ARTHRITIS & RHEUMATOLOGY Vol. 67, No. 1, January 2015, pp 288–295 DOI 10.1002/art.38877 © 2015, American College of Rheumatology
Association of a Mutation in LACC1 With a Monogenic Form of Systemic Juvenile Idiopathic Arthritis Salma M. Wakil,1 Dorota M. Monies,1 Mohamed Abouelhoda,1 Nada Al-Tassan,1 Haya Al-Dusery,2 Ewa A. Naim,1 Banan Al-Younes,1 Jameela Shinwari,2 Futwan A. Al-Mohanna,2 Brian F. Meyer,1 and Sulaiman Al-Mayouf 2 domain–containing 1. The mutation was confirmed by Sanger sequencing and segregated with disease in all 5 families based on an autosomal-recessive pattern of inheritance and complete penetrance. Conclusion. Our findings provide strong genetic evidence of an association of a mutation in LACC1 with systemic JIA in the families studied. Association of LACC1 with Crohn’s disease and leprosy has been reported and justifies investigation of its role in autoinflammatory disorders.
Objective. The pathologic basis of systemic juvenile idiopathic arthritis (JIA) is a subject of some controversy, with evidence for both autoimmune and autoinflammatory etiologies. Several monogenic autoinflammatory disorders have been described, but thus far, systemic JIA has only been attributed to a mutation of MEFV in rare cases and has been weakly associated with the HLA class II locus. This study was undertaken to identify the cause of an autosomal-recessive form of systemic JIA. Methods. We studied 13 patients with systemic JIA from 5 consanguineous families, all from the southern region of Saudi Arabia. We used linkage analysis, homozygosity mapping, and whole-exome sequencing to identify the disease-associated gene and mutation. Results. Linkage analysis localized systemic JIA to a region on chromosome 13 with a maximum logarithm of odds score of 11.33, representing the strongest linkage identified to date for this disorder. Homozygosity mapping reduced the critical interval to a 1.02-Mb region defined proximally by rs9533338 and distally by rs9595049. Whole-exome sequencing identified a homoallelic missense mutation in LACC1, which encodes the enzyme laccase (multicopper oxidoreductase)
Systemic juvenile idiopathic arthritis (JIA) is typically considered to be a subtype of JIA with extraarticular features, including quotidian fever, associated macular rash, lymphadenopathy, organomegaly, polyserositis, and iritis (1). Extraarticular features may overshadow joint inflammation at disease onset, at which time indicators of systemic inflammation, including erythrocyte sedimentation rate, C-reactive protein, and platelet and neutrophil counts, are all typically elevated. Proportionately, systemic JIA accounts for ⬃10% of JIA cases in North America and Europe, whereas in India and Japan, systemic JIA represents ⬃30% and 50% of JIA cases, respectively (2,3). Some insights into the pathophysiology of systemic JIA have been provided by recent immunologic and genetic studies (4–7). However, the primary etiology of systemic JIA remains to be revealed. The understanding of diseases with immunologic etiologies has evolved through consideration of contributions of both the adaptive and innate immune responses. Classic autoimmune diseases, including rheumatoid arthritis and type 1 diabetes mellitus, are typically associated with autoreactive antigen-specific T cells and/or autoantibodies and frequently show strong associations with major histocompatibility complex
Supported by the King Faisal Specialist Hospital and Research Centre (RAC Project 2020023). 1 Salma M. Wakil, PhD, Dorota M. Monies, PhD, Mohamed Abouelhoda, PhD, Nada Al-Tassan, PhD, Ewa A. Naim, MSc, Banan Al-Younes, MSc, Brian F. Meyer, PhD: King Faisal Specialist Hospital and Research Centre, and King Abdulaziz City for Science and Technology, Riyadh, Saudi Arabia; 2Haya Al-Dusery, BSc, Jameela Shinwari, MSc, Futwan A. Al-Mohanna, PhD, Sulaiman Al-Mayouf, MD: King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia. Address correspondence to Sulaiman Al-Mayouf, MD, MBC 03 King Faisal Specialist Hospital and Research Centre, PO Box 3354, Riyadh 11211, Saudi Arabia. E-mail: [email protected]. Submitted for publication June 25, 2014; accepted in revised form September 9, 2014. 288
MONOGENIC SYSTEMIC JUVENILE IDIOPATHIC ARTHRITIS
(MHC) class II alleles (8,9). Alternatively, pathologic conditions in which the effector cells include monocytes, neutrophils, and lymphocytes are collectively known as autoinflammatory diseases (6). The notion of autoinflammatory disorders was first proposed with the discovery that mutations in TNFR1 resulted in a dominantly inherited syndrome (tumor necrosis factor [TNF] receptor–associated periodic syndrome [TRAPS]) characterized by fever and systemic inflammation (10). Positional cloning of the recessively inherited familial Mediterranean fever (FMF) preceded this discovery and identified mutation of MEFV to underlie this disorder (11,12). Autoinflammatory diseases were initially regarded as monogenic disorders based on findings in FMF and TRAPS, and associated with common clinical features including recurrent fevers and systemic inflammation involving the joints, skin, gastrointestinal tract, and eyes (6). Neonatal-onset multisystem inflammatory disease (NOMID), caused by mutations in NLRP3, which encodes cryopyrin, is another classic example of a monogenic autoinflammatory disorder (13). More recently, the category of autoinflammatory syndromes has been expanded to encompass apparently polygenic disorders, such as Crohn’s disease (CD), type 2 diabetes mellitus, and systemic JIA (6). Unlike JIA, strong associations with MHC class II alleles have not been reported in systemic JIA, an observation consistent with other autoinflammatory conditions. Inherited risk factors for systemic JIA include polymorphisms in promoter regions and genes encoding TNF (14), interleukin-6 (IL-6) (15), IL-10 (5), macrophage migration inhibitory factor (16), and the IL-1 family (17). Loss-of-function mutations in P2X7, which encodes an ATP receptor involved in IL-1 regulation (18), and polymorphisms within SLC26A2 (19) have also been reported in association with systemic JIA. Taken together, these findings support a polygenic model of genetic risk for systemic JIA. No significant associations have been made to date between systemic JIA and genes underlying monogenic autoinflammatory disorders (NLRP3, NOD2, MEFV, or PSTPIP1) (20). A notable exception was a subgroup (15%) of Turkish patients with severe systemic JIA and MEFV mutations (21). While there is abundant evidence of dysfunction of innate immunity in systemic JIA, there still remain arguments that dismiss ruling out a role of adaptive immunity (22–24). Furthermore, the largest genomewide association study of systemic JIA published to date (abstract only) describes a clear but weak association of systemic JIA with the HLA class II locus, which is consistent with T cell autoimmunity (24).
