ng w ith a bu nd an t c ys te in e r es id ue s o f h ai r ker at in s. 41: 20 :59 352 81 :A /G rs 37 74 35 48 6 M CM 8 D e n ovo 14 = 0 p. E94 G 23 .1 3 Th e p ro te in e nc od ed b y t hi s g en e i s o ne o f th e h ig hl y c on se rv ed m in i- c hr om os om e m ai nt en an ce p ro te in s ( M C M ) t ha t a re es se nt ia l f or t he i ni tia tio n o f e uk ar yo tic ge no m e r ep lic at io n. D is ea se s a ss oc ia te d w ith M C M 8 i nc lu de P re m at ur e O va ria n F ai lu re 10 a nd A m en or rh ea . 42 15 17:7 34 99 28 7: T/ C rs 20 09 474 87 CA SK IN2 Com pou nd he te ro zy gou s (m ot he r) 15 0 = 0.0 01 p. R62 3Q 18 .14 2 Th is g en e e nc od es a l ar ge p ro te in t ha t c on ta in s si x a nk yr in r ep ea ts , a s w el l a s a S rc h om ol og y 3 ( SH 3) d om ai n a nd t w o s te ril e a lp ha m ot if (S A M ) d om ai ns , w hi ch m ay b e i nv ol ve d i n pr ote pr ote in inte ra ct io ns . 43 17:7 35 00 51 5: G /A rs2 01 52 19 12 CA SK IN2 Com pou nd he te ro zy gou s (fa th er ) 83 = 0 p. P4 54 L 17. 33 16 1: 20 30 18 04 3: G /C rs 61 75 64 14 PP FI A4 Com pou nd he te ro zy gou s (m ot he r) 43 = 0 - 15 .6 6 2 M ay r eg ul at e t he d is as se m bl y o f f oc al ad he si on s. M ay l oc al ize r ec ep to lik e t yr os in e ph os ph at as es t yp e 2 A a t s pe ci fic s ite s o n t he pl as m a m em br an e, p os si bl y r eg ul at in g t he ir in te ra ct io n w ith t he e xt ra ce llu la r e nv iro nm en t an d t he ir a ss oc ia tio n w ith s ub st ra te s. 44 1: 20 30 36 89 4: G /T rs 52 85 73 27 5 PP FI A4 Com pou nd he te ro zy gou s (fa th er ) 9 = 0 p. R1 041 L 33 17 5: 17 60 02 84 0: G /A rs 78 07 69 74 0 CD H R2 D e n ovo 5 = 0 .0000 3 p. = SP LIC E_ SI TE D O N O R 16 .9 1 In te rm ic ro vi lla r a dh es io n m ol ec ul e t ha t co nt ro ls t he p ac ki ng o f m ic ro vi lli a t t he a pi ca l m em br an e o f e pi th el ia l c el ls . 23 ,24 12 :12 97 42 28 :C /T rs 7808 73 69 5 D D X47 D e n ovo 2 = 0 p.P9 0S 24 .5 1 In vo lv ed i n a po pt os is . M ay h av e a r ol e i n r RN A pr oc es si ng a nd m RN A s pl ic in g. A ss oc ia te s w ith p re - r RN A p re cu rs or s. 45: 18 2: 18 82 281 04 :G /A U nk no wn CA LC RL D e n ovo U no bse rv ed p. P2 09 L 29 .6 2 Re ce pt or f or c al ci to ni n g en re la te d p ep tid e (C G RP ) a nd a dr en om ed ul lin . 46 T A B LE 2 (Co nti nue d) (Co nti nue s)
Tr io C hr : p os iti on :al le le s rs n um be r C an di da te r is k ge ne In he rit anc e mo de (p ar en tal al le le ) G nom A D al le le co un t P op ul at io n fr eq uen cy A m in o A ci d cha ng e C A D D - sco re Li te ra tu re ca te go ry Pr ot ei n func tio n 8:1 19 96 02 6: T/ G rs 20 17 34 66 3 U SP 17 L2 Com pou nd he te ro zy gou s (m ot he r) 51 = 0 p. L82 I - 1 H as d eu bi qu iti na tin g a ct iv ity . A ls o r eg ul at es ce ll p ro lif er at io n a nd a po pt os is t hr ou gh de ub iq ui tin at io n o f S U D S3 a r eg ul at or o f hi st one d ea ce ty la tio n. 47 8: 11 99 61 22 :C /T rs 1999 85 47 9 U SP 17 L2 Com pou nd he te ro zy gou s (fa th er ) 76 = 0 p. D5 0N 0.0 34 19 2: 97 79 032 1: A /G rs 77 00 5111 0 ANK RD 36 Com