A novel TNFRSF1A splice mutation associated with ... - The BMJ

22 downloads 2486 Views 212KB Size Report
Dec 17, 2007 - were quantified using BioRad Quantity One 4.6 software and expressed as a ...... Receive free email alerts when new articles cite this article.
ARD Online First, published on December 17, 2007 as 10.1136/ard.2007.078667

A novel TNFRSF1A splice mutation associated with increased NF-κB transcription factor activation in patients with TNFreceptor associated periodic syndrome (TRAPS) 1a*

Churchman Sarah M, 1a*Church Leigh D, 1bSavic Sinisa, 1aCoulthard Lydia R, 1a Hayward Bruce, 2Nedjai Belinda, 2Turner Mark D, 1aMathews Rebeccah J, 3 Baguley Elaine, 2Hitman Graham A, 1bGooi Hock C, 1bWood Philip MD, 1a Emery Paul, 1aMcDermott Michael F. 1a

Sarah M. Churchman, PhD, Leigh D. Church, PhD, Bruce Hayward, PhD, Lydia R. Coulthard BMedSci., Rebeccah J. Matthews BSc., Paul Emery, MA, MD, FRCP, Michael F. McDermott, MRCPI, DMed 1bSinisa Savic, MRCP, MSc: 1aSection of Musculoskeletal Disease, Leeds Institute of Molecular Medicine and 1bDepartment of Clinical Immunology and Allergy, St. James’s University Hospital, Beckett Street, Leeds, LS9 7TF, UK; 2Belinda Nedjai, DEA, Mark D. Turner, PhD, Graham A Hitman, MBBS, MD, FRCP: Centre for Diabetes and Metabolic Medicine, Institute of Cell and Molecular Science, Barts and The London, Queen Mary's School of Medicine and Dentistry, University of London, Whitechapel, London E1 2AT, UK. 3Elaine Baguley, Department of Rheumatology, Hull Royal Infirmary, Hull HU3 2JZ. * These authors have contributed equally to the manuscript. Correspondence to: Michael F. McDermott, Leeds Institute of Molecular Medicine, Wellcome Trust Brennar Building, St. James’s University Hospital, Beckett Street, Leeds, LS9 7TF, UK. Email: [email protected]

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd to permit this article (if accepted) to be published in ARD and any other BMJPGL products and sublicences such use and exploit all subsidiary rights, as set out in our licence (http://ARD.bmjjournals.com/ifora/licence.pdf).

1 Copyright Article author (or their employer) 2007. Produced by BMJ Publishing Group Ltd (& EULAR) under licence.

Abstract Objective. To characterise and investigate the functional consequences of a novel TNFRSF1A splice site mutation causing TRAPS in a 16 year old male patient and his mother. Methods. Mutational DNA screening was performed in the patient and his mother. Western blotting was used to analyse protein expression levels of TNFR1. A multiplex bead immunoassay was used to quantify serum levels of range of cytokines, and an ELISA-based transcription factor assay to measure NF-κB transactivation. Serum levels of soluble TNFR1 (sTNFR1) were measured by ELISA and FACS analysis used to measure monocyte TNFR1 cell surface expression. Results. A novel mutation, c.472+1G>A (C158delinsYERSSPEAKPSPHPRG), involving a splice site in intron 4 of TNFRSF1A, was found in both the proband and affected mother leading to a 45 nucleotide insertion of intronic DNA into the mRNA, resulting in an in-frame insertion of 15 amino acids in the mature TNFR1 protein and a deletion of a cysteine residue C129(158) in cysteine rich domain (CRD) 3. The patients had reduced serum sTNFR1 and surface expression levels of TNFR1, with marked increases in both pro- and antiinflammatory cytokine. Their peripheral blood mononuclear cells (PBMC) had increased basal NF-κB activation compared with healthy controls and also had increased p50 nuclear expression following TNF stimulation compared with PBMC from healthy controls, as well as T50M (T79M) and C88R (C117R) TRAPS patients and RA patients. Conclusion. A novel, TRAPS causing, TNFRSF1A splice site mutation is associated with decreased sTNFR1 levels, cell surface and whole cell extract expression, and increased NFκB transcription factor activation.

