b r i e f c o m m u n i c at i o n s
npg
© 2014 Nature America, Inc. All rights reserved.
Mutations in TJP2 cause progressive cholestatic liver disease Melissa Sambrotta1, Sandra Strautnieks2, Efterpi Papouli3, Peter Rushton4, Barnaby E Clark4, David A Parry5, Clare V Logan5, Lucy J Newbury6, Binita M Kamath7,8, Simon Ling7,8, Tassos Grammatikopoulos1,9, Bart E Wagner10, John C Magee11, Ronald J Sokol12, Giorgina Mieli-Vergani1,9, University of Washington Center for Mendelian Genomics13, Joshua D Smith14, Colin A Johnson5, Patricia McClean15, Michael A Simpson16, A S Knisely2, Laura N Bull17,18 & Richard J Thompson1,9 Elucidating genetic causes of cholestasis has proved to be important in understanding the physiology and pathophysiology of the liver. Here we show that proteintruncating mutations in the tight junction protein 2 gene (TJP2) cause failure of protein localization and disruption of tight-junction structure, leading to severe cholestatic liver disease. These findings contrast with those in the embryoniclethal knockout mouse, highlighting differences in redundancy in junctional complexes between organs and species. Progressive familial intrahepatic cholestasis (PFIC) was first described in 1969 (ref. 1). Early-onset progressive liver disease is common to all affected individuals, and PFIC may be subdivided on the basis of the levels of serum γ-glutamyl transferase activity (GGT)2–4. Whereas most forms of cholestasis manifest elevated levels of GGT, most individuals with PFIC have normal or near-normal GGT. In 1998, two genes were found to bear recessive mutation in different subsets of individuals with normal-GGT PFIC2,4. FIC1 (familial intrahepatic cholestasis protein 1) deficiency is caused by mutations in ATP8B1, and BSEP (bile salt export pump) deficiency is caused by mutations in ABCB11. Studies have since shown that approximately one-third of individuals with normal-GGT PFIC do not have mutations in either gene5. We studied 33 affected children from 29 families. All children had chronic cholestatic liver disease, with low GGT for the degree of cholestasis, and did not have mutations in ABCB11 or ATP8B1. Eighteen of the 29 families were consanguineous. We examined
samples by targeted resequencing, whole-exome sequencing or both approaches. Four separate sequencing projects contributed to this work (Supplementary Fig. 1). The targeted resequencing assay was designed to capture 21 genes known to be associated with cholestasis. In addition to PFIC-associated genes, the design included genes responsible for other inherited disorders in which cholestasis features (Supplementary Table 1). For both next-generation sequencing methods, we mapped reads and called variants using NextGENe software or exome sequencing pipelines with the ExomeDepth program, a copy number variation (CNV) detection tool. We removed common variants by comparing the identified variants with those in the dbSNP, 1000 Genome Project and National Heart, Lung, and Blood Institute (NHLBI) Exome Sequencing Project databases. We also filtered out synonymous mutations and nonsynonymous mutations that were not predicted to have a deleterious effect on protein function by the PolyPhen, SIFT or MutationTaster tools. Targeted resequencing of 18 cases identified 5 individuals who each carried a distinct homozygous mutation in TJP2 (cases 1–5). Independently performed whole-exome sequencing identified a disease-causing mutation in TJP2 in two siblings from a consanguineous family. One of these mutations was also among the five identified by targeted resequencing (case 2a). In light of this knowledge, we discovered homozygous mutations in TJP2 in three further individuals by examining whole-exome sequencing data. No other disease-causing variants were found in these families (Supplementary Tables 2, 3 and 4a–e). Three affected siblings of cases with mutations identified through targeted resequencing were confirmed by Sanger sequencing to have the same mutations as their siblings (Supplementary Fig. 1). Clinical and genetic features of the 12 affected individuals with TJP2 mutations are described in Table 1 and Supplementary Figure 2. A 4-bp deletion in family 1 and two 1-bp deletions in families 2 and 3 were located in exon 5, causing frameshifts and the generation of novel protein-coding sequences ending with the same premature termination codon (PTC). We identified an additional 1-bp deletion in exon 9 in family 4, causing a frameshift and consequent introduction of a downstream PTC. Family 5 harbored a mutation in the splice acceptor site of intron 13; cDNA sequencing confirmed that this mutation resulted in alternative splicing using the contiguous AG bases on the 5′ end of exon 14, causing a 2-base deletion in the
1Institute
of Liver Studies, Division of Transplantation Immunology and Mucosal Biology, King’s College London School of Medicine, London, UK. 2Institute of Liver Studies, King’s College Hospital, London, UK. 3Biomedical Research Centre (BRC) Genomics Core Facility, Guy’s and St Thomas’ National Health Service (NHS) Foundation Trust and King’s College London, London, UK. 4King’s College Hospital NHS Foundation Trust, Department of Haematological Medicine, London, UK. 5Leeds Institute of Molecular Medicine, St James’s University Hospital, University of Leeds, Leeds, UK. 6Renal Sciences, Division of Transplantation Immunology and Mucosal Biology, King’s College London School of Medicine, London, UK. 7Division of Gastroenterology, Hepatology and Nutrition, The Hospital for Sick Children, Toronto, Ontario, Canada. 8Department of Pediatrics, University of Toronto, Toronto, Ontario, Canada. 9Paediatric Liver, GI and Nutrition Centre, King’s College Hospital, London, UK. 10Histopathology Department, Royal Hallamshire Hospital, Sheffield, UK. 11Department of Surgery, University of Michigan Medical School, Ann Arbor, Michigan, USA. 12Department of Pediatrics, Children’s Hospital Colorado and University of Colorado School of Medicine, Aurora, Colorado, USA. 13Further information appears in the Supplementary Note. 14Department of Genome Sciences, University of Washington, Seattle, Washington, USA. 15Children’s Liver and Gastroenterology Unit, Leeds General Infirmary, Leeds, UK. 16Division of Genetics and Molecular Medicine, King’s College London School of Medicine, Guy’s Hospital, London, UK. 17Liver Center Laboratory, Department of Medicine, University of California, San Francisco, San Francisco, California, USA. 18Institute for Human Genetics, University of California, San Francisco, San Francisco, California, USA. Correspondence should be addressed to R.J.T. ([email protected]) or M.S. ([email protected]). Received 21 September 2013; accepted 14 February 2014; published online 9 March 2014; doi:10.1038/ng.2918
326
VOLUME 46 | NUMBER 4 | APRIL 2014 Nature Genetics
7 8
6
5
All cases presented in the first 3 months of life. Eight of 12 have undergone orthotopic liver transplantation. None manifested elevated GGT after the postnatal period. The normal range for GGT at 2 months is T Exon 23 Exon 13 WES WES Yes No
p.Glu318Glyfs*2 c.953–735_2356–249del Exons 6–16
p.Arg664Serfs*2 c.1992–2A>G Intron 13
p.Tyr261Serfs*50 p.Ala454Glyfs*60 c.782delA c.1361delC Exon 5 Exon 9 3 4
7 8
1 1
Stable at age 7 years Subdural hematomas
4 2 3 27 24 23 23 24 2 58 74 44 67 75 22 27 63 109 ND 10 2 2.5 8.5 6 4 4 ND p.Ser296Alafs*15 c.885delC Exon 5
WES TRS and WES TRS TRS SS TRS SS WES SS Yes Yes Yes Yes Yes Yes Yes Yes Yes 2a 2b 3 4a 4b 5a 5b 6a 6b 2
0.5 2 2 1 0.25 2 2 3 2
Notes 6 15 2.6 p.Ala256Thrfs*54 c.766_769delGCCT Exon 5 TRS
Nucleotide change Consanguinity
Yes 3 1 1
Age at GGT measurement (months) Early, postneonatal GGT (IU/l) Age at OLT (years) Predicted amino acid change Exon or intron number Mutation discovery approach Age at presentation (months) Case number Family number
Table 1 Clinical and genetic characteristics of the 12 individuals found to harbor homozygous mutations in TJP2
© 2014 Nature America, Inc. All rights reserved.
