Brief Report
Arthritis & Rheumatism DOI 10.1002/art.38824
Running title: Pathogenic TREX1 deficiency in early-onset cerebral SLE
Whole exome sequencing in early-onset cerebral SLE identifies a pathogenic variant in TREX1. Julia I Ellyard1, Rebekka Jerjen1, Jaime L Martin1, Adrian Lee1, Matthew A Field2, Simon H Jiang1,3, Jean Cappello1, Svenja K Naumann1, T Daniel Andrews2, Hamish S Scott4, Marco G Casarotto5, Christopher C Goodnow2, Jeffrey Chaitow6, Virginia Pascual7, Paul Hertzog8, Stephen I Alexander9, Matthew C Cook2,10#, Carola G Vinuesa1# # M.C.C and C.G.V. contributed equally to this work 1
Department of Pathogens and Immunity, John Curtin School of Medical Research,
Australian National University, Canberra, Australia. 2
Department of Immunology, John Curtin School of Medical Research, Australian
National University, Canberra, Australia. 3
Department of Renal Medicine, The Canberra Hospital, Canberra, ACT, Australia
4
Division of Molecular Pathology, Institute of Medical and Veterinary Science and
The Hanson Institute, and School of Medicine, University of Adelaide, Adelaide, Australia 5
Department of Molecular Bioscience, John Curtin School of Medical Research,
Australian National University, Canberra, Australia. 6
Department of Rheumatology, Sydney Children's Hospitals Network, Sydney, NSW,
Australia. 7
Baylor Institute for Immunology Research, Dallas TX; The University of Texas
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/art.38824 © 2014 American College of Rheumatology Received: Feb 04, 2014; Revised: Jun 05, 2014; Accepted: Aug 07, 2014
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Pathogenic TREX1 deficiency in severe early-onset SLE
Southwestern Medical Center at Dallas, Dallas, TX 8
Monash Institute of Medical Research (MIMR), Clayton, Victoria, Australia
9
Centre for Kidney Research, Children's Hospital at Westmead, Westmead, New
South Wales, Australia. 10
Department of Immunology, The Canberra Hospital, Canberra, ACT, Australia
Corresponding authors: Julia Ellyard. Phone: +61 2 61253519 Fax: +61 2 6125 2595 Email: [email protected]; Carola Vinuesa. Phone: +61 2 61254500 Fax:
+61 2 6125 2595 Email: [email protected]
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Pathogenic TREX1 deficiency in severe early-onset SLE
ABSTRACT Objective: Systemic lupus erythematosus (SLE) is a chronic and heterogeneous autoimmune disease. Both twin and sibling studies indicate a strong genetic contribution to lupus, but in the majority of cases the pathogenic variant remains to be identified. The genetic contribution to disease is likely to be greatest in cases with early onset and severe phenotypes. Whole exome sequencing now offers the possibility of identifying rare alleles responsible for disease in such cases. Methods: We performed whole exome sequencing in a 4-year-old female with early-onset SLE and conducted biochemical analysis of the putative defect. Results: Whole exome sequencing of a 4-year-old female with cerebral lupus identified a rare, homozygous mutation in the Three Prime Repair Exonuclease 1 (TREX1) that was predicted to be highly deleterious. The TREX1 R97H mutant protein had a 20-fold reduction in exonuclease activity and was associated with an elevated IFN-α signature in the patient. The discovery and characterization of a pathogenic TREX1 in our proband has therapeutic implications: the patient is now a candidate for neutralizing anti-IFNα therapy. Conclusion: Our study is the first to demonstrate that whole exome sequencing can be used to identify rare or novel deleterious variants as genetic causes of SLE and, through a personalized approach, improve therapeutic options.
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Pathogenic TREX1 deficiency in severe early-onset SLE
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with complex, heterogeneous pathogenesis. Twin-twin and sibling studies implicate a strong genetic risk for SLE1. Although human and mouse studies have identified several rare gene variants resulting in SLE, the majority of current genetic knowledge has been obtained from genome-wide association studies (GWAS). GWAS has identified several common gene variants associated with modest risk for developing SLE1, yet direct evidence of a pathogenic relationship between many of these variants and disease is lacking.