2

Introduction Tumor necrosis factor receptor-associated periodic syndrome (TRAPS) is an autosomal dominant autoinflammatory disorder caused by mutations in the TNFRSF1A gene,[1-9] characterised by recurrent attacks of fever, abdominal pain, synovial inflammation, conjunctivitis, periorbital oedema and a positive family history in most cases. The TNFRSF1A gene comprises 10 exons, and translates to a 455 amino acid (a.a) precursor where 29 a.a are cleaved from the amino terminus to give the mature TNFR1 protein. More than 50 different pathogenic mutations have been described (INFEVERS database, http://fmf.igh.cnrs.fr/infevers).[10, 11] The majority (over 90%) of TNFRSF1A mutations are single-nucleotide missense mutations within exons 2, 3, 4 and 6; however, one mutation, creating a splice site in intron 2, and two deletions, c.211_213del [5] and c.293_295del, have also been reported (INFEVERS database). Most mutations described to date are located in the extracellular region in cysteine rich domains, with the single exception of I170N (I199N) [The codon number in the parenthesis refers to the conventional codon number including the 29 amino acid leader sequence] located close to the receptor cleavage site, between Asn172 (201) and Val173 (202).[12] The mechanisms by which mutations in the TNFRSF1A gene cause the clinical features of this disease are still largely unknown. Several independent groups have proposed multiple mechanisms, including impaired shedding,[1] abnormal apoptosis,[13] and defective trafficking.[14, 15] Several reports have relied upon expression models with immortalised cell lines or the transfection of truncated mutant TNFR1 constructs to study the cellular mechanisms of this disease. This has lead to several contradictory findings that are likely to be more reflective of the model rather than the particular mutation involved. Furthermore, it is emerging that there are likely to be mutation specific mechanisms at work that are responsible for the wide range in disease severity observed, as demonstrated by low penetrance mutations, such as P46L (P75L) and R92Q (R121Q), and the more severe pathology associated with mutations involving cysteine residues and T50 (T79) mutations.[3, 5] Here we describe a novel mutation, involving a splice site in intron 4 of TNFRSF1A, in a 16-year-old male patient with a long-standing history of multiple previously unexplained episodic symptoms, including recurrent fevers and abdominal pain. Previous investigations had excluded inflammatory bowel disease, familial Mediterranean fever (FMF) and Hyper IgD and periodic fever syndrome (HIDS). Using primary cells, we also report that this TNFRSF1A mutation leads to increased NF-κB activation.

3

Materials and Methods Patients and Health controls The study was approved by the Leeds (East) Research Ethics Committee. Blood samples were obtained from patients and healthy controls, all with informed consent. Disease control groups for the functional studies were; two TRAPS patients with known mutations T50M (T79M) and C88R (C117R) and 3 patients with rheumatoid arthritis (RA). The peripheral blood mononuclear cells (PBMC) were isolated by gradient lymphoprepTM (Invitrogen, UK) centrifugation, counted and then divided for cryopreservation, FACs analysis, RNA and protein extraction. Polymerase chain reaction (PCR) and DNA sequencing DNA was isolated from whole blood using a DNeasy spin column (Qiagen, UK) and PCR was carried out as described previously.[1] Primers designed for cDNA PCR were TNFR 3-6 F: GCTCCAAATGCCGAAAGG and TNFR 3-6 R CGTGCACTCCAGGCTTTTCT (MWG Biotech, Germany). PCR conditions were as described previously.[1] The products were purified using ExoSAP (USB, Germany), and prepared for sequencing using BigDye 3.1 chemistry. Sequencing was carried out on the ABI3130 Genetic Analyser (Applied Biosystems, USA). The cDNA PCR products were cloned into pGEM-TEasy (ProMega, UK) and sequenced as above. Quantitative real time PCR RNA was isolated from the PBMC using a phenol:chloroform extraction method and cDNA reverse transcribed using Superscript II (Invitrogen, UK). Quantitative real time PCR was carried out on AB7900, using Applied Biosystems core kit reagents for 50 cycles. Exon targeting primers were TNFR F: GTGCTTCAATTGCAGCCTCTG and TNFR R: CCTGCATGGCAGGTGCA (Accession no. TNFRSF1A: NM_001065). Product size was determined using 2 % TAE agarose gel electrophoresis. Western blot analysis for TNFR1 expression Protein lysates were prepared from isolated PBMC using a cytsolic/nuclear fractionation kit (Biovision, USA). Lysate were normalised to protein and subjected to 10% sodium dodecylsulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) under reducing conditions and analysed by western blotting (WB). Blots were probed with a goat polyclonal anti-human TNFR1 antibody, raised against an epitope mapping at the N-terminus (clone N20; SCBT, USA), and bound immunoglobulin was detected with an enhanced chemiluminescence kit (GE Healthcare, UK); blots were subsequently re-probed with anti-β actin monoclonal antibody (MoAb) (Sigma-Aldrich, UK), as a loading control. Protein bands were quantified using BioRad Quantity One 4.6 software and expressed as a ratio (TNFR1/βactin). Measurement of sTNFR1 levels in sera The concentrations of sTNFR1 were measured using a commercially available ELISA assay (R&D System, UK), as described.[1] TNFR1 expression analysis by FACS analysis The surface and intracellular expression of TNFR1 was studied by FACS analysis in monocytes from the proband and his mother. PBMC were isolated from blood using Lymphoprep (Nycomed Pharma AS, Norway) and stained with FITC-labelled TNFR1 MoAb (Serotec,UK). To identify the monocyte population, double staining was carried out by