npg
Chronic respiratory disease
B r i e f c o m m u n i c at i o n s
Nature Genetics VOLUME 46 | NUMBER 4 | APRIL 2014
mRNA and thereby introducing a frameshift and a consequent PTC. We identified structural variations with the CNV tool through examination of the whole-exome sequencing data. We found a deletion of 11 exons (exons 6–16) in family 6; splicing was shown to occur between the 2 exons adjacent to the intronic breakpoints. The novel mRNA was confirmed, by cDNA sequencing, to contain a PTC. We identified an additional rearrangement in family 7 through inspection of wholeexome sequencing data, with the final exon of the gene completely deleted. Finally, we identified a nonsense mutation in whole-exome sequencing data in family 8. We confirmed all mutations by PCR and Sanger sequencing, although in family 7 the exact breakpoints have not been found. All mutations are predicted to abolish protein translation, and none have previously been identified. We investigated the effect of the mutations on gene expression in the liver using quantitative RT-PCR (qRT-PCR). We isolated total RNA from liver biopsy specimens for five affected individuals (cases 1, 4a, 5a, 5b and 6a) and from the livers of six allograft donors. GAPDH, encoding glyceraldehyde 3-phosphate dehydrogenase, served as an internal control. We found a mean reduction of 50% in TJP2 mRNA levels in cases compared to controls, suggesting activation of nonsense-mediated decay (Supplementary Fig. 3). Using liver tissue, immunohistochemical studies (cases 1, 3, 4a, 4b, 5a, 5b and 6a) and protein blotting (cases 1, 4a, 5a, 5b and 6a) performed with an antibody against a C-terminal TJP2 epitope unsurprisingly detected no protein (Fig. 1a). Claudins are integral to the tight-junction structure6. They have been demonstrated to bind to the PDZ1 domain of TJP proteins in vitro7. Claudin-1 (CLDN1) and claudin-2 (CLDN2) are known to be expressed in the liver6 and to manifest different subcellular localizations8. Immunohistochemical analysis of CLDN1 expression showed tight-junction marking in controls. Staining was substantially reduced in liver tissue from cases (Fig. 1b), although protein blot analysis showed normal expression levels of CLDN1 (Fig. 1d). As expected, CLDN2 showed a pericana licular staining pattern in controls8. This distribution was preserved in the material from cases, with an apparent increase in CLDN2 abundance (Fig. 1c). However, protein blot analysis showed normal levels of CLDN2 protein relative to control liver tissue (Fig. 1d). These findings suggest that CLDN1, although expressed, fails to localize in the absence of TJP2, whereas CLDN2 localization is maintained. On transmission electron microscopy, tight junctions in liver tissue from cases appeared elongated and lacked the densest part of the zona occludens, as seen in mouse embryos with knockout of Tjp2 (also known as ZO-2)9 (Supplementary Fig. 4). TJP2 is a cytosolic component of several classes of cell-cell junctions10. Through interaction with cytoskeletal proteins and integral membrane proteins, members of the TJP family have an important role in the localization of components of these paracellular structures. Human TJP2 generates two isoforms (A and C), both of which are widely expressed11. A single incompletely penetrant homozygous missense mutation (affecting both isoforms) was previously identified in TJP2 in some Amish individuals with familial hypercholanemia, a disorder manifesting pruritus and fat malabsorption but not progressive liver disease. This mutation impaired binding of TJP2 to claudins in vitro12. In contrast, the mutations identified in the individuals presented here are predicted to abolish the translation of both isoforms of TJP2. Long-term survival in these individuals contrasts with the embryonic lethality seen in the homozygous knockout of the mouse ortholog9 and suggests important interspecies differences. All individuals with TJP2 mutations investigated here had severe liver disease. Nine of 12 have required liver transplantation, 2 had stable liver disease with mild portal hypertension at ages 4 and 7 years, 327
B r i e f c o m m u n i c at i o n s
npg
© 2014 Nature America, Inc. All rights reserved.