Recent advances in whole exome sequencing (WES) are enabling identification of causal mutations underlying many genetic disorders. Where GWAS is limited to common variants, the ability to identify individual specific rare variants by WES can significantly advance the diagnosis and treatment options. We describe a case of severe SLE in a 4 year-old female of consanguineous parentage whom, given her pedigree, age of onset and severity of disease, is highly likely to have a genetic basis to her disease. Using WES, we identified a rare, disease-causing variant in TREX1 and demonstrated the pathophysiological basis of this variant.
PATIENT AND METHODS Patient case report A 3 year-old female of Lebanese ethnicity presented with arthritis, malar rash, diffuse purpura rashes, episodic fever, chronic headache and vomiting. There was no history of seizures or visual disturbance. There was no family history of autoimmunity. Her parents were first cousins (Figure 1A). Serology demonstrated high titer anti-nuclear antibodies (ANA; Hep2 titer 640), anti-double stranded DNA-antibodies (dsDNA;
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15kIU/L), anti-cardiolipin antibodies (IgM titer 27), elevated IgG (17.3g/L) and IgA (2.91g/L), lymphopenia and Coombs’ positive anemia, consistent with SLE. C3 (1.60g/L) and C4 (0.24g/L) levels, renal and liver function tests and cerebrospinal fluid analysis were normal. Initial brain MRI/MRA demonstrated steroid-related volume loss, but no other abnormalities. At 4 years of age she presented with rightsided hemiparesis. An MRI demonstrated occlusion of the left middle cerebral artery and left-sided MCA infarct (Figure 1B and C). MRA revealed widespread and marked irregularities of the medium sized vessels consistent with vasculitis in the context of cerebral SLE.
Genetic studies Informed written consent was obtained according to ethics approval from Australian National University and Canberra Hospital Human Ethics Committees. Genomic DNA was extracted from saliva using OrageneTM DNA Self Collection Kits. Whole exome capture and sequencing was performed by Beijing Genomics Institute and bioinformatics analysis performed at JCSMR, ANU as described in the supplementary data. Genotypes were verified by PCR and Sanger sequencing.
Exonuclease Activity Assay Mutant TREX1R97H was cloned from pML303-human TREX1WT 1-242 (gift of F. Perrino) using PCR site-directed mutagenesis. Exonuclease activity assays using recombinant human TREX1WT and TREX1R97H were performed as previously described2. Exonuclease activity was quantified using ImageQuant TL 8.1 software.
Dimerization studies
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Pathogenic TREX1 deficiency in severe early-onset SLE
Dimerization studies were performed using MBP-tagged and untagged versions of TREX1WT and TREX1R97H as previously described2.
IFN activity Interferon antiviral activity was measured using a cytopathic effect (CPE)-reduction bioassay with WISH cells and Semliki Forest virus: titers were calibrated against an international reference standard.
IFN signature studies RNA was extracted from PBMC samples and cDNA synthesis performed using MLVRT (Invitrogen). Quantitative PCR was performed by SRBR green incorporation using the 7900HT Fast Real-Time PCR System (Applied Biosystems). ∆CT values were normalized to the healthy PBMC negative control. A heat map of the relative expression values was generated with GENE-E software (Broad Institute), using global expression and default color scheme for ratio data preferences.
RESULTS Identification of novel homozygous variant in TREX1 Bioinformatics analysis of the WES from the proband identified a rare (MAFA substitution in the Three Prime Repair Exonuclease 1 (TREX1) (Figure 1D). The resulting arginine to histidine substitution at amino acid position 97 (R97H) was predicted to be deleterious (Polyphen2 score=1; SIFT=0.03). DNA sequence analysis of other family members confirmed homozygosity was confined to the proband (Figure 1A, D). Detailed analysis of the exome data revealed no other immune-related, novel or rare (MAFC resulting in the H335R substitution predicted to be benign (MAF=0.33); and a G>C at IV9-5 with a MAF=0.215/469. Familial genotyping revealed they did not segregate with disease (Figure S1), excluding CECR1 as the cause of the proband’s syndrome.
Mutations in TREX1 have been linked to a spectrum of autoimmune diseases including Aicardi-Goutières Syndrome (AGS)5,6, familial chilblain lupus (CHBL)7,8, and SLE7,9. Homozygosity for TREX1R97H was therefore considered a plausible explanation for this presentation. As this allele had not been described as a cause of pathology we proceeded to functional analysis.