4

simultaneous incubation with a PE-labelled or FITC-labelled antibody respectively, recognizing the monocyte marker CD14 (BD Bioscience, UK). Corresponding isotype control MoAbs (BD Bioscience, UK), were used as negative controls. For analysis of intracellular TNFR1 expression the cells were first fixed and permeabilised (Caltag, UK) prior to staining. TNFR1 shedding was measured following PBMC stimulation with 50 ng/ml phorbal 12-myristate 13-acetate PMA (Sigma, UK)/ 250 ng/ml ionomycin (Sigma, UK) for 4 hrs. Cells were acquired into a FACScan flow cytometer (BD Bioscience, UK). Monocytes were gated by their forward/side scatter profiles and CD14 expression. NF-κB transactivation analysis Nuclear proteins were extracted using the cytosolic/nuclear fractionation kit (Biovision, USA), and quantified using the BCA method. Transactivation of NF-κB p50 and p65 subunits was determined using a p50/p65 NF-κB enzyme-linked immunosorbent assay (ELISA)based transcription factor assay kit (TransAM™ assay; Active Motif Europe, Belgium).Absorbance at 450nm was read on an OpsysMR microplate reader (Dynex Technologies Ltd., UK). Cytokine quantification Serum cytokine levels of TNF, IL-1β, IL-6, IL-10, IL-2, and IL-12 were determined, using a multiplex bead immunoassay kit (Biosource Ltd, UK). Following the last wash step, the beads were resuspended in buffer and analysed in duplicate with a Luminex 100™ instrument (Riverside, USA). Data analysis was performed using the Luminex 100 IS software version 2.3 and results displayed as mean ± SD.

5

Results Novel TNFRSF1A splice site mutation in proband and mother with TRAPS The proband, a 16-year-old male, was well until aged 3 years when he presented with a fever of 4 days duration, abdominal pain and diarrhoea. He had markedly raised inflammatory markers and WBC count of 15.2 x 109/l, with neutrophilia of 11.9 x 109/l and CRP of 265 mg/l. One year later he had developed sore throat, headache, generalised nonmigratory erythematosus rash and fever, and was treated as presumed tonsillitis. Over the following years he had several admissions with episodes of generalised abdominal pain, sometimes accompanied by vomiting and diarrhoea. In between these episodes he was well and developed steadily along the 25th centile for height and weight. From year 2000 he had recurrent episodic fevers, lymphangitis, and pleuritis with groin and musculoskeletal pains affecting his lower limbs; however, he was generally well between the episodes. The possibility of a hereditary periodic fever syndrome was raised in 2004 and he underwent genetic screening for FMF and HIDS, which were again negative; no MEFV mutations were found by the National Amyloidosis Centre, and HIDS on the basis of normal levels of IgD and urinary mevalonic acid. The patient was treated empirically with prophylactic colchicine, and ibuprofen in the event of inflammatory episodes. He remained relatively well, but continued to experience episodic abdominal and musculoskeletal pains. It also transpired that the patient’s mother had been suffering from musculoskeletal pains and fevers, for which she had been treated with oral steroids. Consequently, further investigations (Table 1) and screening for TRAPS was arranged for both patients. We found a novel mutation in the first base pair of intron 4 of TNFRSF1A, c.472+1G>A (C158delinsYERSSPEAKPSPHPRG); based on the crystallographic structure proposed by Banner et al,[16] this would lead to a 45 nucleotide insertion of intronic DNA sequence into the mRNA, which in turn results in an in-frame insertion of 15 a.a in the mature TNFR1 protein and deletion of a cysteine residue: C129(158) in cysteine rich domain (CRD) 3 of the mature TNFR1 protein (Figure 1a). This mutation was not observed in 100 Caucasian controls and results in a clinical presentation of TRAPS in the proband and his mother. The effect of this mutation on transcription was studied by quantitative real time PCR; wild type (WT) cDNA yielded a product with a dissociation temperature of 83.6°C (Figure 1b); the proband’s cDNA, however, yielded a dissociation curve displaying two products, with melting temperatures of 83.6°C and 86.4°C respectively (Figure 1b), indicating that a larger product was present in addition to WT, as a result of aberrant splicing. The presence of the larger product, approximately 130 bp in size, was confirmed on a 2 % TAE agarose gel, in addition to the single 87 bp product of the healthy control (data not shown). To determine the exact nature of this insertion, primers TNFR 3-6 F and R were used to amplify exons 3 to 6 of the cDNA, and the PCR products were sequenced. This showed 2 distinguishable products, identifiable as both the WT allele and an allele with 45bp insertion from the fourth intron. This was also confirmed by subcloning of the two separate products (supplementary figure 1a). The effect of this insertion on the 3D protein structure and folding was examined using the modelling program, Modeller 9v1 (supplementary figure 1b). [17, 18] Comparative analysis of the 3D structure of the mutated protein (green) with wildtype TNFR1 (blue), revealed the loss of the disulfide bond between C117(146) and C129(158) (red) and the introduction of an additional loop (pink). Serum cytokine levels in patients with splice site mutation Sera cytokine profiles in the splice mutation patients were performed and compared with controls. The proband had significantly elevated IL-1β, IL-2, IL-6, IL-10 and IL-12 levels, compared to healthy controls (n=8) (Table 2). TNF was marginally elevated but within the range of controls. In the proband’s mother, who was receiving treatment with steroids at