and 1 died at 13 months. In addition, one had recurrent unexplained hematomas despite normal clotting at 8 years, and one had poorly characterized lung disease. Our immunohistochemical studies showed that CLDN1 fails to localize normally to cholangiocyte-cholangiocyte borders and to biliary canaliculus margins, despite normal protein levels. However, localization of CLDN2 does not seem to be similarly dependent on TJP2. We hypothesize that, in humans, lack of TJP2 alone is insufficient to cause substantial disruption of tight junctions in epithelia outside of the liver. In the unusually hostile environment of the canalicular and cholangiocytic membranes, which are exposed to high concentrations of detergent bile acids, expression of TJP2 may be essential for hepatobiliary integrity13. In mice, Tjp2 has been shown to be nonredundant. We have now shown that this lack of redundancy is, for the human ortholog, probably restricted to the liver and that a complete absence of TJP2 protein causes severe liver disease. URLs. Agilent eArray web portal, https://earray.chem.agilent.com/ earray/. Methods Methods and any associated references are available in the online version of the paper. Requests for software. Bespoke Perl script used for homozygosity mapping, [email protected]. Note: Any Supplementary Information and Source Data files are available in the online version of the paper. Acknowledgments M.S. was funded by an Alex Mowat PhD Studentship. R.J.T. and B.E.C. received funding for this work from a consumables grant awarded by the King’s College Hospital Department of Research and Development. Targeted resequencing and whole-exome sequencing were supported by the National Institute for Health Research (NIHR) Biomedical Research Centre based at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the UK Department of Health. C.A.J. was supported by a Sir Jules Thorn Award for Biomedical Research (JTA/09). Additional funding for this project included US National Institutes of Health (NIH) grant R56 DK094828 to L.N.B. and R.J.T., the University of California, San Francisco (UCSF)–King’s College Health Partners Faculty Fellowship Travel Grant (UCSF Academic Senate) to L.N.B., US NIH grant U01 DK062500 to P. Rosenthal, US NIH grants U01 DK062453
328
Case
Control
TJP2
a
d CLDN1
b
1
4a
5a 5b
1
4a 5a 5b
6a
C
CLDN1
GAPDH 6a
C
CLDN2
c
GAPDH
CLDN2 + BSEP
Figure 1 Protein consequences of TJP2 mutation. (a–c) Immunohisto chemical staining of case 5a and a control for TJP2 (a), CLDN1 (b), and CLDN2 and BSEP (c). TJP2 expression (a, brown) is absent from cholangiocytes and canalicular margins. CLDN1 expression (b, brown) is markedly reduced at both sites. CLDN2 expression (c, brown) clustered within the cytoplasm adjoining bile canaliculi (highlighted by BSEP, red), as has been shown previously. Hematoxylin counterstain; scale bar, 100 µm. (d) Protein blotting for CLDN1 and CLDN2 isolated from liver biopsy tissues (one control (C) and five cases). Normalization was performed using GAPDH as a loading control.
and UL1 TR000154 to R.J.S. and US NIH grant U01 DK062456 to J.C.M. Further whole-exome sequencing was undertaken by the University of Washington Center for Mendelian Genomics (UW CMG) and was funded by the National Human Genome Research Institute and by NHLBI grant 1U54HG006493 to D. Nickerson, J. Shendure and M. Bamshad. AUTHOR CONTRIBUTIONS M.S. performed the majority of the experimental work, analyzed and interpreted data, and wrote the manuscript. S.S., E.P. and C.V.L. performed experiments. P.R. and B.E.C. helped design the targeted capture. D.A.P., J.D.S. and M.A.S. analyzed whole-exome sequencing data. L.J.N. helped with protein blotting. B.E.W. performed electron microscopy. B.M.K., S.L. and P.M. provided cases and sequence data. G.M.-V. and T.G. provided cases. J.C.M. and R.J.S. run ChiLDREN and provided samples and clinical data. The University of Washington Center for Mendelian Genomics performed whole-exome sequencing for ChiLDREN cases. A.S.K. undertook histopathological analysis. C.A.J. and L.N.B. directed the whole-exome sequencing projects. R.J.T. initiated the project, analyzed the data and wrote the manuscript. All authors commented on and edited the final manuscript. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html. Clayton, R.J. et al. Am. J. Dis. Child. 117, 112–124 (1969). Bull, L.N. et al. Nat. Genet. 18, 219–224 (1998). de Vree, J.M. et al. Proc. Natl. Acad. Sci. USA 95, 282–287 (1998). Strautnieks, S.S. et al. Nat. Genet. 20, 233–238 (1998). Davit-Spraul, A. et al. Hepatology 51, 1645–1655 (2010). Mitic, L.L. et al. Am. J. Physiol. Gastrointest. Liver Physiol. 279, G250–G254 (2000). 7. Itoh, M. et al. J. Cell Biol. 147, 1351–1363 (1999). 8. Holczbauer, Á. et al. J. Histochem. Cytochem. 61, 294–305 (2013). 9. Xu, J. et al. Mol. Cell. Biol. 28, 1669–1678 (2008). 10. Fanning, A.S. et al. Mol. Biol. Cell 23, 577–590 (2012). 11. Chlenski, A. et al. Biochim. Biophys. Acta 1493, 319–324 (2000). 12. Carlton, V.E. et al. Nat. Genet. 34, 91–96 (2003). 13. Grosse, B. et al. Hepatology 55, 1249–1259 (2012). 1. 2. 3. 4. 5. 6.
VOLUME 46 | NUMBER 4 | APRIL 2014 Nature Genetics
ONLINE METHODS
npg
© 2014 Nature America, Inc. All rights reserved.
Subjects. Cases were selected from clinical databases in each center or from the Childhood Liver Disease Research and Education Network (ChiLDREN) longitudinal study of genetic causes of intrahepatic cholestasis (LOGIC; ClinicalTrials.gov accession NCT00571272). All had chronic cholestatic liver disease, with GGT low for the degree of cholestasis, and were known to be free of mutations in ABCB11 and ATP8B1. All samples had previously been collected for research purposes, after approval by the ethics committees of King’s College Hospital, Leeds Teaching Hospitals and SickKids Toronto and every institutional review board of the ChiLDREN Network and parental consent. The samples used were all deidentified and coded. In the case of local databases, this was performed after the collection of clinical data; in the ChiLDREN network, this was performed prospectively. Next-generation sequencing. For target enrichment sequencing, the probe library was designed using the Agilent eArray web portal. The 176 kb of targeted regions included 364 translated and untranslated exons for 21 genes involved in the major inherited cholestatic disorders (Supplementary Table 1) and 1–3 kb of sequence upstream of the transcriptional start site of each gene promoter. The bait library was optimized for a 120-mer bait length centered on the region of interest with 4× coverage, with 40 bp overlapping the target region boundaries; masking was enabled to avoid repeat regions. To increase coverage and, consequently, the adequacy of base-calling detection, the designed bait library was divided into six different groups according to the proportion of GC content using the SureSelect Bin Orphan and High-GC Baits Workflow from the Galaxy web-based platform. The relative abundance of each group of baits was boosted according to the GC content. Samples were prepared with the SureSelectXT Target Enrichment kit (Agilent Technologies). Whole-exome sequencing was undertaken using the SureSelectXT Human All Exon V4 kit (Agilent Technologies) and the SeqCap EZ Human Exome Library v3.0 (Roche NimbleGen), according to the manufacturers’ protocols. In both cases, cluster generation was carried out with the C-Bot cluster generation automated system and multiplexed sequencing on HiSeq 2000 and 2500 sequencers with the 2 × 100-bp paired-end module (Illumina). Next-generation sequencing data analysis. Next-generation sequencing data were processed and analyzed using NextGENe software (SoftGenetics) and exome sequencing pipelines. Sequences were all aligned to the hg19/GRCh37 human reference genome. NextGENe software. Reads were converted into FASTA format, using a Phred score of ≥15 and minimum coverage of 25× to filter for quality. Reads were mapped using a preloaded BWT alignment algorithm, and mutations were called only when meeting the following values: mutation frequency at a given position of ≥20% and mutation coverage of ≥5×. Exome sequencing analysis. Slightly different approaches were employed by different laboratories. However, sequence alignment was performed using Novalign or Burrows-Wheeler Aligner (BWA). Alignments were processed in the SAM/BAM format using SAMtools14 and the Picard tool, and single-nucleotide variants (SNVs) and indels were called using the Genome Analysis Toolkit (GATK) UnifiedGenotyper15,16. Afterward, genetic variations were annotated using ANNOVAR. ExomeDepth was used for CNV analysis17. Subsequently, variations were compared to several different databases: NCBI dbSNP Build 129 or 132, the 1000 Genomes Project, the NHLBI Exome Sequencing Project, the Exome Variant Server and internal databases. Variations occurring with allele frequency of greater than 2% in the general population were then filtered out. Potential disease-causing mutations were predicted using PolyPhen, SIFT, MutationTaster and Ensembl’s SNP Effect Predictor18. A bespoke Perl script was used to identify biallelic variants compatible with autosomal recessive inheritance (either homozygous or potentially compound heterozygous). Perl scripts are available upon request.