289
In this study, we describe 13 patients with systemic JIA from 5 consanguineous families with an apparently common founder. All 13 patients were homoallelic for a missense mutation in a novel gene LACC1, which encodes the enzyme laccase (multicopper oxidoreductase) domain–containing 1. The mutation results in the substitution of an evolutionarily invariant cysteine with arginine and lends further support to the characterization of a subset of systemic JIA as a recessive monogenic autoinflammatory syndrome. PATIENTS AND METHODS Patients. The study included 13 patients who underwent treatment and regular followup in the pediatric rheumatology clinics at King Faisal Specialist Hospital and Research Centre (KFSHR&RC) between 1990 and 2013. KFSHR&RC is the major tertiary care center in Saudi Arabia for most subspecialties including pediatric rheumatology. Patients were from 5 extended consanguineous families referred randomly but were all from the southern region of Saudi Arabia. All subjects (patients and family members) were enrolled under an Institutional Review Board–approved protocol (Research Advisory Council #2020023) and provided full informed consent. Linkage and homozygosity mapping. All family members (affected and unaffected) were genotyped using an Affymetrix Axiom array following the manufacturer’s protocol (http://www.affymetrix.com/support/technical/manuals.affx). The resulting genotypes were analyzed for shared runs of homozygosity (ROH) using AutoSNPa (http://dna.leeds.ac.uk/ autosnpa/). Linkage analysis was performed using the Allegro module of the Easy Linkage software package, assuming autosomal-recessive inheritance and 100% penetrance (http:// easylinkage-plus.software.informer.com). Whole-exome sequencing and analysis. Two micrograms of each DNA sample was treated to obtain an Ion Proton precapture library using an AB Library Builder System (Life Technologies). Briefly, DNA was obtained by running the samples through precast 2% agarose gel cassettes (Pippin Prep DNA Size Selection System; Sage Science) for size selection. The purified adapter ligated fragments were subjected to library amplification using Platinum PCR Amplification Mix (Life Technologies) with P1 and P2 amplification primers and 10 amplification cycles to obtain the genomic precapture library. Libraries were quantified using an Agilent Bioanalyzer High-Sensitivity DNA chip. Target enrichment was performed with a TargetSeq human whole-exome kit (Life Technologies). The kit is designed to enrich for ⬃41 Mb of exons, small RNA genes, Catalog of Somatic Mutations in Cancer (COSMIC) variants, and predicted microRNA (miRNA) binding sites in 3⬘-untranslated regions (3⬘-UTRs), covering a total of 46.2 Mb of genomic sequence. The prepared exome library was further used for emulsion polymerase chain reaction (PCR) on an Ion OneTouch System, and templated Ion Sphere particles were enriched using Ion OneTouch ES, according to the recommendations of the manufacturer (Life Technologies) for both procedures. The template-positive Ion PI Ion Sphere particles were processed for sequencing on an Ion Proton instrument (Life Technologies). Approximately 6–8 Gb of sequence was generated per sequencing run. Reads were mapped to Univer-
290
WAKIL ET AL
sity of California, Santa Cruz hg19 (http://genome.ucsc.edu/), and variants were identified using an Ion Torrent pipeline (Life Technologies). The resultant variant caller file was filtered to include only homozygous variants within the predetermined ROH (by linkage and homozygosity mapping) that were absent from dbSNP, the 1,000 Genomes Project database, and ⬃1,000 Arab exomes run in our laboratories. Potential causative variants were validated by Sanger sequencing and further vetted for familial segregation based on autosomalrecessive inheritance. Sanger sequencing. Amplicons containing candidate variants were amplified and sequenced using a BigDye Terminator kit (Applied Biosystems) and run on an ABI 3730xl automated sequencer (Applied Biosystems). SeqScape software version 2.6 (Applied Biosystems) was used to align sequence data.
RESULTS Clinical features of the patients with systemic JIA. The study included a total of 13 children (12 girls) who were classified as having systemic JIA according to the International League of Associations for Rheuma-
tology criteria for JIA (25,26). All affected individuals were from the southern province of Saudi Arabia and came from consanguineous families where parents were first cousins (Figure 1). The mean ⫾ SD age at enrollment was 9.7 ⫾ 3.2 years (range 2–14 years), while the age at disease onset was 3.2 ⫾ 1.8 years. All patients had the characteristic quotidian fever and associated erythematous maculopapular rash. The frequencies of tenosynovitis, serositis and organomegaly, and lymphadenopathy were low (seen in 3, 2, and 5 of 13 patients, respectively). All patients had symmetrical polyarthritis, affecting small and large joints. None had psoriasis, enthesitis, or sacroiliitis, findings suggesting Reiter’s syndrome, inflammatory bowel disease (IBD), or acute anterior uveitis. Interestingly, 9 patients had persistent systemic manifestations, mainly intermittent fever with active polyarthritis, and 4 patients had short-lived systemic clinical manifestations and were diagnosed as having a polyarticular subtype. None of our patients had features suggesting macrophage activation syndrome.
Figure 1. Pedigrees of the 5 consanguineous nuclear families (Fam) of patients with systemic juvenile idiopathic arthritis. All affected subjects (solid symbols) were from consanguineous parents (first cousins). Sanger sequencing was used to confirm genotypes of all individuals for whom DNA was available. Homozygous mutant genotypes (⫺/⫺), carriers (⫹/⫺), and homozygous wild-type genotypes (⫹/⫹) were observed. In all instances, the genotypes were consistent with an autosomal-recessive inheritance and complete penetrance. Open symbols represent unaffected subjects. Numbers below the symbols indicate individuals in the various generations. Subject 25 was not included in the study due to lack of an available DNA sample.
MONOGENIC SYSTEMIC JUVENILE IDIOPATHIC ARTHRITIS
291
Figure 2. A, Genome-wide linkage analysis, revealing a peak with a maximum logarithm of odds (MLS) score of 11.33 on chromosome 13 at rs9567217. Numbers on the x-axis represent chromosomes; numbers on the y-axis are the MLS score. B, AutoSNPa output from chromosome 13, revealing a run of homozygosity (outlined in red), defined proximally by rs9533338 and distally by rs9595049, that is shared by all 13 affected individuals and absent in all unaffected individuals. Black shows homozygous single-nucleotide polymorphism [SNP] runs; yellow shows heterozygous SNP runs.
All had leukocytosis, thrombocytosis, and elevated markers of inflammation. However, 8 patients were positive for antinuclear antibody and negative for extractable nuclear antigen. Additionally, 5 patients had weak rheumatoid factor when tested on one occasion. All patients were treated with nonsteroidal antiinflammatory drugs, systemic corticosteroids, methotrexate, and biologic agents. All patients were treated with etanercept (Amgen) with inadequate response, and 7 were switched to adalimumab (Abbott Laboratories). Furthermore, 5 patients were treated with tocilizumab (Hoffmann-La Roche), and 3 patients received rituximab (Genentech). Unfortunately, remission was not achieved in any of the patients. Imaging studies showed erosive arthritic changes with significant damage, mainly in the wrist and hip joints, with guarded functional capacity as measured by the Childhood Health Assessment Questionnaire (27), with a mean ⫾ SD score of 1.25 ⫾ 0.99.
Results of linkage analysis and homozygosity mapping. Linkage analysis (Figure 2A) localized the disease to a region on chromosome 13 that was defined proximally by rs2802501 and distally by rs34386149 (chromosome 13:41,076,410–51,230,074), with a maximum logarithm of odds (MLS) score of 11.33 at rs9567217 representing the strongest linkage identified to date for systemic JIA. Based on an ROH that was shared by all affected individuals and absent in unaffected individuals (Figure 2B), we refined the critical interval to a 1.02-Mb region defined proximally by rs9533338 and distally by rs9595049 (chromosome 13: 43,570,887–44,593,911) containing 4 annotated genes, DNAJC15, ENOX1, CCDC122, and LACC1. Results of whole-exome sequencing. Exome sequencing of the index case generated 15 Gb of sequence and resulted in, on average, ⬎100⫻ coverage of the ⬃41 Mb of exons, small RNA genes, COSMIC variants, and predicted miRNA binding sites in 3⬘-UTRs, covering a
292
WAKIL ET AL
Figure 3. A, Sequence chromatogram showing the missense mutation c.850T⬎C, p.C284R in LACC1. B, Evolutionary conservation of the cysteine at position 284 of LACC1.