6

the time, the levels of TNF, IL-10, and IL-12 were within the normal range, and only IL-1β, IL-6 and IL-2 levels were elevated compared to controls (Table 2). NF-κB activation in PBMCs from patients with splice site mutation We then investigated the activation of the NF-κB transcription factor, in PBMC from patients with the splice mutation and compared the activation with PBMC from TRAPS patients carrying the T50M (T79M) and C88R (C117R) mutations, respectively, and also patients with the rheumatoid arthritis (RA) (Figure 2). Basal nuclear expression of both p50 and p65 NF-κB subunits were increased more than three fold in the splice mutation patients compared with controls (n=8). The nuclear expression of the p65 subunit in the splice mutations was comparable with that observed in the T50M (T79M) and C88R (C117R) mutations, yet less than that observed for RA patients (n=3). The p50 subunit nuclear expression was strongly elevated in the splice mutations compared with either controls or the T50M (T79M) and C88R (C117R) mutations and even greater than that observed for RA. To determine how responsive the PBMC were to TNF stimulation, the nuclear expression of the p50 and p65 NF-κB subunits was measured following 5, 10 and 15 minute stimulation with 100 ng/ml TNF (Figure 2). PBMC isolated from splice mutation patients revealed a time-dependent increase in nuclear translocation of the p50 and p65 NF-κB subunits following TNF stimulation greater than with the controls. However, the increase in the p65 subunit nuclear expression in the splice mutations was less than that observed for RA patients (Figure 2). Conversely, the p50 nuclear expression was marginally greater in the splice mutation PBMC compared with RA patients (Figure 2). Interestingly, despite having elevated basal p65 nuclear expression in the PBMC carrying the T50M (T79M) and C88R (C117R) mutation, there was no further nuclear increase in p65 or p50 NF-κB subunits following stimulation with TNF in these mutations. Soluble and cellular TNFR1 protein expression in TRAPS patients with splice site mutation Given the multiple mechanisms proposed by which TNFSFR1A mutations can lead to clinical disease including impaired receptor shedding and dysfunctional trafficking, TNFR1 localisation was examined in the splice mutation patients. Flow cytometric (FACS) analysis of the surface expression of TNFR1 on monocytes identified a reduction in the cell surface expression of TNFR1 on monocytes of both the proband, patient A, (MFI=3.64) and his mother, patient B, (MFI=4.43) compared with the healthy controls (MFI=5.20±0.61, n=5) (Figure 3a). To determine whether the splice mutation induced the abnormal TNFR1 localisation that may underlie the reduced surface expression and elevated NF-κB activity, whole cell TNFR1 protein expression levels were analysed by intracellular staining (Figure 3b). The reduction in TNFR1 expression was still evident in the splice mutation patients (patient A, MFI=6.19; patient B, MFI=7.11) compared with healthy controls (MFI=7.92±0.38, n=5). The reduction in TNFR1 protein expression was confirmed by Western blot analysis of lysates from PBMC. Both splice mutation patients had marginally lower detectable TNFR1 compared with controls (Figure 3c); densitometric analysis of the TNFR1 and β-actin (loading control) protein bands confirmed this. This reduction in cell surface expression of TNFR1 and total protein was also reflected in the level of soluble TNFR1 (sTNFR1) in serum. Serum levels of sTNFR1 in the splice mutation patients were decreased by almost a third in both patients compared with serum from controls (1238 pg/ml ± 234.5) (Figure 3d). Analysis of TNFR1 shedding, assessed by

7

FACS following stimulation of monocytes with phorbal 12-myristate 13-acetate (PMA), did not reveal a shedding defect (data not shown).