Functional characterization of TREX1 R97H TREX1 is the primary mammalian 3’-5’ intracellular exonuclease; it degrades endogenous nucleic acids in the cell cytoplasm. The R97 residue is highly conserved throughout evolution (Figure 2A), suggesting an important role in protein function. To examine the effect of the mutation on protein function, we measured exonuclease activity in vitro. Compared to TREX1WT, TREX1R97H had a greatly diminished ability to degrade single-stranded DNA (ssDNA) (Figure 2B). Quantification of the exonuclease activity7,10 revealed an approximately 20-fold reduction in exonuclease activity of TREX1R97H (Table 1).
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Loss of function mutations in TREX1 are known to augment production of type I IFNs11; elevated levels of IFN-α in the CSF and serum are a signature of AGS patients with mutations in TREX15,10. Analysis of the proband’s serum revealed detectable levels of IFN-α (8 IU/ml) compared to undetectable levels (2-fold. Together these findings indicate that TREX1R97H has impaired exonuclease function that results in defective clearance of nucleic acids, triggering signaling pathways that promote ANA production, secretion of type I IFNs and inflammation.
Structural characterization of TREX1R97H To understand the cause of reduced exonuclease activity by TREX1R97H, we performed in silico structural analysis using the program Cn3D (NCBI). TREX1 forms a homodimer with several residues at the dimer interface being important for both dimerization and protein function7. Based on the crystal structure for murine TREX17, the mutated R97 residue was predicted to be located at the dimer interface, opposite an arginine residue at position 114 (R114) (Figure 2D). Although the R97H mutation occurred at the dimer interface, it did not appear to impair its ability to form dimers in vitro (Figure S3). However, it is possible the mutation may affect the overall stability of the TREX1R97H dimer and/or protein.
DISCUSSION In this study we used WES to investigate the genetic basis of a case of severe, early-
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onset SLE, identifying a rare deleterious homozygous variant of TREX1. At the time of discovery this variant was novel, however, it has recently been annotated in the 1000 genome project (MAFA variant results in the R97H amino acid substitution.
Figure 2: R97H mutation impairs TREX1 exonuclease activity. A) Evolutionary conservation of R97 (red) in TREX1. B) Visualization and comparison of degradation of a 30-mer oligonucleotide by TREX1WT and TREX1R97H reveals an approximately 20-fold decrease in endonuclease activity. Results are representative of three experiments. C) Heat map showing relative expression of 11 IFN-inducible genes in PBMCs from proband and two female relatives heterozygous for the TREX1R97H variant. Values were normalized to an unstimulated healthy PBMC sample (Nil). Healthy PBMCs cultured with IFN-α were included as positive control. Fold increases in gene expression are shown for proband (II.V) compared to her sister (II.I). Full data set is shown in Figure S2. D) Structure of murine TREX1 dimer showing the location of R97 (yellow), R99 (green) and R114 (red).
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Acknowledgements The authors wish to thank; A. Wilson, S. Aditya, F. Perrino and C. Orebaugh, and the ACRF Bimolecular Resource Facility. This work was supported by an Elizabeth Blackburn NHMRC Research Fellowship, NHMRC program and project grants to C.G.V., an NHMRC Overseas Biomedical Fellowship to J.I.E. and a RACP/Jacquot research scholarship to S.H.J.