total of 46.2 Mb of genomic sequence. We identified 36,421 variants relative to hg19, with 625 variants on chromosome 13. These were further filtered to exclude all variants present outside of our defined ROH (chromosome 13:43,570,887–44,593,919) within which we identified only 2 variants. Average base coverage of the CDS and flanking regions (⫾20 bp) within this critical interval was 184⫻, with 99.6% of bases having 10 or more reads. When previously reported variants were excluded (present in dbSNP, the 1,000 Genomes Project database, 1,000 Arab exomes, 700 Saudi exomes, and 1,200 chromosomes from the Saudi population), only 1 survived the filtration. It was a homozygous change in exon 4 of LACC1 (NM.001128303). The variant was identified in 114 reads (62 forward and 52 reverse reads) and was predicted to be pathogenic by PolyPhen-2 (http://genetics.bwh.harvard. edu/pph2/) and SIFT (http://sift.bii.a-star.edu.sg/) with scores of 1.0 and 0.0,
respectively. PCR amplification and direct Sanger sequencing of the index case confirmed the mutation c.T850C p.C284R in exon 4 of LACC1 (Figure 3A). The p.C284R mutation is evolutionarily invariant (Figure 3B) and part of the copper reductase domain of LACC1. The mutation segregated with systemic JIA in all 5 families as expected of an autosomal-recessive disorder (Figure 1). All patients shared an identical haplotype at the LACC1 locus, and it is likely that the 5 families have a common founder. DISCUSSION Despite extensive investigation, the pathogenesis of systemic JIA remains unclear (22–24,28), although there is strong support for classifying it as an autoinflammatory disease (29–31). Our findings are the first demonstration of autosomal-recessive inheritance of sys-
MONOGENIC SYSTEMIC JUVENILE IDIOPATHIC ARTHRITIS
temic JIA associated with mutation of LACC1. To date, the apparent monogenic inheritance of systemic JIA has only been reported in a small subset of Turkish patients in whom mutant MEFV alleles, recessive and dominant, were associated with systemic JIA. However, since no multiplex families were described, the clear segregation of disease with MEFV mutations was not established (21), and systemic JIA is currently not viewed as a monogenic disorder. Indeed, it has been differentiated from monogenic autoinflammatory disorders such as FMF (12), TRAPS (10), NOMID (13), and hemophagocytic lymphohistiocytosis (32). LACC1 encodes the enzyme laccase (multicopper oxidoreductase) domain–containing 1. Laccases (benzenediol:oxygen oxidoreductase; EC 1.10.3.2) belong to a group of “blue multicopper oxidases” that includes the mammalian enzymes ceruloplasmin, hephaestin, and bilirubin oxidase among others (33). Laccase is a nonspecific enzyme that catalyzes the oxidation of a broad range of substrates, including polyphenols, aromatic amines, and inorganic ions (34). However, the biologic function of LACC1 is still poorly understood. A review of ENCODE (http://genome.ucsc.edu/ ENCODE/) project data for LACC1, including expression array, DNA hypersensitivity, and ChIP-Seq tracks, provided no obvious insight into the biology of this enzyme. The pedigrees of 5 consanguineous multiplex families of patients with systemic JIA in our study clearly indicate an autosomal-recessive pattern of inheritance in this cohort (Figure 1). Parametric linkage analysis based on autosomal-recessive inheritance resulted in an MLS score of 11.33 at a single ⬃10-Mb locus on chromosome 13, placing monogenic recessive inheritance beyond doubt (Figure 2A). Homozygosity mapping refined this locus further, and whole-exome sequencing identified a single missense variant (c.T850C p.C284R) (Figure 3A) in LACC1, which was predicted to be pathogenic, segregated with disease in all 5 families, and was absent in databases of human variants (dbSNP, the 1,000 Genomes Project database, 1,000 Arab exomes, 700 Saudi exomes, and 1,200 chromosomes from the Saudi population). Evolutionary conservation of the cysteine residue at position 284 and its presence in the multicopper oxidase domain of LACC1 further supported the notion of the pathogenicity of this variant (Figure 3B). However, in the absence of functional data or a compelling biologic rationale for the role of LACC1 in this disease process, it must be acknowledged that the c.T850C p.C284R mutation identified by this study may not be
293
causative. It may simply be in tight linkage disequilibrium with a true causative variant present in intronic, regulatory, or other elements of genes (DNAJC15, ENOX1, CCDC122, and LACC1) in the critical interval (chromosome 13:43,570,887–44,593,919) that remain unsequenced with whole-exome sequencing. In recent years, genome-wide association studies have identified shared risk variants for apparently clinically unrelated diseases (35). A classic example is the association of IL23R variants with leprosy (36), CD (37), ulcerative colitis (38), psoriasis (39), and ankylosing spondylitis (40), ranging from infection to autoimmune disorders, albeit united by inflammation. A genomewide association study undertaken in a Chinese cohort of leprosy patients identified associations in CCDC122, LACC1, NOD2, TNFSF15, HLA–DR, and RIPK2 (41). Interestingly, 3 of the genes are part of the nucleotidebinding oligomerization domain–containing protein 2 (NOD-2) signaling pathway, and 4 (TNFSF15, NOD2, LACC1, and HLA–DR) have been associated with CD (42). Arthropathies are often encountered together with IBD, either ulcerative colitis or CD (43), and IBD is encountered together with chronic granulomatous disease (44), highlighting significant comorbidity in the autoinflammatory disorders. These comorbidities and common associations of leprosy and CD with members of the NOD-2 signaling pathway are consistent with the underlying inflammatory pathology of these disorders and their classification as autoinflammatory syndromes. LACC1 has been associated with leprosy and CD and through the findings of the present study is now strongly linked to systemic JIA. Taken together, these findings implicate LACC1 in the pathology of autoinflammatory diseases. This remains to be confirmed by functional investigations or the identification of additional disease alleles in other patient cohorts with this or similar phenotypes. In summary, we provide robust genetic evidence of a monogenic autosomal-recessive form of systemic JIA associated with mutation of LACC1. It is likely that this represents a subset of systemic JIA, which to date has largely been regarded as a polygenic disorder. The association of LACC1 with systemic JIA, CD, and leprosy implicates it in the pathology of autoinflammatory disorders and presents a strong case for investigating the biology of laccases in inflammation. This study also highlights the value of rare Mendelian traits in elucidating the pathogenesis of common polygenic disorders.
294
WAKIL ET AL
ACKNOWLEDGMENTS We are grateful to all family members for their participation in this study. We thank the Sequencing, Genotyping, and DNA Extraction Core Facilities of the Department of Genetics, Research Centre, King Faisal Specialist Hospital and Research Centre. This project utilized resources of the Saudi Human Genome Project funded by the King Abdulaziz City for Science and Technology, Riyadh, Saudi Arabia. We thank the administration and support staff of this program through which Next Generation Sequencing capabilities were provided. AUTHOR CONTRIBUTIONS All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Al-Mayouf had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. Wakil, Monies, Abouelhoda, Al-Tassan, Al-Dusery, Naim, Al-Younes, Shinwari, Al-Mohanna, Meyer, Al-Mayouf. Acquisition of data. Wakil, Monies, Naim, Al-Younes, Al-Mayouf. Analysis and interpretation of data. Wakil, Monies, Abouelhoda, Al-Tassan, Al-Dusery, Naim, Al-Younes, Shinwari, Al-Mohanna, Meyer, Al-Mayouf.
11.
12. 13.
14.
15.
16.
17.
REFERENCES 1. Petty RE, Southwood TR, Manners P, Baum J, Glass DN, Goldenberg J, et al. International League of Associations for Rheumatology classification of juvenile idiopathic arthritis: second revision, Edmonton, 2001. J Rheumatol 2004;31:390–2. 2. Fujikawa S, Okuni M. Clinical analysis of 570 cases with juvenile rheumatoid arthritis: results of a nationwide retrospective survey in Japan. Acta Paediatr Jpn 1997;39:245–9. 3. Sawhney S, Magalhaes CS. Paediatric rheumatology: a global perspective. Best Pract Res Clin Rheumatol 2006;20:201–21. 4. Bleesing J, Prada A, Siegel DM, Villanueva J, Olson J, Ilowite NT, et al. The diagnostic significance of soluble CD163 and soluble interleukin-2 receptor ␣-chain in macrophage activation syndrome and untreated new-onset systemic juvenile idiopathic arthritis. Arthritis Rheum 2007;56:965–71. 5. Fife MS, Gutierrez A, Ogilvie EM, Stock CJ, Samuel JM, Thomson W, et al. Novel IL10 gene family associations with systemic juvenile idiopathic arthritis. Arthritis Res Ther 2006;8:R148. 6. Masters SL, Simon A, Aksentijevich I, Kastner DL. Horror autoinflammaticus: the molecular pathophysiology of autoinflammatory disease. Annu Rev Immunol 2009;27:621–68. 7. Moller JC, Paul D, Ganser G, Range U, Gahr M, Kelsch R, et al. IL10 promoter polymorphisms are associated with systemic onset juvenile idiopathic arthritis (SoJIA). Clin Exp Rheumatol 2010;28: 912–8. 8. Nejentsev S, Howson JM, Walker NM, Szeszko J, Field SF, Stevens HE, et al. Localization of type 1 diabetes susceptibility to the MHC class I genes HLA-B and HLA-A. Nature 2007;450: 887–92. 9. Raychaudhuri S, Sandor C, Stahl EA, Freudenberg J, Lee HS, Jia X, et al. Five amino acids in three HLA proteins explain most of the association between MHC and seropositive rheumatoid arthritis. Nat Genet 2012;44:291–6. 10. McDermott MF, Aksentijevich I, Galon J, McDermott EM, Ogunkolade BW, Centola M, et al. Germline mutations in the extracellular domains of the 55 kDa TNF receptor, TNFR1, define
18.