8

Discussion The majority of TNFRSF1A gene mutations identified underlying TRAPS are caused by missense mutations affecting cysteine residues involved in disulfide bonds and other residues in CRD1 and 2 that are predicted to have a pronounced affect on the overall secondary protein structure of TNFR1. Here we have identified a novel TNFRSF1A mutation that is different to most TRAPS mutations as it introduces an insertion of 15 a.a and the deletion of a crucial cysteine residue C129(158) in CRD3 of TNFR1 protein. The only other known mutation in CRD3 causing TRAPS is an F112I mutation, found in affected members of a Finnish family.[19] The mutation present in our patients at the 5’ end of intron 4 (c.472+1G>A), occurs at a critical splice junction recognition site, causing the nascent mRNA to be spliced incorrectly and results in the inclusion a portion of intron 4 in the mRNA. In the genomic DNA, a “gt” occurs 45 bases distal to the mutation in the intron sequence, thereby presenting a potential alternative splice site. The use of this site was confirmed by both the increased product size observed on quantitative real time PCR (gel and dissociation curve), and by DNA sequencing. The predicted protein, has an insertion of 15 a.a, which would cause a size increase in the protein product from the wild type 455 a.a./50.5 kDa to 470 a.a./52.2 kDa. Based on the crystal structure of TNFR1 proposed by Banner et al [16] this insertion at the splice site would cause an extended loop in the modelled 3D protein structure (supplementary figure 1) and the deletion of a cysteine residue at position C129(158), thus disrupting the disulfide bond between C117(146) and C129(158) in CRD3. Most of the previously described TRAPS-associated TNFRSF1A gene mutations occur within CRD1 and CRD2. CRD1, 2 and 3 share much homology including six cysteine residues which form 3 disulfide bonds in each domain. The disulfide bonds formed by cysteine residues are crucial to the correct folding and secondary structure of TNFR1. TRAPS patients with more clinical severity have been found to have mutations at key cysteine residues in CRD1 and CRD2 and it has been proposed that the substitution of cysteines for certain extracellular residues may lead to interchain disulfide-linked homo-dimerisation and constitutive activation of the receptor.[14, 15, 20] Crystallographic studies of TNFR1 bound with lymphotoxin have predicted that CDR2 and CRD3 are largely responsible for mediating ligand binding.[16] The deletion of C129(158) disrupts the second disulfide bond in CRD3 which is homologous to the disulfide bond formed by C73:C88 (C102:C117) in CRD2. Mutations of these cysteines are known to have significant TRAPS pathology. The loss of C129(158) in CRD3 by this splice site mutation may indeed cause constitutive activation in these patients, as reflected by their elevated serum cytokine levels and enhanced basal and TNF-induced NF-κB activation. The elevated level of IL-1β observed in both patients (especially the proband), which would point to IL-1 being a key mediator in the pathogenesis of TRAPS, and fits the observation that IL-1ra treatment may be an alternative to anti-TNF therapy in TRAPS. [21] Another significant observation was that TNFR1 cell surface expression, serum levels and cellular content, in the splice mutation, were all reduced. The reduced TNFR1 surface expression in these patients would argue against this mutation inducing a shedding defect, as found in some TNFRSF1A mutations including T50M (T79M) and C52F (C81F),[1, 3] and to a lesser extent H22Y (H51Y),C33Y (C62Y), and P46L (P75L).[3, 22] However, the degree of defective TNFR1 shedding in TRAPS patients has been found to be quite variable between different mutations.[5] Furthermore, no shedding defect has been observed in some TRAPS patients in the presence of a reduced plasma concentration of sTNFR1, demonstrating further inconsistencies between TRAPS mutations. Analysis of a shedding defect in our patients did not reveal any significant defect (data not shown). The possibility that the reduced expression