doi:10.1038/ng.2918
CNVs were removed if the read ratio was between 0.2 and 1.5, if they were known19 or if the Bayes factor was ≤10. Sanger sequencing validation. Sanger sequencing validation was performed for all individuals found to harbor a TJP2 mutation and for their affected siblings. Forward and reverse primers were manually designed for the flanking mutated regions (primer sequences are listed with annealing temperature conditions in Supplementary Table 5a). PCR amplification was optimized in accordance with the standard PCR protocol using FastStart Taq DNA Polymerase, dNTPack (Roche Applied Science). Sequencing reactions were performed using the BigDye v.1.1 Terminator cycle sequencing kit and the ABI Prism 3130xl Genetic Analyzer (Life Technologies). To investigate the effect of splice-site mutations and frameshift deletions on mRNA splicing, Sanger sequencing was also performed on cDNA. Forward and reverse primers were manually designed, some within exons and others across exon-intron boundaries (primer sequences are listed with annealing temperature conditions in Supplementary Table 5b). TJP2 mRNA expression. Frozen liver from five liver biopsy specimens and six liver-donor controls was used. Total RNA was isolated from 30 mg of liver tissue using TRIzol (Invitrogen); cDNA was synthesized using 1 µg/µl RNA and the high-capacity RNA-to-cDNA kit (Life Technologies), following the manufacturers’ protocols. To evaluate TJP2 mRNA expression, qRT-PCR was carried out using a TaqMan Gene Expression assay with glyceraldehyde 3-phosphate dehydrogenase (GADPH) as an endogenous control. Primers and probe sets for TJP2 and GAPDH were purchased from Life Technologies. Reactions were performed in triplicate with an ABI Prism 7000 Sequence Detection System, following the manufacturer’s instructions. Data were analyzed by the comparative Ct method (2−∆∆Ct ). Protein blot analysis. Proteins were extracted from 50 mg of frozen liver tissue and homogenized in 200 µl of radioimmunoprecipitation assay buffer containing 1% nonyl phenoxypolyethoxylethanol, 0.5% sodium deoxycholate and 0.1% SDS with protease inhibitors added just before use (10 µl/ml phenylmethylsulfonyl fluoride, 30 µl/ml aprotinin, 10 µl/ml sodium orthovanadate). Protein concentrations were determined using Lowry’s method with a BSA protein standard. Protein samples were normalized to a concentration of 1 µg/µl and heated with 10% protein blot buffer (0.5 M Tris, pH 6.8, 0.35 M SDS, 30% glycerol, 0.6 M dithiothreitol, 0.175 M bromophenol blue) and 10% β-mercaptoethanol for 5 min at 100 °C. Samples were then submitted to electro phoretic separation on 8% SDS-polyacrylamide gels for CLDN1 and CLDN2 and on 15% SDS-polyacrylamide gels for TJP2. Proteins were transferred onto Hybond enhanced chemiluminescence nitrocellulose membranes (GE Healthcare) and blocked with 10% nonfat milk diluted in 0.1% TBS and 0.05% Tween-20 (TBST) for 1 h. Primary immunoblotting was carried out using antibodies to CLDN1 (1:5,000 dilution; 18-7362, Zymed Laboratories, Invitrogen), CLDN2 (1:5,000 dilution; 32-5600, Invitrogen), TJP2 (1:5,000 dilution; LS-B2185, Source BioScience) and GAPDH as loading control (1:20,000 dilution; MAB374, Millipore) and incubated overnight at 4 °C. Membranes were washed with 0.1% TBST for 3 × 10 min. Secondary immunoblotting was carried out with horseradish peroxidase (HRP)-conjugated rabbit-specific antibody (1:10,000 dilution; sc-3837, Santa Cruz Biotechnology) or HRPconjugated mouse-specific antibody (1:20,000 dilution; NA931V, GE Healthcare) for a 1-h incubation at room temperature. Membranes were washed with 0.1% TBST for 3 × 10 min. The Amersham ECL Prime chemiluminescence detection system was used to visualize protein bands (GE Healthcare). Immunohistochemistry. Archival material obtained for the purposes of clinical diagnosis and treatment, fixed in formalin and processed into paraffin wax by routine methods, from individuals with mutation in TJP2 and
Nature Genetics
from individuals with cholestatic liver disease ascribed to known mutation in other genes (ATP8B1 or ABCB11) or considered to be idiopathic (extrahepatic biliary atresia) was sectioned at 4 µm and routinely immunostained (BondMax, Leica Biosystems) with the antibodies used for protein blotting against the TJP2, CLDN1 and CLDN2 proteins (at dilutions of 1:40, 1:100 and 1:16,000, respectively), using hematoxylin counterstaining. The antibody to BSEP was rabbit polyclonal (HPA019035, Sigma-Aldrich; dilution of 1:1,500).
14. Li, H. et al. Bioinformatics 25, 2078–2079 (2009). 15. DePristo, M.A. et al. Nat. Genet. 43, 491–498 (2011). 16. McKenna, A. et al. Genome Res. 20, 1297–1303 (2010). 17. Plagnol, V. et al. Bioinformatics 28, 2747–2754 (2012). 18. McLaren, W. et al. Bioinformatics 26, 2069–2070 (2010). 19. Conrad, D.F. et al. Nature 464, 704–712 (2010).
npg
© 2014 Nature America, Inc. All rights reserved.
Transmission electron microscopy. Tissue specimens were primarily fixed in paraformaldehyde and glutaraldehyde, post-fixed in osmium tetroxide (OsO4)
and embedded in resin. Ultrathin sections were stained with uranyl acetate and lead citrate.