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REFERENCES 1. Sestak AL, Fürnrohr BG, Harley JB, Merrill JT, Namjou B. The genetics of systemic lupus erythematosus and implications for targeted therapy. Ann Rheum Dis 2011;70 Suppl 1:i37–43. 2. Orebaugh CD, Fye JM, Harvey S, Hollis T, Perrino FW. The TREX1 Exonuclease R114H Mutation in Aicardi-Goutieres Syndrome and Lupus Reveals Dimeric Structure Requirements for DNA Degradation Activity. J Biol Chem 2011;286:40246-40254. 3. Zhou Q, Yang D, Ombrello AK, Zavialov AV, Toro C, Zavialov AV, et al. Earlyonset stroke and vasculopathy associated with mutations in ADA2. N Engl J Med 2014;370:911–920. 4. Navon Elkan P, Pierce SB, Segel R, Walsh T, Barash J, Padeh S, et al. Mutant adenosine deaminase 2 in a polyarteritis nodosa vasculopathy. N Engl J Med 2014;370:921–931. 5. Goutières F, Aicardi J, Barth PG, Lebon P. Aicardi-Goutières syndrome: an update and results of interferon-alpha studies. Ann Neurol 1998;44:900–907. 6. Crow YJ, Hayward BE, Parmar R, Robins P, Leitch A, Ali M, et al. Mutations in the gene encoding the 3′-5′ DNA exonuclease TREX1 cause Aicardi-Goutières syndrome at the AGS1 locus. Nat Genet 2006;38:917–920. 7. de Silva U, Choudhury S, Bailey SL, Harvey S, Perrino FW, Hollis T. The crystal structure of TREX1 explains the 3' nucleotide specificity and reveals a polyproline II helix for protein partnering. J Biol Chem 2007;282:10537–10543. 8. Lee-Kirsch MA, Chowdhury D, Harvey S, Gong M, Senenko L, Engel K, et al. A mutation in TREX1 that impairs susceptibility to granzyme A-mediated cell death underlies familial chilblain lupus. J Mol Med 2007;85:531–537. 9. Lee-Kirsch MA, Gong M, Chowdhury D, Senenko L, Engel K, Lee Y-A, et al. Mutations in the gene encoding the 3′-5′ DNA exonuclease TREX1 are associated with systemic lupus erythematosus. Nat Genet 2007;39:1065–1067. 10. Lehtinen DA, Harvey S, Mulcahy MJ, Hollis T, Perrino FW. The TREX1 doublestranded DNA degradation activity is defective in dominant mutations associated with autoimmune disease. J Biol Chem 2008;283:31649–31656. 11. Stetson DB, Ko JS, Heidmann T, Medzhitov R. Trex1 prevents cell-intrinsic initiation of autoimmunity. Cell 2008;134:587–598. 12. Olivieri I, Cattalini M, Tonduti D, La Piana R, Uggetti C, Galli J, et al. Dysregulation of the immune system in Aicardi-Goutières syndrome: another example in a TREX1-mutated patient. Lupus 2013;22:1064–1069. 13. Bhattacharyya S, Zhao Y, Kay TWH, Muglia LJ. Glucocorticoids target suppressor of cytokine signaling 1 (SOCS1) and type 1 interferons to regulate Toll-
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like receptor-induced STAT1 activation. Proc Natl Acad Sci USA 2011;108:9554– 9559. 14. Vogt J, Agrawal S, Ibrahim Z, Southwood TR, Philip S, Macpherson L, et al. Striking intrafamilial phenotypic variability in Aicardi-Goutières syndrome associated with the recurrent Asian founder mutation in RNASEH2C. Am J Med Genet A 2013;161A:338–342. 15. Namjou B, Kothari PH, Kelly JA, Glenn SB, Ojwang JO, Adler A, et al. Evaluation of the TREX1 gene in a large multi-ancestral lupus cohort. Genes Immun 2011;12:270–279.
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Figure 1 254x338mm (72 x 72 DPI)
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Figure 2 254x338mm (72 x 72 DPI)
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Figure S2: Relative expression of 11 IFN-α inducible genes ex vivo in PBMCs from the proband and two female relatives (I.II, II.I) as measured by real-time PCR. PBMCs from a healthy donor with (IFN) and without (Nil) IFN-α stimulation were included as positive and negative controls respectively. All samples were normalised to expression of PBMCs without IFN-α expression.