19.
20.
21.
22.
23. 24.
25.
26.
27.
28.
a family of dominantly inherited autoinflammatory syndromes. Cell 1999;97:133–44. International FMF Consortium. Ancient missense mutations in a new member of the RoRet gene family are likely to cause familial Mediterranean fever. Cell 1997;90:797–807. French FMF Consortium. A candidate gene for familial Mediterranean fever. Nat Genet 1997;17:25–31. Aksentijevich I, Nowak M, Mallah M, Chae JJ, Watford WT, Hofmann SR, et al. De novo CIAS1 mutations, cytokine activation, and evidence for genetic heterogeneity in patients with neonatal-onset multisystem inflammatory disease (NOMID): a new member of the expanding family of pyrin-associated autoinflammatory diseases. Arthritis Rheum 2002;46:3340–8. Date Y, Seki N, Kamizono S, Higuchi T, Hirata T, Miyata K, et al. Identification of a genetic risk factor for systemic juvenile rheumatoid arthritis in the 5⬘-flanking region of the TNF␣ gene and HLA genes. Arthritis Rheum 1999;42:2577–82. Fishman D, Faulds G, Jeffery R, Mohamed-Ali V, Yudkin JS, Humphries S, et al. The effect of novel polymorphisms in the interleukin-6 (IL-6) gene on IL-6 transcription and plasma IL-6 levels, and an association with systemic-onset juvenile chronic arthritis. J Clin Invest 1998;102:1369–76. Donn RP, Shelley E, Ollier WE, Thomson W, and the British Paediatric Rheumatology Study Group. A novel 5⬘-flanking region polymorphism of macrophage migration inhibitory factor is associated with systemic-onset juvenile idiopathic arthritis. Arthritis Rheum 2001;44:1782–5. Stock CJ, Ogilvie EM, Samuel JM, Fife M, Lewis CM, Woo P. Comprehensive association study of genetic variants in the IL-1 gene family in systemic juvenile idiopathic arthritis. Genes Immun 2008;9:349–57. Lamb R, Thomson W, British Society of Paediatric and Adolescent Rheumatology, Ogilvie EM, Donn R. Positive association of SLC26A2 gene polymorphisms with susceptibility to systemiconset juvenile idiopathic arthritis. Arthritis Rheum 2007;56: 1286–91. Gattorno M, Piccini A, Lasiglie D, Tassi S, Brisca G, Carta S, et al. The pattern of response to anti–interleukin-1 treatment distinguishes two subsets of patients with systemic-onset juvenile idiopathic arthritis. Arthritis Rheum 2008;58:1505–15. Day TG, Ramanan AV, Hinks A, Lamb R, Packham J, Wise C, et al. Autoinflammatory genes and susceptibility to psoriatic juvenile idiopathic arthritis. Arthritis Rheum 2008;58:2142–6. Ayaz NA, Ozen S, Bilginer Y, Erguven M, Taskiran E, Yilmaz E, et al. MEFV mutations in systemic onset juvenile idiopathic arthritis. Rheumatology (Oxford) 2009;48:23–5. Rigante D, Cantarini L. The systemic-onset variant of juvenile idiopathic arthritis needs to be recorded as an autoinflammatory syndrome: comment on the review by Nigrovic [letter]. Arthritis Rheumatol 2014;66:2645. Nigrovic PA. Reply [letter]. Arthritis Rheumatol 2014;66:2645–6. Ombrello MJ, Remmers EF, Tachmazidou I, Grom AA, Foll D, Martini A, et al. Genome wide association meta-analysis implicates HLA-DRB1, the BTNL2/HLA-DRA region, and a novel susceptibility locus on chromosome 1 in systemic juvenile idiopathic arthritis [abstract]. Arthritis Rheum 2013;65 Suppl:S937. Fink CW. Proposal for the development of classification criteria for idiopathic arthritides of childhood. J Rheumatol 1995;22: 1566–9. Petty RE, Southwood TR, Baum J, Bhettay E, Glass DN, Manners P, et al. Revision of the proposed classification criteria for juvenile idiopathic arthritis: Durban, 1997. J Rheumatol 1998;25:1991–4. Singh G, Athreya BH, Fries JF, Goldsmith DP. Measurement of health status in children with juvenile rheumatoid arthritis. Arthritis Rheum 1994;37:1761–9. Mellins ED, Macaubas C, Grom AA. Pathogenesis of systemic
MONOGENIC SYSTEMIC JUVENILE IDIOPATHIC ARTHRITIS
29.
30.
31.
32.
33.
34. 35.
36.
juvenile idiopathic arthritis: some answers, more questions. Nat Rev Rheumatol 2011;7:416–26. Griffin TA, Barnes MG, Ilowite NT, Olson JC, Sherry DD, Gottlieb BS, et al. Gene expression signatures in polyarticular juvenile idiopathic arthritis demonstrate disease heterogeneity and offer a molecular classification of disease subsets. Arthritis Rheum 2009;60:2113–23. Ogilvie EM, Khan A, Hubank M, Kellam P, Woo P. Specific gene expression profiles in systemic juvenile idiopathic arthritis. Arthritis Rheum 2007;56:1954–65. Pascual V, Allantaz F, Arce E, Punaro M, Banchereau J. Role of interleukin-1 (IL-1) in the pathogenesis of systemic onset juvenile idiopathic arthritis and clinical response to IL-1 blockade. J Exp Med 2005;201:1479–86. Grom AA. Natural killer cell dysfunction: a common pathway in systemic-onset juvenile rheumatoid arthritis, macrophage activation syndrome, and hemophagocytic lymphohistiocytosis? [review]. Arthritis Rheum 2004;50:689–98. Sakurai T, Kataoka K. Basic and applied features of multicopper oxidases, CueO, bilirubin oxidase, and laccase. Chem Rec 2007;7: 220–9. Baldrian P. Fungal laccases: occurrence and properties. FEMS Microbiol Rev 2006;30:215–42. Liu H, Irwanto A, Tian H, Fu X, Yu Y, Yu G, et al. Identification of IL18RAP/IL18R1 and IL12B as leprosy risk genes demonstrates shared pathogenesis between inflammation and infectious diseases. Am J Hum Genet 2012;91:935–41. Zhang F, Liu H, Chen S, Low H, Sun L, Cui Y, et al. Identification
295
37. 38.
39. 40.
41. 42. 43.
44.
of two new loci at IL23R and RAB32 that influence susceptibility to leprosy. Nat Genet 2011;43:1247–51. Barrett JC, Hansoul S, Nicolae DL, Cho JH, Duerr RH, Rioux JD, et al. Genome-wide association defines more than 30 distinct susceptibility loci for Crohn’s disease. Nat Genet 2008;40:955–62. Anderson CA, Boucher G, Lees CW, Franke A, D’Amato M, Taylor KD, et al. Meta-analysis identifies 29 additional ulcerative colitis risk loci, increasing the number of confirmed associations to 47. Nat Genet 2011;43:246–52. Nair RP, Duffin KC, Helms C, Ding J, Stuart PE, Goldgar D, et al. Genome-wide scan reveals association of psoriasis with IL-23 and NF-B pathways. Nat Genet 2009;41:199–204. Evans DM, Spencer CC, Pointon JJ, Su Z, Harvey D, Kochan G, et al. Interaction between ERAP1 and HLA-B27 in ankylosing spondylitis implicates peptide handling in the mechanism for HLA-B27 in disease susceptibility. Nat Genet 2011;43:761–7. Zhang FR, Huang W, Chen SM, Sun LD, Liu H, Li Y, et al. Genomewide association study of leprosy. N Engl J Med 2009;361: 2609–18. Schurr E, Gros P. A common genetic fingerprint in leprosy and Crohn’s disease? N Engl J Med 2009;361:2666–8. Salvarani C, Vlachonikolis IG, van der Heijde DM, Fornaciari G, Macchioni P, Beltrami M, et al. Musculoskeletal manifestations in a population-based cohort of inflammatory bowel disease patients. Scand J Gastroenterol 2001;36:1307–13. Marks DJ, Miyagi K, Rahman FZ, Novelli M, Bloom SL, Segal AW. Inflammatory bowel disease in CGD reproduces the clinicopathological features of Crohn’s disease. Am J Gastroenterol 2009;104:117–24.