Nature Genetics
doi:10.1038/ng.2918
npg
© 2014 Nature America, Inc. All rights reserved.
Mutations in TJP2 cause progressive cholestatic liver disease Melissa Sambrotta1, Sandra Strautnieks2, Efterpi Papouli3, Peter Rushton4, Barnaby E Clark4, David A Parry5, Clare V Logan5, Lucy J Newbury6, Binita M Kamath7,8, Simon Ling7,8, Tassos Grammatikopoulos1,9, Bart E Wagner10, John C Magee11, Ronald J Sokol12, Giorgina Mieli-Vergani1,9, University of Washington Center for Mendelian Genomics13, Joshua D Smith14, Colin A Johnson5, Patricia McClean15, Michael A Simpson16, A S Knisely2, Laura N Bull17,18 & Richard J Thompson1,9 Elucidating genetic causes of cholestasis has proved to be important in understanding the physiology and pathophysiology of the liver. Here we show that proteintruncating mutations in the tight junction protein 2 gene (TJP2) cause failure of protein localization and disruption of tight-junction structure, leading to severe cholestatic liver disease. These findings contrast with those in the embryoniclethal knockout mouse, highlighting differences in redundancy in junctional complexes between organs and species. Progressive familial intrahepatic cholestasis (PFIC) was first described in 1969 (ref. 1). Early-onset progressive liver disease is common to all affected individuals, and PFIC may be subdivided on the basis of the levels of serum γ-glutamyl transferase activity (GGT)2–4. Whereas most forms of cholestasis manifest elevated levels of GGT, most individuals with PFIC have normal or near-normal GGT. In 1998, two genes were found to bear recessive mutation in different subsets of individuals with normal-GGT PFIC2,4. FIC1 (familial intrahepatic cholestasis protein 1) deficiency is caused by mutations in ATP8B1, and BSEP (bile salt export pump) deficiency is caused by mutations in ABCB11. Studies have since shown that approximately one-third of individuals with normal-GGT PFIC do not have mutations in either gene5. We studied 33 affected children from 29 families. All children had chronic cholestatic liver disease, with low GGT for the degree of cholestasis, and did not have mutations in ABCB11 or ATP8B1. Eighteen of the 29 families were consanguineous. We examined
samples by targeted resequencing, whole-exome sequencing or both approaches. Four separate sequencing projects contributed to this work (Supplementary Fig. 1). The targeted resequencing assay was designed to capture 21 genes known to be associated with cholestasis. In addition to PFIC-associated genes, the design included genes responsible for other inherited disorders in which cholestasis features (Supplementary Table 1). For both next-generation sequencing methods, we mapped reads and called variants using NextGENe software or exome sequencing pipelines with the ExomeDepth program, a copy number variation (CNV) detection tool. We removed common variants by comparing the identified variants with those in the dbSNP, 1000 Genome Project and National Heart, Lung, and Blood Institute (NHLBI) Exome Sequencing Project databases. We also filtered out synonymous mutations and nonsynonymous mutations that were not predicted to have a deleterious effect on protein function by the PolyPhen, SIFT or MutationTaster tools. Targeted resequencing of 18 cases identified 5 individuals who each carried a distinct homozygous mutation in TJP2 (cases 1–5). Independently performed whole-exome sequencing identified a disease-causing mutation in TJP2 in two siblings from a consanguineous family. One of these mutations was also among the five identified by targeted resequencing (case 2a). In light of this knowledge, we discovered homozygous mutations in TJP2 in three further individuals by examining whole-exome sequencing data. No other disease-causing variants were found in these families (Supplementary Tables 2, 3 and 4a–e). Three affected siblings of cases with mutations identified through targeted resequencing were confirmed by Sanger sequencing to have the same mutations as their siblings (Supplementary Fig. 1). Clinical and genetic features of the 12 affected individuals with TJP2 mutations are described in Table 1 and Supplementary Figure 2. A 4-bp deletion in family 1 and two 1-bp deletions in families 2 and 3 were located in exon 5, causing frameshifts and the generation of novel protein-coding sequences ending with the same premature termination codon (PTC). We identified an additional 1-bp deletion in exon 9 in family 4, causing a frameshift and consequent introduction of a downstream PTC. Family 5 harbored a mutation in the splice acceptor site of intron 13; cDNA sequencing confirmed that this mutation resulted in alternative splicing using the contiguous AG bases on the 5′ end of exon 14, causing a 2-base deletion in the
1Institute
of Liver Studies, Division of Transplantation Immunology and Mucosal Biology, King’s College London School of Medicine, London, UK. 2Institute of Liver Studies, King’s College Hospital, London, UK. 3Biomedical Research Centre (BRC) Genomics Core Facility, Guy’s and St Thomas’ National Health Service (NHS) Foundation Trust and King’s College London, London, UK. 4King’s College Hospital NHS Foundation Trust, Department of Haematological Medicine, London, UK. 5Leeds Institute of Molecular Medicine, St James’s University Hospital, University of Leeds, Leeds, UK. 6Renal Sciences, Division of Transplantation Immunology and Mucosal Biology, King’s College London School of Medicine, London, UK. 7Division of Gastroenterology, Hepatology and Nutrition, The Hospital for Sick Children, Toronto, Ontario, Canada. 8Department of Pediatrics, University of Toronto, Toronto, Ontario, Canada. 9Paediatric Liver, GI and Nutrition Centre, King’s College Hospital, London, UK. 10Histopathology Department, Royal Hallamshire Hospital, Sheffield, UK. 11Department of Surgery, University of Michigan Medical School, Ann Arbor, Michigan, USA. 12Department of Pediatrics, Children’s Hospital Colorado and University of Colorado School of Medicine, Aurora, Colorado, USA. 13Further information appears in the Supplementary Note. 14Department of Genome Sciences, University of Washington, Seattle, Washington, USA. 15Children’s Liver and Gastroenterology Unit, Leeds General Infirmary, Leeds, UK. 16Division of Genetics and Molecular Medicine, King’s College London School of Medicine, Guy’s Hospital, London, UK. 17Liver Center Laboratory, Department of Medicine, University of California, San Francisco, San Francisco, California, USA. 18Institute for Human Genetics, University of California, San Francisco, San Francisco, California, USA. Correspondence should be addressed to R.J.T. ([email protected]) or M.S. ([email protected]). Received 21 September 2013; accepted 14 February 2014; published online 9 March 2014; doi:10.1038/ng.2918
326
VOLUME 46 | NUMBER 4 | APRIL 2014 Nature Genetics
7 8
6
5
All cases presented in the first 3 months of life. Eight of 12 have undergone orthotopic liver transplantation. None manifested elevated GGT after the postnatal period. The normal range for GGT at 2 months is T Exon 23 Exon 13 WES WES Yes No
p.Glu318Glyfs*2 c.953–735_2356–249del Exons 6–16
p.Arg664Serfs*2 c.1992–2A>G Intron 13
p.Tyr261Serfs*50 p.Ala454Glyfs*60 c.782delA c.1361delC Exon 5 Exon 9 3 4
7 8
1 1
Stable at age 7 years Subdural hematomas
4 2 3 27 24 23 23 24 2 58 74 44 67 75 22 27 63 109 ND 10 2 2.5 8.5 6 4 4 ND p.Ser296Alafs*15 c.885delC Exon 5
WES TRS and WES TRS TRS SS TRS SS WES SS Yes Yes Yes Yes Yes Yes Yes Yes Yes 2a 2b 3 4a 4b 5a 5b 6a 6b 2
0.5 2 2 1 0.25 2 2 3 2
Notes 6 15 2.6 p.Ala256Thrfs*54 c.766_769delGCCT Exon 5 TRS
Nucleotide change Consanguinity
Yes 3 1 1
Age at GGT measurement (months) Early, postneonatal GGT (IU/l) Age at OLT (years) Predicted amino acid change Exon or intron number Mutation discovery approach Age at presentation (months) Case number Family number
Table 1 Clinical and genetic characteristics of the 12 individuals found to harbor homozygous mutations in TJP2
© 2014 Nature America, Inc. All rights reserved.