9

we observed might result from the loss of the antibody recognizing epitope in the mutated TNFR1 receptor rather than loss of surface protein expression cannot be formally ruled out. The overall reduced serum and TNFR1 cell surface expression observed in monocytes carrying the splice mutation is in keeping with several other TRAPS mutations, where similar findings have been reported.[1, 3, 5, 14, 15] This supports the hypothesis that impaired intracellular trafficking of the misfolded mutated TNFR1 protein leads to its retention in the endoplasmic reticulum (ER) and ultimate disposal through a process of ER associated degradation (ERAD).[14, 15] Intriguingly, we did not observe any increase in cellular TNFR1 expression in patients carrying the splice mutation. Instead, total TNFR1 expression, as determined by intracellular staining and WB, was reduced in the splice patients compared with healthy controls, albeit to a lesser extent to that observed for surface expression. The possibility that differences observed between the splice mutation patients and healthy controls could be due to loss of epitope binding must always be considered, but as a polyclonal TNFR1 antibody was used for WB, this would argue against this explanation. Although testing the ER retention hypothesis in primary cells is challenging, as there are currently no mutation-specific TNFR1 antibodies available to distinguish between WT and mutant TNFR1, future studies are planned using confocal microscopy to examine total TNFR1 intracellular localisation. The mechanism whereby this splice mutation leads to the pro-inflammatory phenotype, characteristic of TRAPS, is unclear. An important yet paradoxical feature of this splice mutation that despite having low cell surface TNFR1 expression, PBMC still retain the ability to respond to TNF compared with PBMC carrying the T50M (T79M) or C88R (C117R) mutation. The reasons why the splice mutation behaves so differently from T50M and C88R (T79M and C117R) in this regard is unclear. However, insertion of 15 a.a at the splice site in these patients is likely to have significant structural implications for the receptor. Interestingly, there is increasing evidence that the p50 NF-κB homodimer can associate with the B cell lymphoma 3 (Bcl-3) transcriptional coactivator to confer transactivation capabilities [23] thereby providing a potential mechanism by which increased activation of the p50 homodimer can be associated with promotion rather than inhibition of the proinflammatory responses and elevated serum cytokine levels we have observed. In conclusion, much work over the past 5 years has been focused on investigating how mutations in the first 2 CRDs translate into receptor dysfunction. Identification of this novel splice site mutation affecting CRD3 suggests that these investigations should extend to include mutations in this domain. Considering the biochemical significance of the cysteine residues in all CRDs for proper structure and folding of this protein, in addition to the proposed role CRD3 plays in ligand binding, it is somewhat surprising that the patients carrying this mutation do not present with a more severe pathology. Further studies of this mutation and others are in progress to unravel the complex cellular mechanisms underlying the varying TRAPS pathologies and observed heterogeneity between different TNFRSF1A mutations. Acknowledgements: We are particularly grateful to the patient and mother who agreed to participate in the study. Supported in part by grants from the Wellcome Trust, Sir Jules Thorn “Seed Corn” Fund, Arthritis Research Campaign, Charitable Foundation of the Leeds Teaching Hospitals, and Bart’s and the London Charitable Foundation.

10

References