Input
TREX1-MBP
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Supplementary methods Whole exome sequencing and analysis Whole exome capture and sequencing was performed by Beijing Genomics Institute using the SureSelect Human All Exon Kit (Agilent) and the Illumina Hiseq 2000 platform. Raw data were processed by Illumina basecalling Software 1.7. Coverage statistics for the raw exome data are shown in Table S1. Bioinformatic analysis was performed at JCSMR, ANU. Raw sequence reads were aligned to the reference genome (Hg19) and single nucleotide variants (SNVs) and small indels called using SAMTools. Results were filtered against known variants (dbSNP 132). Consanguinity and an absence of other affected siblings lead us to focus on novel or rare (MAF
Arthritis & Rheumatism DOI 10.1002/art.38824
Running title: Pathogenic TREX1 deficiency in early-onset cerebral SLE
Whole exome sequencing in early-onset cerebral SLE identifies a pathogenic variant in TREX1. Julia I Ellyard1, Rebekka Jerjen1, Jaime L Martin1, Adrian Lee1, Matthew A Field2, Simon H Jiang1,3, Jean Cappello1, Svenja K Naumann1, T Daniel Andrews2, Hamish S Scott4, Marco G Casarotto5, Christopher C Goodnow2, Jeffrey Chaitow6, Virginia Pascual7, Paul Hertzog8, Stephen I Alexander9, Matthew C Cook2,10#, Carola G Vinuesa1# # M.C.C and C.G.V. contributed equally to this work 1
Department of Pathogens and Immunity, John Curtin School of Medical Research,
Australian National University, Canberra, Australia. 2
Department of Immunology, John Curtin School of Medical Research, Australian
National University, Canberra, Australia. 3
Department of Renal Medicine, The Canberra Hospital, Canberra, ACT, Australia
4
Division of Molecular Pathology, Institute of Medical and Veterinary Science and
The Hanson Institute, and School of Medicine, University of Adelaide, Adelaide, Australia 5
Department of Molecular Bioscience, John Curtin School of Medical Research,
Australian National University, Canberra, Australia. 6
Department of Rheumatology, Sydney Children's Hospitals Network, Sydney, NSW,
Australia. 7
Baylor Institute for Immunology Research, Dallas TX; The University of Texas
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/art.38824 © 2014 American College of Rheumatology Received: Feb 04, 2014; Revised: Jun 05, 2014; Accepted: Aug 07, 2014
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Pathogenic TREX1 deficiency in severe early-onset SLE
Southwestern Medical Center at Dallas, Dallas, TX 8
Monash Institute of Medical Research (MIMR), Clayton, Victoria, Australia
9
Centre for Kidney Research, Children's Hospital at Westmead, Westmead, New
South Wales, Australia. 10
Department of Immunology, The Canberra Hospital, Canberra, ACT, Australia
Corresponding authors: Julia Ellyard. Phone: +61 2 61253519 Fax: +61 2 6125 2595 Email: [email protected]; Carola Vinuesa. Phone: +61 2 61254500 Fax:
+61 2 6125 2595 Email: [email protected]
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ABSTRACT Objective: Systemic lupus erythematosus (SLE) is a chronic and heterogeneous autoimmune disease. Both twin and sibling studies indicate a strong genetic contribution to lupus, but in the majority of cases the pathogenic variant remains to be identified. The genetic contribution to disease is likely to be greatest in cases with early onset and severe phenotypes. Whole exome sequencing now offers the possibility of identifying rare alleles responsible for disease in such cases. Methods: We performed whole exome sequencing in a 4-year-old female with early-onset SLE and conducted biochemical analysis of the putative defect. Results: Whole exome sequencing of a 4-year-old female with cerebral lupus identified a rare, homozygous mutation in the Three Prime Repair Exonuclease 1 (TREX1) that was predicted to be highly deleterious. The TREX1 R97H mutant protein had a 20-fold reduction in exonuclease activity and was associated with an elevated IFN-α signature in the patient. The discovery and characterization of a pathogenic TREX1 in our proband has therapeutic implications: the patient is now a candidate for neutralizing anti-IFNα therapy. Conclusion: Our study is the first to demonstrate that whole exome sequencing can be used to identify rare or novel deleterious variants as genetic causes of SLE and, through a personalized approach, improve therapeutic options.
John Wiley & Sons
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ELLYARD et al
Pathogenic TREX1 deficiency in severe early-onset SLE
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with complex, heterogeneous pathogenesis. Twin-twin and sibling studies implicate a strong genetic risk for SLE1. Although human and mouse studies have identified several rare gene variants resulting in SLE, the majority of current genetic knowledge has been obtained from genome-wide association studies (GWAS). GWAS has identified several common gene variants associated with modest risk for developing SLE1, yet direct evidence of a pathogenic relationship between many of these variants and disease is lacking.