npg
Chronic respiratory disease
B r i e f c o m m u n i c at i o n s
Nature Genetics VOLUME 46 | NUMBER 4 | APRIL 2014
mRNA and thereby introducing a frameshift and a consequent PTC. We identified structural variations with the CNV tool through examination of the whole-exome sequencing data. We found a deletion of 11 exons (exons 6–16) in family 6; splicing was shown to occur between the 2 exons adjacent to the intronic breakpoints. The novel mRNA was confirmed, by cDNA sequencing, to contain a PTC. We identified an additional rearrangement in family 7 through inspection of wholeexome sequencing data, with the final exon of the gene completely deleted. Finally, we identified a nonsense mutation in whole-exome sequencing data in family 8. We confirmed all mutations by PCR and Sanger sequencing, although in family 7 the exact breakpoints have not been found. All mutations are predicted to abolish protein translation, and none have previously been identified. We investigated the effect of the mutations on gene expression in the liver using quantitative RT-PCR (qRT-PCR). We isolated total RNA from liver biopsy specimens for five affected individuals (cases 1, 4a, 5a, 5b and 6a) and from the livers of six allograft donors. GAPDH, encoding glyceraldehyde 3-phosphate dehydrogenase, served as an internal control. We found a mean reduction of 50% in TJP2 mRNA levels in cases compared to controls, suggesting activation of nonsense-mediated decay (Supplementary Fig. 3). Using liver tissue, immunohistochemical studies (cases 1, 3, 4a, 4b, 5a, 5b and 6a) and protein blotting (cases 1, 4a, 5a, 5b and 6a) performed with an antibody against a C-terminal TJP2 epitope unsurprisingly detected no protein (Fig. 1a). Claudins are integral to the tight-junction structure6. They have been demonstrated to bind to the PDZ1 domain of TJP proteins in vitro7. Claudin-1 (CLDN1) and claudin-2 (CLDN2) are known to be expressed in the liver6 and to manifest different subcellular localizations8. Immunohistochemical analysis of CLDN1 expression showed tight-junction marking in controls. Staining was substantially reduced in liver tissue from cases (Fig. 1b), although protein blot analysis showed normal expression levels of CLDN1 (Fig. 1d). As expected, CLDN2 showed a pericana licular staining pattern in controls8. This distribution was preserved in the material from cases, with an apparent increase in CLDN2 abundance (Fig. 1c). However, protein blot analysis showed normal levels of CLDN2 protein relative to control liver tissue (Fig. 1d). These findings suggest that CLDN1, although expressed, fails to localize in the absence of TJP2, whereas CLDN2 localization is maintained. On transmission electron microscopy, tight junctions in liver tissue from cases appeared elongated and lacked the densest part of the zona occludens, as seen in mouse embryos with knockout of Tjp2 (also known as ZO-2)9 (Supplementary Fig. 4). TJP2 is a cytosolic component of several classes of cell-cell junctions10. Through interaction with cytoskeletal proteins and integral membrane proteins, members of the TJP family have an important role in the localization of components of these paracellular structures. Human TJP2 generates two isoforms (A and C), both of which are widely expressed11. A single incompletely penetrant homozygous missense mutation (affecting both isoforms) was previously identified in TJP2 in some Amish individuals with familial hypercholanemia, a disorder manifesting pruritus and fat malabsorption but not progressive liver disease. This mutation impaired binding of TJP2 to claudins in vitro12. In contrast, the mutations identified in the individuals presented here are predicted to abolish the translation of both isoforms of TJP2. Long-term survival in these individuals contrasts with the embryonic lethality seen in the homozygous knockout of the mouse ortholog9 and suggests important interspecies differences. All individuals with TJP2 mutations investigated here had severe liver disease. Nine of 12 have required liver transplantation, 2 had stable liver disease with mild portal hypertension at ages 4 and 7 years, 327
B r i e f c o m m u n i c at i o n s
npg
© 2014 Nature America, Inc. All rights reserved.