1. McDermott MF, Aksentijevich I, Galon J, McDermott EM, Ogunkolade BW, Centola M Germline mutations in the extracellular domains of the 55 kDa TNF receptor, TNFR1, define a family of dominantly inherited autoinflammatory syndromes. Cell 1999; (1):133-144. 2. Tchernitchko D, Chiminqgi M, Galacteros F, Prehu C, Segbena Y, Coulibaly H Unexpected high frequency of P46L TNFRSF1A allele in sub-Saharan West African populations. Eur J Hum Genet 2005; (4):513515. 3. Aksentijevich I, Galon J, Soares M, Mansfield E, Hull K, Oh HH The tumor-necrosis-factor receptorassociated periodic syndrome: New mutations in TNFRSF1A, ancestral origins, genotype-phenotype studies, and evidence for further genetic heterogeneity of periodic fevers. Am J Hum Genet 2001; (2):301-314. 4. Dode C, Andre M, Bienvenu T, Hausfater P, Pecheux C, Bienvenu J The enlarging clinical, genetic, and population spectrum of tumor necrosis factor receptor-associated periodic syndrome. Arthritis Rheum 2002; (8):2181-2188. 5. Aganna E, Hammond L, Hawkins PN, Aldea A, McKee SA, van Amstel HKP Heterogeneity among patients with tumor necrosis factor receptor-associated periodic syndrome phenotypes. Arthritis Rheum 2003; (9):2632-2644. 6. Kusuhara K, Nomura A, Nakao F, Hara T. Tumour necrosis factor receptor-associated periodic syndrome with a novel mutation in the TNFRSF1A gene in a Japanese family. Eur J Pediatr 2004; (1):30-2. 7. Stojanov S, Lohse P, McDermott MF, Renner ED, Kery A, Mirakian R Periodic fever due to a novel TNFRSF1A mutation in a heterozygous Chinese carrier of MEFV E148Q. Rheumatology (Oxford) 2004; (4):5267. 8. Aganna E, Zeharia A, Hitman GA, Basel-Vanagaite L, Allotey RA, Booth DR An Israeli Arab patient with a de novo TNFRSF1A mutation causing tumor necrosis factor receptor-associated periodic syndrome. Arthritis Rheum 2002; (1):245-249. 9. Church LD, Churchman SM, Hawkins PN, McDermott MF. Hereditary auto-inflammatory disorders and biologics. Springer Semin Immunopathol 2006; (4):494-508. 10. Sarrauste de Menthiere C, Terriere S, Pugnere D, Ruiz M, Demaille J, Touitou I. INFEVERS: the Registry for FMF and hereditary inflammatory disorders mutations. Nucleic Acids Res 2003; (1):282-5. 11. Touitou I, Lesage S, McDermott M, Cuisset L, Hoffman H, Dode C Infevers: an evolving mutation database for auto-inflammatory syndromes. Hum Mutat 2004; (3):194-8. 12. Kriegel MA, Huffmeier U, Scherb E, Scheidig C, Geiler T, Kalden JR Tumor necrosis factor receptorassociated periodic syndrome characterized by a mutation affecting the cleavage site of the receptor: implications for pathogenesis. Arthritis Rheum 2003; (8):2386-8. 13. D'Osualdo A, Ferlito F, Prigione I, Obici L, Meini A, Zulian F Neutrophils from patients with TNFRSF1A mutations display resistance to tumor necrosis factor-induced apoptosis - Pathogenetic and clinical implications. Arthritis Rheum 2006; (3):998-1008. 14. Lobito AA, Kimberley FC, Muppidi JR, Komarow H, Jackson AJ, Hull KM Abnormal disulfide-linked oligomerization results in ER retention and altered signaling by TNFR1 mutants in TNFR1-associated periodic fever syndrome (TRAPS). Blood 2006; (4):1320-1327. 15. Nedjai B, Hitman,G.A., Yousaf,N., Chernajovsky,Y., Stjernberg,S., Pettersson,T., Ranki,A., Hawkins,P., Arkwright,P.D., McDermott,M.F. and Turner,M.D. Abnormal tumor necrosis factor receptor I cell surface expression and NF-κB activation in tumor necrosis factor receptor-associated periodic syndrome. Arthritis Rheum 2007; . 16. Banner DW, Darcy A, Janes W, Gentz R, Schoenfeld HJ, Broger C Crystal-Structure of the Soluble Human 55 Kd Tnf Receptor-Human Tnf-Beta Complex - Implications for Tnf Receptor Activation. Cell 1993; (3):431-445. 17. Marti-Renom MA, Stuart AC, Fiser A, Sanchez R, Melo F, Sali A. Comparative protein structure modeling of genes and genomes. Ann Rev Biophys Biomol Struct 2000; :291-325. 18. Sali A, Blundell TL. Comparative Protein Modeling by Satisfaction of Spatial Restraints. J Mol Biol 1993; (3):779-815. 19. Nevala H, Karenko L, Stjernberg S, Raatikainen M, Suomalainen H, Lagerstedt A A novel mutation in the third extracellular domain of the tumor necrosis factor receptor 1 in a Finnish family with autosomaldominant recurrent fever. Arthritis Rheum 2002; (4):1061-1066. 20. Santoro M, Carlomagno F, Romano A, Bottaro DP, Dathan NA, Grieco M Activation of Ret as a Dominant Transforming Gene by Germline Mutations of Men2a and Men2b. Science 1995; (5196):381-383. , et al.