Recent advances in whole exome sequencing (WES) are enabling identification of causal mutations underlying many genetic disorders. Where GWAS is limited to common variants, the ability to identify individual specific rare variants by WES can significantly advance the diagnosis and treatment options. We describe a case of severe SLE in a 4 year-old female of consanguineous parentage whom, given her pedigree, age of onset and severity of disease, is highly likely to have a genetic basis to her disease. Using WES, we identified a rare, disease-causing variant in TREX1 and demonstrated the pathophysiological basis of this variant.
PATIENT AND METHODS Patient case report A 3 year-old female of Lebanese ethnicity presented with arthritis, malar rash, diffuse purpura rashes, episodic fever, chronic headache and vomiting. There was no history of seizures or visual disturbance. There was no family history of autoimmunity. Her parents were first cousins (Figure 1A). Serology demonstrated high titer anti-nuclear antibodies (ANA; Hep2 titer 640), anti-double stranded DNA-antibodies (dsDNA;
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15kIU/L), anti-cardiolipin antibodies (IgM titer 27), elevated IgG (17.3g/L) and IgA (2.91g/L), lymphopenia and Coombs’ positive anemia, consistent with SLE. C3 (1.60g/L) and C4 (0.24g/L) levels, renal and liver function tests and cerebrospinal fluid analysis were normal. Initial brain MRI/MRA demonstrated steroid-related volume loss, but no other abnormalities. At 4 years of age she presented with rightsided hemiparesis. An MRI demonstrated occlusion of the left middle cerebral artery and left-sided MCA infarct (Figure 1B and C). MRA revealed widespread and marked irregularities of the medium sized vessels consistent with vasculitis in the context of cerebral SLE.
Genetic studies Informed written consent was obtained according to ethics approval from Australian National University and Canberra Hospital Human Ethics Committees. Genomic DNA was extracted from saliva using OrageneTM DNA Self Collection Kits. Whole exome capture and sequencing was performed by Beijing Genomics Institute and bioinformatics analysis performed at JCSMR, ANU as described in the supplementary data. Genotypes were verified by PCR and Sanger sequencing.
Exonuclease Activity Assay Mutant TREX1R97H was cloned from pML303-human TREX1WT 1-242 (gift of F. Perrino) using PCR site-directed mutagenesis. Exonuclease activity assays using recombinant human TREX1WT and TREX1R97H were performed as previously described2. Exonuclease activity was quantified using ImageQuant TL 8.1 software.
Dimerization studies
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Pathogenic TREX1 deficiency in severe early-onset SLE
Dimerization studies were performed using MBP-tagged and untagged versions of TREX1WT and TREX1R97H as previously described2.
IFN activity Interferon antiviral activity was measured using a cytopathic effect (CPE)-reduction bioassay with WISH cells and Semliki Forest virus: titers were calibrated against an international reference standard.
IFN signature studies RNA was extracted from PBMC samples and cDNA synthesis performed using MLVRT (Invitrogen). Quantitative PCR was performed by SRBR green incorporation using the 7900HT Fast Real-Time PCR System (Applied Biosystems). ∆CT values were normalized to the healthy PBMC negative control. A heat map of the relative expression values was generated with GENE-E software (Broad Institute), using global expression and default color scheme for ratio data preferences.
RESULTS Identification of novel homozygous variant in TREX1 Bioinformatics analysis of the WES from the proband identified a rare (MAFA substitution in the Three Prime Repair Exonuclease 1 (TREX1) (Figure 1D). The resulting arginine to histidine substitution at amino acid position 97 (R97H) was predicted to be deleterious (Polyphen2 score=1; SIFT=0.03). DNA sequence analysis of other family members confirmed homozygosity was confined to the proband (Figure 1A, D). Detailed analysis of the exome data revealed no other immune-related, novel or rare (MAFC resulting in the H335R substitution predicted to be benign (MAF=0.33); and a G>C at IV9-5 with a MAF=0.215/469. Familial genotyping revealed they did not segregate with disease (Figure S1), excluding CECR1 as the cause of the proband’s syndrome.
Mutations in TREX1 have been linked to a spectrum of autoimmune diseases including Aicardi-Goutières Syndrome (AGS)5,6, familial chilblain lupus (CHBL)7,8, and SLE7,9. Homozygosity for TREX1R97H was therefore considered a plausible explanation for this presentation. As this allele had not been described as a cause of pathology we proceeded to functional analysis.