and 1 died at 13 months. In addition, one had recurrent unexplained hematomas despite normal clotting at 8 years, and one had poorly characterized lung disease. Our immunohistochemical studies showed that CLDN1 fails to localize normally to cholangiocyte-cholangiocyte borders and to biliary canaliculus margins, despite normal protein levels. However, localization of CLDN2 does not seem to be similarly dependent on TJP2. We hypothesize that, in humans, lack of TJP2 alone is insufficient to cause substantial disruption of tight junctions in epithelia outside of the liver. In the unusually hostile environment of the canalicular and cholangiocytic membranes, which are exposed to high concentrations of detergent bile acids, expression of TJP2 may be essential for hepatobiliary integrity13. In mice, Tjp2 has been shown to be nonredundant. We have now shown that this lack of redundancy is, for the human ortholog, probably restricted to the liver and that a complete absence of TJP2 protein causes severe liver disease. URLs. Agilent eArray web portal, https://earray.chem.agilent.com/ earray/. Methods Methods and any associated references are available in the online version of the paper. Requests for software. Bespoke Perl script used for homozygosity mapping, [email protected]. Note: Any Supplementary Information and Source Data files are available in the online version of the paper. Acknowledgments M.S. was funded by an Alex Mowat PhD Studentship. R.J.T. and B.E.C. received funding for this work from a consumables grant awarded by the King’s College Hospital Department of Research and Development. Targeted resequencing and whole-exome sequencing were supported by the National Institute for Health Research (NIHR) Biomedical Research Centre based at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the UK Department of Health. C.A.J. was supported by a Sir Jules Thorn Award for Biomedical Research (JTA/09). Additional funding for this project included US National Institutes of Health (NIH) grant R56 DK094828 to L.N.B. and R.J.T., the University of California, San Francisco (UCSF)–King’s College Health Partners Faculty Fellowship Travel Grant (UCSF Academic Senate) to L.N.B., US NIH grant U01 DK062500 to P. Rosenthal, US NIH grants U01 DK062453
328
Case
Control
TJP2
a
d CLDN1
b
1
4a
5a 5b
1
4a 5a 5b
6a
C
CLDN1
GAPDH 6a
C
CLDN2
c
GAPDH
CLDN2 + BSEP
Figure 1 Protein consequences of TJP2 mutation. (a–c) Immunohisto chemical staining of case 5a and a control for TJP2 (a), CLDN1 (b), and CLDN2 and BSEP (c). TJP2 expression (a, brown) is absent from cholangiocytes and canalicular margins. CLDN1 expression (b, brown) is markedly reduced at both sites. CLDN2 expression (c, brown) clustered within the cytoplasm adjoining bile canaliculi (highlighted by BSEP, red), as has been shown previously. Hematoxylin counterstain; scale bar, 100 µm. (d) Protein blotting for CLDN1 and CLDN2 isolated from liver biopsy tissues (one control (C) and five cases). Normalization was performed using GAPDH as a loading control.
and UL1 TR000154 to R.J.S. and US NIH grant U01 DK062456 to J.C.M. Further whole-exome sequencing was undertaken by the University of Washington Center for Mendelian Genomics (UW CMG) and was funded by the National Human Genome Research Institute and by NHLBI grant 1U54HG006493 to D. Nickerson, J. Shendure and M. Bamshad. AUTHOR CONTRIBUTIONS M.S. performed the majority of the experimental work, analyzed and interpreted data, and wrote the manuscript. S.S., E.P. and C.V.L. performed experiments. P.R. and B.E.C. helped design the targeted capture. D.A.P., J.D.S. and M.A.S. analyzed whole-exome sequencing data. L.J.N. helped with protein blotting. B.E.W. performed electron microscopy. B.M.K., S.L. and P.M. provided cases and sequence data. G.M.-V. and T.G. provided cases. J.C.M. and R.J.S. run ChiLDREN and provided samples and clinical data. The University of Washington Center for Mendelian Genomics performed whole-exome sequencing for ChiLDREN cases. A.S.K. undertook histopathological analysis. C.A.J. and L.N.B. directed the whole-exome sequencing projects. R.J.T. initiated the project, analyzed the data and wrote the manuscript. All authors commented on and edited the final manuscript. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html. Clayton, R.J. et al. Am. J. Dis. Child. 117, 112–124 (1969). Bull, L.N. et al. Nat. Genet. 18, 219–224 (1998). de Vree, J.M. et al. Proc. Natl. Acad. Sci. USA 95, 282–287 (1998). Strautnieks, S.S. et al. Nat. Genet. 20, 233–238 (1998). Davit-Spraul, A. et al. Hepatology 51, 1645–1655 (2010). Mitic, L.L. et al. Am. J. Physiol. Gastrointest. Liver Physiol. 279, G250–G254 (2000). 7. Itoh, M. et al. J. Cell Biol. 147, 1351–1363 (1999). 8. Holczbauer, Á. et al. J. Histochem. Cytochem. 61, 294–305 (2013). 9. Xu, J. et al. Mol. Cell. Biol. 28, 1669–1678 (2008). 10. Fanning, A.S. et al. Mol. Biol. Cell 23, 577–590 (2012). 11. Chlenski, A. et al. Biochim. Biophys. Acta 1493, 319–324 (2000). 12. Carlton, V.E. et al. Nat. Genet. 34, 91–96 (2003). 13. Grosse, B. et al. Hepatology 55, 1249–1259 (2012). 1. 2. 3. 4. 5. 6.
VOLUME 46 | NUMBER 4 | APRIL 2014 Nature Genetics
ONLINE METHODS
npg
© 2014 Nature America, Inc. All rights reserved.
Subjects. Cases were selected from clinical databases in each center or from the Childhood Liver Disease Research and Education Network (ChiLDREN) longitudinal study of genetic causes of intrahepatic cholestasis (LOGIC; ClinicalTrials.gov accession NCT00571272). All had chronic cholestatic liver disease, with GGT low for the degree of cholestasis, and were known to be free of mutations in ABCB11 and ATP8B1. All samples had previously been collected for research purposes, after approval by the ethics committees of King’s College Hospital, Leeds Teaching Hospitals and SickKids Toronto and every institutional review board of the ChiLDREN Network and parental consent. The samples used were all deidentified and coded. In the case of local databases, this was performed after the collection of clinical data; in the ChiLDREN network, this was performed prospectively. Next-generation sequencing. For target enrichment sequencing, the probe library was designed using the Agilent eArray web portal. The 176 kb of targeted regions included 364 translated and untranslated exons for 21 genes involved in the major inherited cholestatic disorders (Supplementary Table 1) and 1–3 kb of sequence upstream of the transcriptional start site of each gene promoter. The bait library was optimized for a 120-mer bait length centered on the region of interest with 4× coverage, with 40 bp overlapping the target region boundaries; masking was enabled to avoid repeat regions. To increase coverage and, consequently, the adequacy of base-calling detection, the designed bait library was divided into six different groups according to the proportion of GC content using the SureSelect Bin Orphan and High-GC Baits Workflow from the Galaxy web-based platform. The relative abundance of each group of baits was boosted according to the GC content. Samples were prepared with the SureSelectXT Target Enrichment kit (Agilent Technologies). Whole-exome sequencing was undertaken using the SureSelectXT Human All Exon V4 kit (Agilent Technologies) and the SeqCap EZ Human Exome Library v3.0 (Roche NimbleGen), according to the manufacturers’ protocols. In both cases, cluster generation was carried out with the C-Bot cluster generation automated system and multiplexed sequencing on HiSeq 2000 and 2500 sequencers with the 2 × 100-bp paired-end module (Illumina). Next-generation sequencing data analysis. Next-generation sequencing data were processed and analyzed using NextGENe software (SoftGenetics) and exome sequencing pipelines. Sequences were all aligned to the hg19/GRCh37 human reference genome. NextGENe software. Reads were converted into FASTA format, using a Phred score of ≥15 and minimum coverage of 25× to filter for quality. Reads were mapped using a preloaded BWT alignment algorithm, and mutations were called only when meeting the following values: mutation frequency at a given position of ≥20% and mutation coverage of ≥5×. Exome sequencing analysis. Slightly different approaches were employed by different laboratories. However, sequence alignment was performed using Novalign or Burrows-Wheeler Aligner (BWA). Alignments were processed in the SAM/BAM format using SAMtools14 and the Picard tool, and single-nucleotide variants (SNVs) and indels were called using the Genome Analysis Toolkit (GATK) UnifiedGenotyper15,16. Afterward, genetic variations were annotated using ANNOVAR. ExomeDepth was used for CNV analysis17. Subsequently, variations were compared to several different databases: NCBI dbSNP Build 129 or 132, the 1000 Genomes Project, the NHLBI Exome Sequencing Project, the Exome Variant Server and internal databases. Variations occurring with allele frequency of greater than 2% in the general population were then filtered out. Potential disease-causing mutations were predicted using PolyPhen, SIFT, MutationTaster and Ensembl’s SNP Effect Predictor18. A bespoke Perl script was used to identify biallelic variants compatible with autosomal recessive inheritance (either homozygous or potentially compound heterozygous). Perl scripts are available upon request.