pou nd he te ro zy gou s (m ot he r) 16 = 0 p. A 24 0T 11 .8 1 2 Pr ot ei n C od in g g en e. D is ea se s a ss oc ia te d w ith A N K RD 36 i nc lu de G ia nt A xo na l N eu ro pa th y. 48: 2: 97 82 38 66: C /T rs5 67 23 43 99 ANK RD 36 Com pou nd he te ro zy gou s (fa th er ) 12 = 0 p. T4 28 M 18 .47 7: 21 80 50 95 :A /G rs 35 865 357 D N AH 11 Com pou nd he te ro zy gou s (m ot he r) 30 87 = 0.0 11 p. R 29 97Q 35 3 Fo rc e g en er at in g p ro te in o f r es pi ra to ry c ili a. Pr od uc es f or ce t ow ar ds t he m in us e nd s o f micr ot ub ul es . 49 D is ea se s a ss oc ia te d w ith D N A H 11 i nc lu de C ili ar y D ys ki ne si a, P rim ar y an d Pr im ar y C ili ar y D ys ki ne si a. 7: 21 90 16 05 :G /C rs 75 10 35 61 7 D N AH 11 Com pou nd he te ro zy gou s (fa th er ) U no bse rv ed p. K3 77 9N 19 .0 7 1: 14 527 32 95 :C /T rs 782 81 93 94 N OT CH 2N LA D e n ovo 4 = 0 p. T5 0 M 24 .1 1 H um an - s pe ci fic p ro te in t ha t p ro m ot es n eu ra l pr og en ito r p ro lif er at io n a nd e vo lu tio na ry ex pa ns io n o f t he b ra in n eo co rt ex b y re gu la tin g t he N ot ch s ig na lli ng p at hw ay vi a d ire ct i nt er ac tio n w ith N O TC H 2. 50 N O TC H 2 v ar ia nt s a re a ss oc ia te d w ith A la gi lle sy nd ro m e, i nc lu di ng c ho le st as is p he no ty pe s (w w w .o m im .o rg /e nt ry / 6 00 27 5). 21 10 :64 92 78 37 :C /T rs7 15 08 95 7 JM JD 1C Com pou nd he te ro zy gou s (fa th er ) 11 62 = 0.0 04 1 p. E2 53 1K 26 .5 3 A c an di da te h is to ne d em et hy la se t ho ug ht t o be a c ac tiv at or f or k ey t ra ns cr ip tio n f ac to rs . Pl ay s a r ol e i n t he D N A - d am ag e r es po ns e pa th w ay. 51 10 :6 497 48 07 :C /G rs 20 00 162 10 JM JD 1C Com pou nd he te ro zy gou s (m ot he r) 13 2 = 0.0 00 47 p. D 37 4H 26 .2 14 :59 10 49 43 :C /T U nk no wn D AC T1 Com pou nd he te ro zy gou s (m ot he r) 5 = 0 p. T8 M 23 .6 3 In te ra ct s w ith , a nd p os iti ve ly r eg ul at es , di sh ev el le m ed ia te d s ig na lli ng p at hw ay s du rin g de ve lo pmen t. 52 A ss oc ia te d w ith T ow ne B ro ck s s yn dr om 2 (O M IM 6 17 4 66 ). 14 :5 91 13 37 6: T/ C rs 20 097 78 26 D AC T1 Com pou nd he te ro zy gou s (fa th er ) 16 6 = 0.0 01 p. W67 9R 25 .5 22 8: 982 89 23 8: T/ C rs 15 10 15 59 6 TS PY L5 Com pou nd he te ro zy gou s (fa th er ) 10 53 = 0.0 04 p. S27 9G 17. 38 3 In vo lv ed i n m od ul at io n o f c el l g ro w th a nd ce llu la r r es po ns e t o g am m a r ad ia tio n p ro ba bl y vi a r eg ul at io n o f t he A kt s ig na lli ng p at hw ay . In vo lv ed i n r eg ul at io n o f p 53 /T P5 3. 53: 8: 982 90 01 2: A /C rs 79 679 52 0 TS PY L5 Com pou nd he te ro zy gou s (m ot he r) 48 5 = 0.0 03 p. A 21 S 23 .3 T A B LE 2 (Co nti nue d) (Co nti nue s)