Association of a Mutation in LACC1 With a Monogenic Form of Systemic Juvenile Idiopathic Arthritis Salma M. Wakil,1 Dorota M. Monies,1 Mohamed Abouelhoda,1 Nada Al-Tassan,1 Haya Al-Dusery,2 Ewa A. Naim,1 Banan Al-Younes,1 Jameela Shinwari,2 Futwan A. Al-Mohanna,2 Brian F. Meyer,1 and Sulaiman Al-Mayouf 2 domain–containing 1. The mutation was confirmed by Sanger sequencing and segregated with disease in all 5 families based on an autosomal-recessive pattern of inheritance and complete penetrance. Conclusion. Our findings provide strong genetic evidence of an association of a mutation in LACC1 with systemic JIA in the families studied. Association of LACC1 with Crohn’s disease and leprosy has been reported and justifies investigation of its role in autoinflammatory disorders.
Objective. The pathologic basis of systemic juvenile idiopathic arthritis (JIA) is a subject of some controversy, with evidence for both autoimmune and autoinflammatory etiologies. Several monogenic autoinflammatory disorders have been described, but thus far, systemic JIA has only been attributed to a mutation of MEFV in rare cases and has been weakly associated with the HLA class II locus. This study was undertaken to identify the cause of an autosomal-recessive form of systemic JIA. Methods. We studied 13 patients with systemic JIA from 5 consanguineous families, all from the southern region of Saudi Arabia. We used linkage analysis, homozygosity mapping, and whole-exome sequencing to identify the disease-associated gene and mutation. Results. Linkage analysis localized systemic JIA to a region on chromosome 13 with a maximum logarithm of odds score of 11.33, representing the strongest linkage identified to date for this disorder. Homozygosity mapping reduced the critical interval to a 1.02-Mb region defined proximally by rs9533338 and distally by rs9595049. Whole-exome sequencing identified a homoallelic missense mutation in LACC1, which encodes the enzyme laccase (multicopper oxidoreductase)
Systemic juvenile idiopathic arthritis (JIA) is typically considered to be a subtype of JIA with extraarticular features, including quotidian fever, associated macular rash, lymphadenopathy, organomegaly, polyserositis, and iritis (1). Extraarticular features may overshadow joint inflammation at disease onset, at which time indicators of systemic inflammation, including erythrocyte sedimentation rate, C-reactive protein, and platelet and neutrophil counts, are all typically elevated. Proportionately, systemic JIA accounts for ⬃10% of JIA cases in North America and Europe, whereas in India and Japan, systemic JIA represents ⬃30% and 50% of JIA cases, respectively (2,3). Some insights into the pathophysiology of systemic JIA have been provided by recent immunologic and genetic studies (4–7). However, the primary etiology of systemic JIA remains to be revealed. The understanding of diseases with immunologic etiologies has evolved through consideration of contributions of both the adaptive and innate immune responses. Classic autoimmune diseases, including rheumatoid arthritis and type 1 diabetes mellitus, are typically associated with autoreactive antigen-specific T cells and/or autoantibodies and frequently show strong associations with major histocompatibility complex
Supported by the King Faisal Specialist Hospital and Research Centre (RAC Project 2020023). 1 Salma M. Wakil, PhD, Dorota M. Monies, PhD, Mohamed Abouelhoda, PhD, Nada Al-Tassan, PhD, Ewa A. Naim, MSc, Banan Al-Younes, MSc, Brian F. Meyer, PhD: King Faisal Specialist Hospital and Research Centre, and King Abdulaziz City for Science and Technology, Riyadh, Saudi Arabia; 2Haya Al-Dusery, BSc, Jameela Shinwari, MSc, Futwan A. Al-Mohanna, PhD, Sulaiman Al-Mayouf, MD: King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia. Address correspondence to Sulaiman Al-Mayouf, MD, MBC 03 King Faisal Specialist Hospital and Research Centre, PO Box 3354, Riyadh 11211, Saudi Arabia. E-mail: [email protected]. Submitted for publication June 25, 2014; accepted in revised form September 9, 2014. 288
MONOGENIC SYSTEMIC JUVENILE IDIOPATHIC ARTHRITIS
(MHC) class II alleles (8,9). Alternatively, pathologic conditions in which the effector cells include monocytes, neutrophils, and lymphocytes are collectively known as autoinflammatory diseases (6). The notion of autoinflammatory disorders was first proposed with the discovery that mutations in TNFR1 resulted in a dominantly inherited syndrome (tumor necrosis factor [TNF] receptor–associated periodic syndrome [TRAPS]) characterized by fever and systemic inflammation (10). Positional cloning of the recessively inherited familial Mediterranean fever (FMF) preceded this discovery and identified mutation of MEFV to underlie this disorder (11,12). Autoinflammatory diseases were initially regarded as monogenic disorders based on findings in FMF and TRAPS, and associated with common clinical features including recurrent fevers and systemic inflammation involving the joints, skin, gastrointestinal tract, and eyes (6). Neonatal-onset multisystem inflammatory disease (NOMID), caused by mutations in NLRP3, which encodes cryopyrin, is another classic example of a monogenic autoinflammatory disorder (13). More recently, the category of autoinflammatory syndromes has been expanded to encompass apparently polygenic disorders, such as Crohn’s disease (CD), type 2 diabetes mellitus, and systemic JIA (6). Unlike JIA, strong associations with MHC class II alleles have not been reported in systemic JIA, an observation consistent with other autoinflammatory conditions. Inherited risk factors for systemic JIA include polymorphisms in promoter regions and genes encoding TNF (14), interleukin-6 (IL-6) (15), IL-10 (5), macrophage migration inhibitory factor (16), and the IL-1 family (17). Loss-of-function mutations in P2X7, which encodes an ATP receptor involved in IL-1 regulation (18), and polymorphisms within SLC26A2 (19) have also been reported in association with systemic JIA. Taken together, these findings support a polygenic model of genetic risk for systemic JIA. No significant associations have been made to date between systemic JIA and genes underlying monogenic autoinflammatory disorders (NLRP3, NOD2, MEFV, or PSTPIP1) (20). A notable exception was a subgroup (15%) of Turkish patients with severe systemic JIA and MEFV mutations (21). While there is abundant evidence of dysfunction of innate immunity in systemic JIA, there still remain arguments that dismiss ruling out a role of adaptive immunity (22–24). Furthermore, the largest genomewide association study of systemic JIA published to date (abstract only) describes a clear but weak association of systemic JIA with the HLA class II locus, which is consistent with T cell autoimmunity (24).