Functional characterization of TREX1 R97H TREX1 is the primary mammalian 3’-5’ intracellular exonuclease; it degrades endogenous nucleic acids in the cell cytoplasm. The R97 residue is highly conserved throughout evolution (Figure 2A), suggesting an important role in protein function. To examine the effect of the mutation on protein function, we measured exonuclease activity in vitro. Compared to TREX1WT, TREX1R97H had a greatly diminished ability to degrade single-stranded DNA (ssDNA) (Figure 2B). Quantification of the exonuclease activity7,10 revealed an approximately 20-fold reduction in exonuclease activity of TREX1R97H (Table 1).
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Loss of function mutations in TREX1 are known to augment production of type I IFNs11; elevated levels of IFN-α in the CSF and serum are a signature of AGS patients with mutations in TREX15,10. Analysis of the proband’s serum revealed detectable levels of IFN-α (8 IU/ml) compared to undetectable levels (2-fold. Together these findings indicate that TREX1R97H has impaired exonuclease function that results in defective clearance of nucleic acids, triggering signaling pathways that promote ANA production, secretion of type I IFNs and inflammation.
Structural characterization of TREX1R97H To understand the cause of reduced exonuclease activity by TREX1R97H, we performed in silico structural analysis using the program Cn3D (NCBI). TREX1 forms a homodimer with several residues at the dimer interface being important for both dimerization and protein function7. Based on the crystal structure for murine TREX17, the mutated R97 residue was predicted to be located at the dimer interface, opposite an arginine residue at position 114 (R114) (Figure 2D). Although the R97H mutation occurred at the dimer interface, it did not appear to impair its ability to form dimers in vitro (Figure S3). However, it is possible the mutation may affect the overall stability of the TREX1R97H dimer and/or protein.
DISCUSSION In this study we used WES to investigate the genetic basis of a case of severe, early-
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onset SLE, identifying a rare deleterious homozygous variant of TREX1. At the time of discovery this variant was novel, however, it has recently been annotated in the 1000 genome project (MAFA variant results in the R97H amino acid substitution.
Figure 2: R97H mutation impairs TREX1 exonuclease activity. A) Evolutionary conservation of R97 (red) in TREX1. B) Visualization and comparison of degradation of a 30-mer oligonucleotide by TREX1WT and TREX1R97H reveals an approximately 20-fold decrease in endonuclease activity. Results are representative of three experiments. C) Heat map showing relative expression of 11 IFN-inducible genes in PBMCs from proband and two female relatives heterozygous for the TREX1R97H variant. Values were normalized to an unstimulated healthy PBMC sample (Nil). Healthy PBMCs cultured with IFN-α were included as positive control. Fold increases in gene expression are shown for proband (II.V) compared to her sister (II.I). Full data set is shown in Figure S2. D) Structure of murine TREX1 dimer showing the location of R97 (yellow), R99 (green) and R114 (red).
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Acknowledgements The authors wish to thank; A. Wilson, S. Aditya, F. Perrino and C. Orebaugh, and the ACRF Bimolecular Resource Facility. This work was supported by an Elizabeth Blackburn NHMRC Research Fellowship, NHMRC program and project grants to C.G.V., an NHMRC Overseas Biomedical Fellowship to J.I.E. and a RACP/Jacquot research scholarship to S.H.J.