Tr io C hr : p os iti on :al le le s rs n um be r C an di da te r is k ge ne In he rit anc e mo de (p ar en tal al le le ) G nom A D al le le co un t P op ul at io n fr eq uen cy A m in o A ci d cha ng e C A D D - sco re Li te ra tu re ca te go ry Pr ot ei n func tio n 23 4: 10 38 32 61 1: G /A rs 75 59 99 26 SL C9 B1 D e n ovo 2 = 0 .0000 11 p. R3 05 * 36 . 1 So di um /h yd ro ge n e xc ha ng er a nd tr an sm emb ra ne p ro te in . 54 24 6: 12 37 86 03 3:. /A rs 20 14 31 15 9 TR DN D e n ovo U no bse rv ed p. S2 97 Ff s* 32 n. a. 2 C on tr ib ut es t o r eg ul at io n o f l um in al C a2 + re le as e v ia t he s ar co pl as m ic r et ic ul um ca lc iu m rel ea se c ha nnel s. 55 A ss oc ia te d w ith ve nt ric ul ar t ac hy ca rd ia ( O M IM 6 15 4 41 ) 25 12 :1 32 19 60 4: T/ C rs 36 75 47 95 2 FA M 23 4B Com pou nd he te ro zy gou s (m ot he r) 23 = 0 p. R 295 W 35 2 Pr ot ei n C od in g g en e. D is ea se s a ss oc ia te d w ith F A M 23 4B i nc lu de T em ta m y Sy nd ro m e an d A ut os om al D om in an t N on - Sy nd ro m ic I nt el le ct ua l D is ab ili ty . 56: 12 :1 32 21 60 7: T/ A rs 14 02 71 82 5 FA M 23 4B Com pou nd he te ro zy gou s (fa th er ) 15 0 = 0.0 01 p. F444 I 25 .6 26 18 :2 72 26 03 :G /A U nk no wn SM CH D1 D e n ovo U no bse rv ed p. D 849 N 31 3 In vo lv ed i n D N A m an ag em en t a nd p la ys a n es se nt ia l r ol e i n X c hr om os om e i na ct iv at io n. 57 11 :1 26 16 29 48 :G /A rs 18 51 14 12 5 TIR AP D e n ovo 49 = 0 p. R 21 5H 23 .6 1 A da pt er i nv ol ve d i n T LR 2 a nd T LR 4 s ig na lli ng pa th w ay s i n t he i nn at e i m m un e r es po ns e. A ct s v ia I R A K 2 a nd T R A 6, l ea di ng t o t he ac tiv at io n o f N ka pp B , M A PK 1, M A PK 3 an d J N K , a nd r es ul tin g i n c yt ok in e s ec re tio n an d the in fla mm at or y re sp on se . 58 28 12 :1 05 15 115 9: G/ A U nk no wn CH ST 11 D e n ovo U no bse rv ed p. G 213 S 32 . 3 C at al ys es t he t ra ns fe r o f s ul ph at e i n ch on dro itin . 59 D is ea se s a ss oc ia te d w ith C H ST 11 i nc lu de M uc in os es a nd C os te llo Sy nd ro m e ( O M IM 6 18 1 67 ). 29 X :696 23 81 4: A /G U nk no wn KI F4 A D e n ovo U no bse rv ed p. N 907 S 10 .11 3 Ir on - s ul ph ur ( Fe - S ) c lu st er b in di ng m ot or pr ot ei n t ha t h as a r ol e i n c hr om os om e se gr eg at io n d ur in g m ito si s. 60 19 :3 54 34 41 1: C /T rs 36 765 19 57 ZN F3 0 Com pou nd he te ro zy gou s (m ot he r) 43 = 0 p. C1 82 R 23 .2 2 M ay b e i nv ol ve d i n t ra ns cr ip tio na l reg ula tio n. 61 D is ea se s a ss oc ia te d w ith Z N F3 0 in cl ud e C hr om os om e 1 9Q 13 .1 1 D el et io n Sy nd ro m e an d B ru ga da S yn dr om e. 19 :3 54 35 632 :C /T rs 14 02 15 76 0 ZN F3 0 Com pou nd he te ro zy gou s (fa th er ) 542 = 0.0 02 p. R5 89W 25 .8 A bb re vi at io ns : C A D D - s co re , Co m bi ne d A nno ta tio D ep en den t D epl et io n sc or eCh r, Ch ro m os ome . T A B LE 2 (Co nti nue d)
very beginning of the MARCH1- gene (transcript position 4) in pa-tient #4, a boy of 8 with PSC– autoimmune hepatitis overlap syn-drome, also called autoimmune sclerosing cholangitis. Such an early stop- gain results in loss of the corresponding protein from this al-lele and is predicted to be highly pathogenic. As the inheritance mode of this gene is still unknown, we cannot predict the biologi-cal effect of this variant. Functional studies of MARCH1 suggested that this gene mediates the immunosuppressive effect of the anti- inflammatory cytokine interleukin 10 (IL10) on antigen presentation in monocytes.18,19 Knockdown of MARCH1 strongly inhibited IL- 10–