289
In this study, we describe 13 patients with systemic JIA from 5 consanguineous families with an apparently common founder. All 13 patients were homoallelic for a missense mutation in a novel gene LACC1, which encodes the enzyme laccase (multicopper oxidoreductase) domain–containing 1. The mutation results in the substitution of an evolutionarily invariant cysteine with arginine and lends further support to the characterization of a subset of systemic JIA as a recessive monogenic autoinflammatory syndrome. PATIENTS AND METHODS Patients. The study included 13 patients who underwent treatment and regular followup in the pediatric rheumatology clinics at King Faisal Specialist Hospital and Research Centre (KFSHR&RC) between 1990 and 2013. KFSHR&RC is the major tertiary care center in Saudi Arabia for most subspecialties including pediatric rheumatology. Patients were from 5 extended consanguineous families referred randomly but were all from the southern region of Saudi Arabia. All subjects (patients and family members) were enrolled under an Institutional Review Board–approved protocol (Research Advisory Council #2020023) and provided full informed consent. Linkage and homozygosity mapping. All family members (affected and unaffected) were genotyped using an Affymetrix Axiom array following the manufacturer’s protocol (http://www.affymetrix.com/support/technical/manuals.affx). The resulting genotypes were analyzed for shared runs of homozygosity (ROH) using AutoSNPa (http://dna.leeds.ac.uk/ autosnpa/). Linkage analysis was performed using the Allegro module of the Easy Linkage software package, assuming autosomal-recessive inheritance and 100% penetrance (http:// easylinkage-plus.software.informer.com). Whole-exome sequencing and analysis. Two micrograms of each DNA sample was treated to obtain an Ion Proton precapture library using an AB Library Builder System (Life Technologies). Briefly, DNA was obtained by running the samples through precast 2% agarose gel cassettes (Pippin Prep DNA Size Selection System; Sage Science) for size selection. The purified adapter ligated fragments were subjected to library amplification using Platinum PCR Amplification Mix (Life Technologies) with P1 and P2 amplification primers and 10 amplification cycles to obtain the genomic precapture library. Libraries were quantified using an Agilent Bioanalyzer High-Sensitivity DNA chip. Target enrichment was performed with a TargetSeq human whole-exome kit (Life Technologies). The kit is designed to enrich for ⬃41 Mb of exons, small RNA genes, Catalog of Somatic Mutations in Cancer (COSMIC) variants, and predicted microRNA (miRNA) binding sites in 3⬘-untranslated regions (3⬘-UTRs), covering a total of 46.2 Mb of genomic sequence. The prepared exome library was further used for emulsion polymerase chain reaction (PCR) on an Ion OneTouch System, and templated Ion Sphere particles were enriched using Ion OneTouch ES, according to the recommendations of the manufacturer (Life Technologies) for both procedures. The template-positive Ion PI Ion Sphere particles were processed for sequencing on an Ion Proton instrument (Life Technologies). Approximately 6–8 Gb of sequence was generated per sequencing run. Reads were mapped to Univer-
290
WAKIL ET AL
sity of California, Santa Cruz hg19 (http://genome.ucsc.edu/), and variants were identified using an Ion Torrent pipeline (Life Technologies). The resultant variant caller file was filtered to include only homozygous variants within the predetermined ROH (by linkage and homozygosity mapping) that were absent from dbSNP, the 1,000 Genomes Project database, and ⬃1,000 Arab exomes run in our laboratories. Potential causative variants were validated by Sanger sequencing and further vetted for familial segregation based on autosomalrecessive inheritance. Sanger sequencing. Amplicons containing candidate variants were amplified and sequenced using a BigDye Terminator kit (Applied Biosystems) and run on an ABI 3730xl automated sequencer (Applied Biosystems). SeqScape software version 2.6 (Applied Biosystems) was used to align sequence data.
RESULTS Clinical features of the patients with systemic JIA. The study included a total of 13 children (12 girls) who were classified as having systemic JIA according to the International League of Associations for Rheuma-
tology criteria for JIA (25,26). All affected individuals were from the southern province of Saudi Arabia and came from consanguineous families where parents were first cousins (Figure 1). The mean ⫾ SD age at enrollment was 9.7 ⫾ 3.2 years (range 2–14 years), while the age at disease onset was 3.2 ⫾ 1.8 years. All patients had the characteristic quotidian fever and associated erythematous maculopapular rash. The frequencies of tenosynovitis, serositis and organomegaly, and lymphadenopathy were low (seen in 3, 2, and 5 of 13 patients, respectively). All patients had symmetrical polyarthritis, affecting small and large joints. None had psoriasis, enthesitis, or sacroiliitis, findings suggesting Reiter’s syndrome, inflammatory bowel disease (IBD), or acute anterior uveitis. Interestingly, 9 patients had persistent systemic manifestations, mainly intermittent fever with active polyarthritis, and 4 patients had short-lived systemic clinical manifestations and were diagnosed as having a polyarticular subtype. None of our patients had features suggesting macrophage activation syndrome.
Figure 1. Pedigrees of the 5 consanguineous nuclear families (Fam) of patients with systemic juvenile idiopathic arthritis. All affected subjects (solid symbols) were from consanguineous parents (first cousins). Sanger sequencing was used to confirm genotypes of all individuals for whom DNA was available. Homozygous mutant genotypes (⫺/⫺), carriers (⫹/⫺), and homozygous wild-type genotypes (⫹/⫹) were observed. In all instances, the genotypes were consistent with an autosomal-recessive inheritance and complete penetrance. Open symbols represent unaffected subjects. Numbers below the symbols indicate individuals in the various generations. Subject 25 was not included in the study due to lack of an available DNA sample.
MONOGENIC SYSTEMIC JUVENILE IDIOPATHIC ARTHRITIS
291
Figure 2. A, Genome-wide linkage analysis, revealing a peak with a maximum logarithm of odds (MLS) score of 11.33 on chromosome 13 at rs9567217. Numbers on the x-axis represent chromosomes; numbers on the y-axis are the MLS score. B, AutoSNPa output from chromosome 13, revealing a run of homozygosity (outlined in red), defined proximally by rs9533338 and distally by rs9595049, that is shared by all 13 affected individuals and absent in all unaffected individuals. Black shows homozygous single-nucleotide polymorphism [SNP] runs; yellow shows heterozygous SNP runs.
All had leukocytosis, thrombocytosis, and elevated markers of inflammation. However, 8 patients were positive for antinuclear antibody and negative for extractable nuclear antigen. Additionally, 5 patients had weak rheumatoid factor when tested on one occasion. All patients were treated with nonsteroidal antiinflammatory drugs, systemic corticosteroids, methotrexate, and biologic agents. All patients were treated with etanercept (Amgen) with inadequate response, and 7 were switched to adalimumab (Abbott Laboratories). Furthermore, 5 patients were treated with tocilizumab (Hoffmann-La Roche), and 3 patients received rituximab (Genentech). Unfortunately, remission was not achieved in any of the patients. Imaging studies showed erosive arthritic changes with significant damage, mainly in the wrist and hip joints, with guarded functional capacity as measured by the Childhood Health Assessment Questionnaire (27), with a mean ⫾ SD score of 1.25 ⫾ 0.99.
Results of linkage analysis and homozygosity mapping. Linkage analysis (Figure 2A) localized the disease to a region on chromosome 13 that was defined proximally by rs2802501 and distally by rs34386149 (chromosome 13:41,076,410–51,230,074), with a maximum logarithm of odds (MLS) score of 11.33 at rs9567217 representing the strongest linkage identified to date for systemic JIA. Based on an ROH that was shared by all affected individuals and absent in unaffected individuals (Figure 2B), we refined the critical interval to a 1.02-Mb region defined proximally by rs9533338 and distally by rs9595049 (chromosome 13: 43,570,887–44,593,911) containing 4 annotated genes, DNAJC15, ENOX1, CCDC122, and LACC1. Results of whole-exome sequencing. Exome sequencing of the index case generated 15 Gb of sequence and resulted in, on average, ⬎100⫻ coverage of the ⬃41 Mb of exons, small RNA genes, COSMIC variants, and predicted miRNA binding sites in 3⬘-UTRs, covering a
292
WAKIL ET AL
Figure 3. A, Sequence chromatogram showing the missense mutation c.850T⬎C, p.C284R in LACC1. B, Evolutionary conservation of the cysteine at position 284 of LACC1.