doi:10.1038/ng.2918
CNVs were removed if the read ratio was between 0.2 and 1.5, if they were known19 or if the Bayes factor was ≤10. Sanger sequencing validation. Sanger sequencing validation was performed for all individuals found to harbor a TJP2 mutation and for their affected siblings. Forward and reverse primers were manually designed for the flanking mutated regions (primer sequences are listed with annealing temperature conditions in Supplementary Table 5a). PCR amplification was optimized in accordance with the standard PCR protocol using FastStart Taq DNA Polymerase, dNTPack (Roche Applied Science). Sequencing reactions were performed using the BigDye v.1.1 Terminator cycle sequencing kit and the ABI Prism 3130xl Genetic Analyzer (Life Technologies). To investigate the effect of splice-site mutations and frameshift deletions on mRNA splicing, Sanger sequencing was also performed on cDNA. Forward and reverse primers were manually designed, some within exons and others across exon-intron boundaries (primer sequences are listed with annealing temperature conditions in Supplementary Table 5b). TJP2 mRNA expression. Frozen liver from five liver biopsy specimens and six liver-donor controls was used. Total RNA was isolated from 30 mg of liver tissue using TRIzol (Invitrogen); cDNA was synthesized using 1 µg/µl RNA and the high-capacity RNA-to-cDNA kit (Life Technologies), following the manufacturers’ protocols. To evaluate TJP2 mRNA expression, qRT-PCR was carried out using a TaqMan Gene Expression assay with glyceraldehyde 3-phosphate dehydrogenase (GADPH) as an endogenous control. Primers and probe sets for TJP2 and GAPDH were purchased from Life Technologies. Reactions were performed in triplicate with an ABI Prism 7000 Sequence Detection System, following the manufacturer’s instructions. Data were analyzed by the comparative Ct method (2−∆∆Ct ). Protein blot analysis. Proteins were extracted from 50 mg of frozen liver tissue and homogenized in 200 µl of radioimmunoprecipitation assay buffer containing 1% nonyl phenoxypolyethoxylethanol, 0.5% sodium deoxycholate and 0.1% SDS with protease inhibitors added just before use (10 µl/ml phenylmethylsulfonyl fluoride, 30 µl/ml aprotinin, 10 µl/ml sodium orthovanadate). Protein concentrations were determined using Lowry’s method with a BSA protein standard. Protein samples were normalized to a concentration of 1 µg/µl and heated with 10% protein blot buffer (0.5 M Tris, pH 6.8, 0.35 M SDS, 30% glycerol, 0.6 M dithiothreitol, 0.175 M bromophenol blue) and 10% β-mercaptoethanol for 5 min at 100 °C. Samples were then submitted to electro phoretic separation on 8% SDS-polyacrylamide gels for CLDN1 and CLDN2 and on 15% SDS-polyacrylamide gels for TJP2. Proteins were transferred onto Hybond enhanced chemiluminescence nitrocellulose membranes (GE Healthcare) and blocked with 10% nonfat milk diluted in 0.1% TBS and 0.05% Tween-20 (TBST) for 1 h. Primary immunoblotting was carried out using antibodies to CLDN1 (1:5,000 dilution; 18-7362, Zymed Laboratories, Invitrogen), CLDN2 (1:5,000 dilution; 32-5600, Invitrogen), TJP2 (1:5,000 dilution; LS-B2185, Source BioScience) and GAPDH as loading control (1:20,000 dilution; MAB374, Millipore) and incubated overnight at 4 °C. Membranes were washed with 0.1% TBST for 3 × 10 min. Secondary immunoblotting was carried out with horseradish peroxidase (HRP)-conjugated rabbit-specific antibody (1:10,000 dilution; sc-3837, Santa Cruz Biotechnology) or HRPconjugated mouse-specific antibody (1:20,000 dilution; NA931V, GE Healthcare) for a 1-h incubation at room temperature. Membranes were washed with 0.1% TBST for 3 × 10 min. The Amersham ECL Prime chemiluminescence detection system was used to visualize protein bands (GE Healthcare). Immunohistochemistry. Archival material obtained for the purposes of clinical diagnosis and treatment, fixed in formalin and processed into paraffin wax by routine methods, from individuals with mutation in TJP2 and
Nature Genetics
from individuals with cholestatic liver disease ascribed to known mutation in other genes (ATP8B1 or ABCB11) or considered to be idiopathic (extrahepatic biliary atresia) was sectioned at 4 µm and routinely immunostained (BondMax, Leica Biosystems) with the antibodies used for protein blotting against the TJP2, CLDN1 and CLDN2 proteins (at dilutions of 1:40, 1:100 and 1:16,000, respectively), using hematoxylin counterstaining. The antibody to BSEP was rabbit polyclonal (HPA019035, Sigma-Aldrich; dilution of 1:1,500).
14. Li, H. et al. Bioinformatics 25, 2078–2079 (2009). 15. DePristo, M.A. et al. Nat. Genet. 43, 491–498 (2011). 16. McKenna, A. et al. Genome Res. 20, 1297–1303 (2010). 17. Plagnol, V. et al. Bioinformatics 28, 2747–2754 (2012). 18. McLaren, W. et al. Bioinformatics 26, 2069–2070 (2010). 19. Conrad, D.F. et al. Nature 464, 704–712 (2010).
npg
© 2014 Nature America, Inc. All rights reserved.
Transmission electron microscopy. Tissue specimens were primarily fixed in paraformaldehyde and glutaraldehyde, post-fixed in osmium tetroxide (OsO4)
and embedded in resin. Ultrathin sections were stained with uranyl acetate and lead citrate.
Nature Genetics
doi:10.1038/ng.2918