dependent down- regulation of cell surface HLA- DR.19 The exact
contribution of the MARCH1 gene regulation to immunopathology remains to be explored.
We identified variants in genes ABCB6 and SLC9B1, which are (according to www.pathc ards.genec ards.org) related to the ‘trans-port of glucose and other sugars, bile salts and organic acids, metal ions and amine compounds’ pathway. Patient #3, a girl with both PSC and IBD diagnosed at age 7, had compound heterozygous
ABCB6 variants. The gene encodes a member of the ATP- binding
cassette (ABC) transporter superfamily and is known to bind heme and porphyrins and function in their ATP- dependent uptake in the mitochondria.20,21 Both variants are predicted to be damaging and
to disrupt the highly conserved ABC transport and ABC transmem-brane regions of the protein, respectively. However, whether this genetic variant is truly involved in alteration of bile salt homeostasis has to be investigated further.
Another possible cause of development of PSC is the epithe-lial cell lining of the bile ducts. Bile salts are toxic in high concen-trations.22 Damage of the epithelial border may result in leakage
of bile and could be an important driver of toxicity. In patient #17 we identified one de novo intronic variant positioned exactly at a splice- donor consensus sequence site in CDHR2, predicted to dis-rupt splicing of the transcript and causing loss of function. CDHR2 plays a central role in the integrity of epithelial tissues such as the bile duct epithelium.23,24
Uncovering the functional consequences of the newly discov-ered candidate variants, in particular for the genes without a known function, and the pathways involved in the onset of PSC will require detailed functional experiments involving different functional read- outs, given the broad nature of the identified genes, and further ver-ification of our findings in independent cohorts.
4.3 | From theory to definitive proof
This is the first explorative study of high- impact rare coding vari-ants in young patients with PSC which could help to identify caus-ative genes. To prove that our set of candidate genes contains a causative gene, replication in an independent patient cohort is a first step. One method to replicate low- frequency and rare variants in complex immune diseases is by targeted genotyping using the Illumina exome chip array (HumanExome BeadChips, Illumina, Inc, San Diego, CA). This next- generation genotyping array includes sev-eral regions on the genome that are known to play a role in immune mediated disease based on the results from existing re- sequencing datasets. In a first attempt to further study the contribution of rare variants in PSC, our research group is in the process of replication genotyping of identified variants in a large international exome array case- control study and identified several genetic loci con-taining rare variants that are associated with PSC. Adding our new candidate risk genes and variants to such arrays may contribute to efficient replication.