total of 46.2 Mb of genomic sequence. We identified 36,421 variants relative to hg19, with 625 variants on chromosome 13. These were further filtered to exclude all variants present outside of our defined ROH (chromosome 13:43,570,887–44,593,919) within which we identified only 2 variants. Average base coverage of the CDS and flanking regions (⫾20 bp) within this critical interval was 184⫻, with 99.6% of bases having 10 or more reads. When previously reported variants were excluded (present in dbSNP, the 1,000 Genomes Project database, 1,000 Arab exomes, 700 Saudi exomes, and 1,200 chromosomes from the Saudi population), only 1 survived the filtration. It was a homozygous change in exon 4 of LACC1 (NM.001128303). The variant was identified in 114 reads (62 forward and 52 reverse reads) and was predicted to be pathogenic by PolyPhen-2 (http://genetics.bwh.harvard. edu/pph2/) and SIFT (http://sift.bii.a-star.edu.sg/) with scores of 1.0 and 0.0,
respectively. PCR amplification and direct Sanger sequencing of the index case confirmed the mutation c.T850C p.C284R in exon 4 of LACC1 (Figure 3A). The p.C284R mutation is evolutionarily invariant (Figure 3B) and part of the copper reductase domain of LACC1. The mutation segregated with systemic JIA in all 5 families as expected of an autosomal-recessive disorder (Figure 1). All patients shared an identical haplotype at the LACC1 locus, and it is likely that the 5 families have a common founder. DISCUSSION Despite extensive investigation, the pathogenesis of systemic JIA remains unclear (22–24,28), although there is strong support for classifying it as an autoinflammatory disease (29–31). Our findings are the first demonstration of autosomal-recessive inheritance of sys-
MONOGENIC SYSTEMIC JUVENILE IDIOPATHIC ARTHRITIS
temic JIA associated with mutation of LACC1. To date, the apparent monogenic inheritance of systemic JIA has only been reported in a small subset of Turkish patients in whom mutant MEFV alleles, recessive and dominant, were associated with systemic JIA. However, since no multiplex families were described, the clear segregation of disease with MEFV mutations was not established (21), and systemic JIA is currently not viewed as a monogenic disorder. Indeed, it has been differentiated from monogenic autoinflammatory disorders such as FMF (12), TRAPS (10), NOMID (13), and hemophagocytic lymphohistiocytosis (32). LACC1 encodes the enzyme laccase (multicopper oxidoreductase) domain–containing 1. Laccases (benzenediol:oxygen oxidoreductase; EC 1.10.3.2) belong to a group of “blue multicopper oxidases” that includes the mammalian enzymes ceruloplasmin, hephaestin, and bilirubin oxidase among others (33). Laccase is a nonspecific enzyme that catalyzes the oxidation of a broad range of substrates, including polyphenols, aromatic amines, and inorganic ions (34). However, the biologic function of LACC1 is still poorly understood. A review of ENCODE (http://genome.ucsc.edu/ ENCODE/) project data for LACC1, including expression array, DNA hypersensitivity, and ChIP-Seq tracks, provided no obvious insight into the biology of this enzyme. The pedigrees of 5 consanguineous multiplex families of patients with systemic JIA in our study clearly indicate an autosomal-recessive pattern of inheritance in this cohort (Figure 1). Parametric linkage analysis based on autosomal-recessive inheritance resulted in an MLS score of 11.33 at a single ⬃10-Mb locus on chromosome 13, placing monogenic recessive inheritance beyond doubt (Figure 2A). Homozygosity mapping refined this locus further, and whole-exome sequencing identified a single missense variant (c.T850C p.C284R) (Figure 3A) in LACC1, which was predicted to be pathogenic, segregated with disease in all 5 families, and was absent in databases of human variants (dbSNP, the 1,000 Genomes Project database, 1,000 Arab exomes, 700 Saudi exomes, and 1,200 chromosomes from the Saudi population). Evolutionary conservation of the cysteine residue at position 284 and its presence in the multicopper oxidase domain of LACC1 further supported the notion of the pathogenicity of this variant (Figure 3B). However, in the absence of functional data or a compelling biologic rationale for the role of LACC1 in this disease process, it must be acknowledged that the c.T850C p.C284R mutation identified by this study may not be
293
causative. It may simply be in tight linkage disequilibrium with a true causative variant present in intronic, regulatory, or other elements of genes (DNAJC15, ENOX1, CCDC122, and LACC1) in the critical interval (chromosome 13:43,570,887–44,593,919) that remain unsequenced with whole-exome sequencing. In recent years, genome-wide association studies have identified shared risk variants for apparently clinically unrelated diseases (35). A classic example is the association of IL23R variants with leprosy (36), CD (37), ulcerative colitis (38), psoriasis (39), and ankylosing spondylitis (40), ranging from infection to autoimmune disorders, albeit united by inflammation. A genomewide association study undertaken in a Chinese cohort of leprosy patients identified associations in CCDC122, LACC1, NOD2, TNFSF15, HLA–DR, and RIPK2 (41). Interestingly, 3 of the genes are part of the nucleotidebinding oligomerization domain–containing protein 2 (NOD-2) signaling pathway, and 4 (TNFSF15, NOD2, LACC1, and HLA–DR) have been associated with CD (42). Arthropathies are often encountered together with IBD, either ulcerative colitis or CD (43), and IBD is encountered together with chronic granulomatous disease (44), highlighting significant comorbidity in the autoinflammatory disorders. These comorbidities and common associations of leprosy and CD with members of the NOD-2 signaling pathway are consistent with the underlying inflammatory pathology of these disorders and their classification as autoinflammatory syndromes. LACC1 has been associated with leprosy and CD and through the findings of the present study is now strongly linked to systemic JIA. Taken together, these findings implicate LACC1 in the pathology of autoinflammatory diseases. This remains to be confirmed by functional investigations or the identification of additional disease alleles in other patient cohorts with this or similar phenotypes. In summary, we provide robust genetic evidence of a monogenic autosomal-recessive form of systemic JIA associated with mutation of LACC1. It is likely that this represents a subset of systemic JIA, which to date has largely been regarded as a polygenic disorder. The association of LACC1 with systemic JIA, CD, and leprosy implicates it in the pathology of autoinflammatory disorders and presents a strong case for investigating the biology of laccases in inflammation. This study also highlights the value of rare Mendelian traits in elucidating the pathogenesis of common polygenic disorders.
294
WAKIL ET AL
ACKNOWLEDGMENTS We are grateful to all family members for their participation in this study. We thank the Sequencing, Genotyping, and DNA Extraction Core Facilities of the Department of Genetics, Research Centre, King Faisal Specialist Hospital and Research Centre. This project utilized resources of the Saudi Human Genome Project funded by the King Abdulaziz City for Science and Technology, Riyadh, Saudi Arabia. We thank the administration and support staff of this program through which Next Generation Sequencing capabilities were provided. AUTHOR CONTRIBUTIONS All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Al-Mayouf had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. Wakil, Monies, Abouelhoda, Al-Tassan, Al-Dusery, Naim, Al-Younes, Shinwari, Al-Mohanna, Meyer, Al-Mayouf. Acquisition of data. Wakil, Monies, Naim, Al-Younes, Al-Mayouf. Analysis and interpretation of data. Wakil, Monies, Abouelhoda, Al-Tassan, Al-Dusery, Naim, Al-Younes, Shinwari, Al-Mohanna, Meyer, Al-Mayouf.
11.
12. 13.
14.
15.
16.
17.
REFERENCES 1. Petty RE, Southwood TR, Manners P, Baum J, Glass DN, Goldenberg J, et al. International League of Associations for Rheumatology classification of juvenile idiopathic arthritis: second revision, Edmonton, 2001. J Rheumatol 2004;31:390–2. 2. Fujikawa S, Okuni M. Clinical analysis of 570 cases with juvenile rheumatoid arthritis: results of a nationwide retrospective survey in Japan. Acta Paediatr Jpn 1997;39:245–9. 3. Sawhney S, Magalhaes CS. Paediatric rheumatology: a global perspective. Best Pract Res Clin Rheumatol 2006;20:201–21. 4. Bleesing J, Prada A, Siegel DM, Villanueva J, Olson J, Ilowite NT, et al. The diagnostic significance of soluble CD163 and soluble interleukin-2 receptor ␣-chain in macrophage activation syndrome and untreated new-onset systemic juvenile idiopathic arthritis. Arthritis Rheum 2007;56:965–71. 5. Fife MS, Gutierrez A, Ogilvie EM, Stock CJ, Samuel JM, Thomson W, et al. Novel IL10 gene family associations with systemic juvenile idiopathic arthritis. Arthritis Res Ther 2006;8:R148. 6. Masters SL, Simon A, Aksentijevich I, Kastner DL. Horror autoinflammaticus: the molecular pathophysiology of autoinflammatory disease. Annu Rev Immunol 2009;27:621–68. 7. Moller JC, Paul D, Ganser G, Range U, Gahr M, Kelsch R, et al. IL10 promoter polymorphisms are associated with systemic onset juvenile idiopathic arthritis (SoJIA). Clin Exp Rheumatol 2010;28: 912–8. 8. Nejentsev S, Howson JM, Walker NM, Szeszko J, Field SF, Stevens HE, et al. Localization of type 1 diabetes susceptibility to the MHC class I genes HLA-B and HLA-A. Nature 2007;450: 887–92. 9. Raychaudhuri S, Sandor C, Stahl EA, Freudenberg J, Lee HS, Jia X, et al. Five amino acids in three HLA proteins explain most of the association between MHC and seropositive rheumatoid arthritis. Nat Genet 2012;44:291–6. 10. McDermott MF, Aksentijevich I, Galon J, McDermott EM, Ogunkolade BW, Centola M, et al. Germline mutations in the extracellular domains of the 55 kDa TNF receptor, TNFR1, define
18.