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REFERENCES 1. Sestak AL, Fürnrohr BG, Harley JB, Merrill JT, Namjou B. The genetics of systemic lupus erythematosus and implications for targeted therapy. Ann Rheum Dis 2011;70 Suppl 1:i37–43. 2. Orebaugh CD, Fye JM, Harvey S, Hollis T, Perrino FW. The TREX1 Exonuclease R114H Mutation in Aicardi-Goutieres Syndrome and Lupus Reveals Dimeric Structure Requirements for DNA Degradation Activity. J Biol Chem 2011;286:40246-40254. 3. Zhou Q, Yang D, Ombrello AK, Zavialov AV, Toro C, Zavialov AV, et al. Earlyonset stroke and vasculopathy associated with mutations in ADA2. N Engl J Med 2014;370:911–920. 4. Navon Elkan P, Pierce SB, Segel R, Walsh T, Barash J, Padeh S, et al. Mutant adenosine deaminase 2 in a polyarteritis nodosa vasculopathy. N Engl J Med 2014;370:921–931. 5. Goutières F, Aicardi J, Barth PG, Lebon P. Aicardi-Goutières syndrome: an update and results of interferon-alpha studies. Ann Neurol 1998;44:900–907. 6. Crow YJ, Hayward BE, Parmar R, Robins P, Leitch A, Ali M, et al. Mutations in the gene encoding the 3′-5′ DNA exonuclease TREX1 cause Aicardi-Goutières syndrome at the AGS1 locus. Nat Genet 2006;38:917–920. 7. de Silva U, Choudhury S, Bailey SL, Harvey S, Perrino FW, Hollis T. The crystal structure of TREX1 explains the 3' nucleotide specificity and reveals a polyproline II helix for protein partnering. J Biol Chem 2007;282:10537–10543. 8. Lee-Kirsch MA, Chowdhury D, Harvey S, Gong M, Senenko L, Engel K, et al. A mutation in TREX1 that impairs susceptibility to granzyme A-mediated cell death underlies familial chilblain lupus. J Mol Med 2007;85:531–537. 9. Lee-Kirsch MA, Gong M, Chowdhury D, Senenko L, Engel K, Lee Y-A, et al. Mutations in the gene encoding the 3′-5′ DNA exonuclease TREX1 are associated with systemic lupus erythematosus. Nat Genet 2007;39:1065–1067. 10. Lehtinen DA, Harvey S, Mulcahy MJ, Hollis T, Perrino FW. The TREX1 doublestranded DNA degradation activity is defective in dominant mutations associated with autoimmune disease. J Biol Chem 2008;283:31649–31656. 11. Stetson DB, Ko JS, Heidmann T, Medzhitov R. Trex1 prevents cell-intrinsic initiation of autoimmunity. Cell 2008;134:587–598. 12. Olivieri I, Cattalini M, Tonduti D, La Piana R, Uggetti C, Galli J, et al. Dysregulation of the immune system in Aicardi-Goutières syndrome: another example in a TREX1-mutated patient. Lupus 2013;22:1064–1069. 13. Bhattacharyya S, Zhao Y, Kay TWH, Muglia LJ. Glucocorticoids target suppressor of cytokine signaling 1 (SOCS1) and type 1 interferons to regulate Toll-
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like receptor-induced STAT1 activation. Proc Natl Acad Sci USA 2011;108:9554– 9559. 14. Vogt J, Agrawal S, Ibrahim Z, Southwood TR, Philip S, Macpherson L, et al. Striking intrafamilial phenotypic variability in Aicardi-Goutières syndrome associated with the recurrent Asian founder mutation in RNASEH2C. Am J Med Genet A 2013;161A:338–342. 15. Namjou B, Kothari PH, Kelly JA, Glenn SB, Ojwang JO, Adler A, et al. Evaluation of the TREX1 gene in a large multi-ancestral lupus cohort. Genes Immun 2011;12:270–279.
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Figure 1 254x338mm (72 x 72 DPI)
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Figure 2 254x338mm (72 x 72 DPI)
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Figure S2: Relative expression of 11 IFN-α inducible genes ex vivo in PBMCs from the proband and two female relatives (I.II, II.I) as measured by real-time PCR. PBMCs from a healthy donor with (IFN) and without (Nil) IFN-α stimulation were included as positive and negative controls respectively. All samples were normalised to expression of PBMCs without IFN-α expression.
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Supplementary methods Whole exome sequencing and analysis Whole exome capture and sequencing was performed by Beijing Genomics Institute using the SureSelect Human All Exon Kit (Agilent) and the Illumina Hiseq 2000 platform. Raw data were processed by Illumina basecalling Software 1.7. Coverage statistics for the raw exome data are shown in Table S1. Bioinformatic analysis was performed at JCSMR, ANU. Raw sequence reads were aligned to the reference genome (Hg19) and single nucleotide variants (SNVs) and small indels called using SAMTools. Results were filtered against known variants (dbSNP 132). Consanguinity and an absence of other affected siblings lead us to focus on novel or rare (MAF