Furthermore, replication in other early- onset PSC cohorts is needed. In 2013, the BROAD Institute partnered with researchers worldwide to develop a collaborative exome sequencing network in IBD, and this initiative is currently ongoing. A similar project is now up and running in PSC with the aim to meta- analyse the exomes of more than 1000 patients of European ancestry. This will enable modelling of the combined contribution of polygenic and oligogenic variants to the inheritance of PSC.
Convincing evidence to prove that a gene is causal in a disease is identifying a similar genetic variant in the same gene in another patient. In monogenic diseases, a freely accessible Web- based tool called GeneMatcher (www.genem atcher.org) is used to identify ad-ditional individuals with rare phenotypes who have variants in the same candidate gene.25 A similar tool does not yet exist for complex
genetic diseases but data sharing such as GeneMatcher might be useful here as well.
F I G U R E 2 Function of the 12 prioritized candidate risk genes
for early- onset PSC. The core indicates the protein functions and the inner ring shows the 12 candidate risk genes. Information on genetic functions comes from multiple databases including Genecards (www.genec ards.com), Reactome (www.react ome.org) and OMIM (Online Mendelian Inheritance in Man; www.omim.org)
4.4 | Implications for clinical practice
At this moment, there is no curative therapy available for PSC. Quality- of- life undermining complications may eventually justify a liver transplantation. The ultimate goal of investigating the genetic basis of this disease is to help reveal mechanisms of disease pa-thology and guide the selection of new targets for drug discovery. Each genetic risk locus can be seen as a potential drug target and the starting point of new treatment opportunities. This has success-fully been demonstrated in the field of IBD, in which small- molecule inhibitors were used to recapitulate the anti- inflammatory function of CARD9 variants associated with protection from IBD.26 Scientists
now recognize that genes with evidence for causality in disease are more promising for identification of new drug targets, and this has led to an increased interest in disease- associated genes with variants that reduce gene function, such as nonsense, frameshift or essential splice- site variants.27 Our study is one of the first steps on this road
to drug discovery for patients with PSC.
5 | CONCLUSION
We identified 54 rare protein- altering genetic variants in 36 genes, of which 12 were prioritized as candidate risk genes. The functional consequences of these variants and their causality to PSC will need to be studied in replication cohorts and with functional testing.
ACKNOWLEDGEMENTS
We thank Krista van Dijk- Bos and Kristin Abbott for assistance with variant filtering and interpretation. We thank Kate Mc Intyre for edi-torial assistance.
CONFLIC TS OF INTEREST
None.
AUTHOR CONTRIBUTIONS
SMH, RKW, PFvR and CCvD contributed to study concept and de-sign, interpretation of data and drafting of the manuscript. BAEdK, MEJ, BGPK, TdM, VMW and ON contributed to the collection of patient data. SMH obtained the data. Whole- exome sequencing and quality control were performed by MJD, CS, RJX and MAR. SMH and CCvD analysed the data. HJV, RB, DBHJ, NF and MV contributed important intellectual content. PFvR and CCvD had full responsibil-ity for the study.
ETHIC S APPROVAL STATEMENT
The Medical Ethical Committee of the UMCG approved the study protocol (METC 2016/289), and secondary approval was obtained from all participating centres.
PATIENT CONSENT STATEMENT
All participating parents and teenagers 12- 19 years old gave in-formed consent prior study inclusion.
CONFERENCE PRESENTATION
This study was selected for a lecture presentation during the
Digestive Disease Week® in May 2019 in San Diego.
ORCID
Sjoukje- Marije Haisma https://orcid.org/0000-0003-4814-7776
Maria E. Joosse https://orcid.org/0000-0001-7420-7125
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How to cite this article: Haisma S- M, Weersma RK, Joosse
ME, et al. Exome sequencing in patient- parent trios suggests new candidate genes for early- onset primary sclerosing cholangitis. Liver Int. 2021;00:1– 14. https://doi.org/10.1111/ liv.14831