19.
20.
21.
22.
23. 24.
25.
26.
27.
28.
a family of dominantly inherited autoinflammatory syndromes. Cell 1999;97:133–44. International FMF Consortium. Ancient missense mutations in a new member of the RoRet gene family are likely to cause familial Mediterranean fever. Cell 1997;90:797–807. French FMF Consortium. A candidate gene for familial Mediterranean fever. Nat Genet 1997;17:25–31. Aksentijevich I, Nowak M, Mallah M, Chae JJ, Watford WT, Hofmann SR, et al. De novo CIAS1 mutations, cytokine activation, and evidence for genetic heterogeneity in patients with neonatal-onset multisystem inflammatory disease (NOMID): a new member of the expanding family of pyrin-associated autoinflammatory diseases. Arthritis Rheum 2002;46:3340–8. Date Y, Seki N, Kamizono S, Higuchi T, Hirata T, Miyata K, et al. Identification of a genetic risk factor for systemic juvenile rheumatoid arthritis in the 5⬘-flanking region of the TNF␣ gene and HLA genes. Arthritis Rheum 1999;42:2577–82. Fishman D, Faulds G, Jeffery R, Mohamed-Ali V, Yudkin JS, Humphries S, et al. The effect of novel polymorphisms in the interleukin-6 (IL-6) gene on IL-6 transcription and plasma IL-6 levels, and an association with systemic-onset juvenile chronic arthritis. J Clin Invest 1998;102:1369–76. Donn RP, Shelley E, Ollier WE, Thomson W, and the British Paediatric Rheumatology Study Group. A novel 5⬘-flanking region polymorphism of macrophage migration inhibitory factor is associated with systemic-onset juvenile idiopathic arthritis. Arthritis Rheum 2001;44:1782–5. Stock CJ, Ogilvie EM, Samuel JM, Fife M, Lewis CM, Woo P. Comprehensive association study of genetic variants in the IL-1 gene family in systemic juvenile idiopathic arthritis. Genes Immun 2008;9:349–57. Lamb R, Thomson W, British Society of Paediatric and Adolescent Rheumatology, Ogilvie EM, Donn R. Positive association of SLC26A2 gene polymorphisms with susceptibility to systemiconset juvenile idiopathic arthritis. Arthritis Rheum 2007;56: 1286–91. Gattorno M, Piccini A, Lasiglie D, Tassi S, Brisca G, Carta S, et al. The pattern of response to anti–interleukin-1 treatment distinguishes two subsets of patients with systemic-onset juvenile idiopathic arthritis. Arthritis Rheum 2008;58:1505–15. Day TG, Ramanan AV, Hinks A, Lamb R, Packham J, Wise C, et al. Autoinflammatory genes and susceptibility to psoriatic juvenile idiopathic arthritis. Arthritis Rheum 2008;58:2142–6. Ayaz NA, Ozen S, Bilginer Y, Erguven M, Taskiran E, Yilmaz E, et al. MEFV mutations in systemic onset juvenile idiopathic arthritis. Rheumatology (Oxford) 2009;48:23–5. Rigante D, Cantarini L. The systemic-onset variant of juvenile idiopathic arthritis needs to be recorded as an autoinflammatory syndrome: comment on the review by Nigrovic [letter]. Arthritis Rheumatol 2014;66:2645. Nigrovic PA. Reply [letter]. Arthritis Rheumatol 2014;66:2645–6. Ombrello MJ, Remmers EF, Tachmazidou I, Grom AA, Foll D, Martini A, et al. Genome wide association meta-analysis implicates HLA-DRB1, the BTNL2/HLA-DRA region, and a novel susceptibility locus on chromosome 1 in systemic juvenile idiopathic arthritis [abstract]. Arthritis Rheum 2013;65 Suppl:S937. Fink CW. Proposal for the development of classification criteria for idiopathic arthritides of childhood. J Rheumatol 1995;22: 1566–9. Petty RE, Southwood TR, Baum J, Bhettay E, Glass DN, Manners P, et al. Revision of the proposed classification criteria for juvenile idiopathic arthritis: Durban, 1997. J Rheumatol 1998;25:1991–4. Singh G, Athreya BH, Fries JF, Goldsmith DP. Measurement of health status in children with juvenile rheumatoid arthritis. Arthritis Rheum 1994;37:1761–9. Mellins ED, Macaubas C, Grom AA. Pathogenesis of systemic
MONOGENIC SYSTEMIC JUVENILE IDIOPATHIC ARTHRITIS
29.
30.
31.
32.
33.
34. 35.
36.
juvenile idiopathic arthritis: some answers, more questions. Nat Rev Rheumatol 2011;7:416–26. Griffin TA, Barnes MG, Ilowite NT, Olson JC, Sherry DD, Gottlieb BS, et al. Gene expression signatures in polyarticular juvenile idiopathic arthritis demonstrate disease heterogeneity and offer a molecular classification of disease subsets. Arthritis Rheum 2009;60:2113–23. Ogilvie EM, Khan A, Hubank M, Kellam P, Woo P. Specific gene expression profiles in systemic juvenile idiopathic arthritis. Arthritis Rheum 2007;56:1954–65. Pascual V, Allantaz F, Arce E, Punaro M, Banchereau J. Role of interleukin-1 (IL-1) in the pathogenesis of systemic onset juvenile idiopathic arthritis and clinical response to IL-1 blockade. J Exp Med 2005;201:1479–86. Grom AA. Natural killer cell dysfunction: a common pathway in systemic-onset juvenile rheumatoid arthritis, macrophage activation syndrome, and hemophagocytic lymphohistiocytosis? [review]. Arthritis Rheum 2004;50:689–98. Sakurai T, Kataoka K. Basic and applied features of multicopper oxidases, CueO, bilirubin oxidase, and laccase. Chem Rec 2007;7: 220–9. Baldrian P. Fungal laccases: occurrence and properties. FEMS Microbiol Rev 2006;30:215–42. Liu H, Irwanto A, Tian H, Fu X, Yu Y, Yu G, et al. Identification of IL18RAP/IL18R1 and IL12B as leprosy risk genes demonstrates shared pathogenesis between inflammation and infectious diseases. Am J Hum Genet 2012;91:935–41. Zhang F, Liu H, Chen S, Low H, Sun L, Cui Y, et al. Identification
295
37. 38.
39. 40.
41. 42. 43.
44.
of two new loci at IL23R and RAB32 that influence susceptibility to leprosy. Nat Genet 2011;43:1247–51. Barrett JC, Hansoul S, Nicolae DL, Cho JH, Duerr RH, Rioux JD, et al. Genome-wide association defines more than 30 distinct susceptibility loci for Crohn’s disease. Nat Genet 2008;40:955–62. Anderson CA, Boucher G, Lees CW, Franke A, D’Amato M, Taylor KD, et al. Meta-analysis identifies 29 additional ulcerative colitis risk loci, increasing the number of confirmed associations to 47. Nat Genet 2011;43:246–52. Nair RP, Duffin KC, Helms C, Ding J, Stuart PE, Goldgar D, et al. Genome-wide scan reveals association of psoriasis with IL-23 and NF-B pathways. Nat Genet 2009;41:199–204. Evans DM, Spencer CC, Pointon JJ, Su Z, Harvey D, Kochan G, et al. Interaction between ERAP1 and HLA-B27 in ankylosing spondylitis implicates peptide handling in the mechanism for HLA-B27 in disease susceptibility. Nat Genet 2011;43:761–7. Zhang FR, Huang W, Chen SM, Sun LD, Liu H, Li Y, et al. Genomewide association study of leprosy. N Engl J Med 2009;361: 2609–18. Schurr E, Gros P. A common genetic fingerprint in leprosy and Crohn’s disease? N Engl J Med 2009;361:2666–8. Salvarani C, Vlachonikolis IG, van der Heijde DM, Fornaciari G, Macchioni P, Beltrami M, et al. Musculoskeletal manifestations in a population-based cohort of inflammatory bowel disease patients. Scand J Gastroenterol 2001;36:1307–13. Marks DJ, Miyagi K, Rahman FZ, Novelli M, Bloom SL, Segal AW. Inflammatory bowel disease in CGD reproduces the clinicopathological features of Crohn’s disease. Am J Gastroenterol 2009;104:117–24.