97

, et al.

13

, et al.

69

, et al.

46

, et al.

48

163

, et al.

43

, et al.

46

27

31

, et al.

24

, et al.

48

,

et

al.

54

, et al.

108

in press

, et al.

73

29

234

, et al.

46

, et al.

267

11

21. Simon A, Bodar EJ, van der Hilst JCH, van der Meer JWM, Fiselier TJW, Cuppen M Beneficial response to interleukin I receptor antagonist in traps. Am J Med 2004; (3):208-210. 22. Arkwright PD, McDermottt MF, Houten SM, Frenkel J, Waterhan HR, Aganna E Hyper IgD syndrome (HIDS) associated with in vitro evidence of defective monocyte TNFRSF1A shedding and partial response to TNF receptor blockade with etanercept. Clin Exp Immunol 2002; (3):484-488. 23. Mathas S, Johrens K, Joos S, Lietz A, Hummel F, Janz M Elevated NF-kappa B p50 complex formation and Bcl-3 expression in classical Hodgkin, anaplastic large-cell, and other peripheral T-cell lymphomas. Blood 2005; (13):4287-4293. , et al.

117

, et al.

130

, et al.

106

12

Figure Legends Figure 1. Identification of novel splice site mutation in intron 4 of TNFRSF1A gene. a) Forward and reverse electropherograms of both wild type (left panel) and mutant (c472+1G>A) (right panel) genomic sequences. b) Dissociation curves from the quantitative real time PCR amplification of TNFRSF1A mRNA, showing one product peak in the wild type and two product peaks in proband (patient A) and mother (patient B) carrying splice site mutation. Figure 2. PBMC expressing the TNFRSF1A splice site mutation show elevated basal and TNF induced NF-κB activation. Basal and TNF induced activation of p50 and p65 NFκB subunits in PBMC from the 2 splice mutation patients was examined by TransAM™ analysis and compared with PBMC isolated from T50M and C88R TRAPS patients, healthy controls (n=8), RA patients (n=3). Isolated PBMC were stimulated with 100ng/ml TNF for 0, 5, 10, and 15 mins before cytosolic and nuclear extracts were prepared. The splice mutation patients had elevated basal nuclear expression of p50 and p65 NF-κB subunits and displayed a marked time-dependent increase TNF-induced nuclear translocation compared with the healthy controls. Figure 3. Patients carrying TNFRSF1A splice site mutation have decreased TNFR1 protein expression and reduced serum sTNFR1 levels. a) Surface and b) intracellular staining of TNFR1 expression on monocytes from splice mutation patients and a representative healthy control was examined by flow cytometry. The healthy control is represented by a dotted line, whilst the proband (patient A, Dark) and his mother (patient B, Light) are represented by solid lines. The isotype control is represented by the shaded histogram. c) Expression of TNFR1 in PBMC from splice mutation patients and a representative healthy control, examined by WB on cytosolic protein fractions. The splice mutation patients have a decrease in surface and total TNFR1 expression compared to healthy controls. d) Expression of sTNFR1 in serum from splice mutation patients and healthy controls, examined by ELISA. sTNFR1 were decreased in both splice mutation patients (black bars) compared with 8 healthy controls (clear bar). Supplementary figure 1. Sequence of insertion generated from alternative splice site and 3 dimensional model of mutant TNFR1 protein. a) The cDNA amplified products were sub-cloned into pGem-T-Easy vectors and sequenced whereby both the wild type and longer mutation products were identified. The 45 base pair insertion in mRNA produced from the mutant genomic DNA is shown. b) The 3 dimensional model of the wildtype (blue) and splice site mutation (green) TNFR1 protein structure generated by Modeller 9v1 software. The cysteine residues in the disulfide bond affected are shown in red on the wildtype protein and the resulting unpaired cysteine residue (yellow) in the splice site mutation. The intronic insertion is shown in pink.

13

Table Legends Table 1. Clinical features of TRAPS splice mutation patients, and two patients carrying T50M and C88R TNFRSF1A gene mutations. Table 2. Serum cytokine levels are raised in patients carrying TNFRSF1A splice mutation. Mean ±SD serum cytokine levels (pg/ml) were measured for 8 healthy controls and the two splice mutation patients, using a multiplex bead array are shown.

14

Table 1. Clinical history

Splice A

Splice B

T50M

C88R

Age of onset (years) Age of diagnosis (years) Family history Fever Skin rash Periorbital oedema Abdominal pain Musculoskeletal symptoms Pleuritis Lymphadenopathy Aphtous ulcers

3 16 Yes Yes No No Yes Yes Yes Yes Yes

Late childhood 46 Yes None documented No Yes (as a child) No Yes No No No

3 9 Yes Yes Yes No No Yes No No No

5 57 Possible Yes No No Yes Yes Yes No No

Relevant investigations

Splice A

Splice B

T50M

C88R

Neutrophil leucocytosis Elevated CRP Elevated PV ANA/RF/C3/C4 Amyloid screen

Yes Yes Yes Negative Negative

Yes Yes Yes Negative Negative

Yes Yes Yes Negative Negative

Yes Yes Yes Negative Negative

15

Table 2. Patients

TNF

IL-1β

IL-6

IL-10

IL-2

IL-12

Healthy controls (n=8) (S.D) Splice patient A Splice patient B

122.8 (±167.7) 236.8 128.6

101.6 (±167.2) 1740.4 344.1

129.6 (±42.88) 231.8 190.4

231.9 (±233.9) 815.6 416.1

117.4 (±77.16) 966.4 267.8

315.8 (±188.9) 925.6 178.6

16

Figure 1. A WT

Patient

Electropherogram (forward)

INTRON

EXON

INTRON

EXON

Electropherogram (Reverse)

INTRON

EXON

INTRON

EXON

B Patient A

WT

Patient B

Derivative

1.90 E-1 1.40 E-1 9.00 E-2 4.00 E-2 -1.00 E-2 60

70 80 90 Temperature (C)

60

70 80 90 Temperature (C)

60

70 80 90 Temperature (C)

Figure 2.

NF-κ B activation (OD450nm)

p65 2.0

1.5

1.0

0.5

0.0 0 5 10 15 0

Healthy Controls

5 10 15 0

Splice patient A

5 10 15 0

Splice patient B

5 10 15 0

T50M patient

5 10 15 0

C88R patient

5 10 15

RA

NF-κ B activation (OD450nm)

p50 2.5 2.0 1.5 1.0 0.5 0.0 0 5 10 15 0

Healthy Controls

5 10 15 0

Splice patient A

5 10 15 0

Splice patient B

5 10 15 0

T50M patient

5 10 15 0

C88R patient

5 10 15

RA

Figure 3. A

Events

64

0

100

101

102

103

104

TNFR1 Healthy control

B

Patient B

Patient A

Events

64

0

101

100

102

103

104

TNFR1 Healthy control

Patient A

Patient B

C Healthy control

Patient A

Patient B

0.28

0.28

TNFR1

β−actin TNFR1/actin ratio

D

0.35

1400

sTNFR1 (pg/ml)

1200 1000 800 600 400 200 0 Healthy controls Healthy

Patient A Patient

Patient B Patient

controls

A

B