Response to anakinra in a de novo case of neonatal-onset multisystem inflammatory disease

ARTHRITIS & RHEUMATISM Vol. 50, No. 8, August 2004, pp 2706–2709 © 2004, American College of Rheumatology

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ranean European Caucasian), and same origin (Spanish grandparents). The presence of RA or other autoimmune diseases in the controls or their first-order relatives (parents, siblings, or descendants) was treated as a potential confounder and was a criterion for exclusion from the study. After application of the inclusion and exclusion criteria, the final number of “hypernormal” controls (i.e., individuals who were demographically similar to the patients, but without RA or any other autoimmune disease in the individual or a first-order relative), was 178. Genotyping of samples was performed as previously described (13). Allele frequencies for CRHRA1 and CRHRA2 were calculated (Table 1), and Hardy-Weinberg equilibrium and linkage disequilibrium between the two loci were evaluated using the ARLEQUIN program (19). For each marker, statistically significant differences between cases and controls were analyzed by the CLUMP program (20), which uses a Monte Carlo approach to assess the significance of the difference in allele frequencies between cases and controls when loci with multiple alleles are studied. Haplotype case–control studies were performed using the FASTEHPLUS program (21), which this is based on the EH program and performs model-free analysis and permutation tests of allelic association (22). Power estimates were performed with the Genetic Power Calculator (http://www.statgen.iop.kcl.ac.uk), using the following parameters: disease prevalence 0.005, D⬘ between disease and marker alleles 0.8, 2-fold increased risk of disease in heterozygotes under a multiplicative model, ␣ ⫽ 0.05. We estimate that with a sample of the size used in this study, there is 94% power to detect association when disease and marker allele frequency (A) ⫽ 0.25, 90% power when A ⫽ 0.15, and 78% power when A ⫽ 0.1. Control and RA samples were in Hardy-Weinberg equilibrium for both of the markers (P ⬎ 0.05; 10,000 dememorization steps), and there was significant linkage disequilibrium between them (P ⬍ 0.0001). CLUMP analysis revealed that neither CRHRA1 nor CRHRA2 showed significant allele frequency differences between the cases and the controls (Table 1). Extended analysis of pairs of allelic combinations from the two sites near the CRH gene by the FASTEHPLUS program, did not improve the significance (data not shown). Results of the present study reinforce our previous findings of a lack of association between the CRH locus and RA. To our knowledge, this is the first published report of a population-based association study with the microsatellites CRHRA1 and CRHRA2. Over the last decade, family-based association studies have become popular in the study of complex diseases because they are robust to population subdivision and admixture (14). However, very few reports have described confounding by population stratification, and recent work with experimental data suggests that it is not a substantial source of bias (23). Furthermore, Ardlie et al point out that with proper study design, population stratification could be a negligible factor in investigations of US and European populations (24). For the above reasons, the shift toward the less powerful but more robust family-based association studies in the study of complex diseases (such as RA) in large populations (e.g., English and Spanish populations) does not seem justi-

DOI 10.1002/art.20377

Lack of association between the corticotropin-releasing hormone locus and rheumatoid arthritis Rheumatoid arthritis (RA) is a systemic autoimmune disease with an estimated prevalence of ⬃0.5% in the Spanish population (1). As in most complex diseases, the genetic component for host susceptibility is still unknown. Genomewide linkage studies have proven unable to pinpoint with enough confidence genomic regions other than HLA, thus leaving the main portion of the genetic influence unidentified (2–5). For this reason, the “positional candidate” strategy has been regarded as the dominant approach to investigate genetic associations with RA. Understanding of the interactions between neuroendocrine and immune-mediated inflammatory reactions has improved enormously in recent years. In particular, there is mounting evidence that corticotropin-releasing hormone (CRH), through its role as central regulator of the hypothalamic–pituitary–adrenal axis, could be crucial in the onset of RA (6,7). Single-nucleotide polymorphisms in the promoter region have been studied in relation to RA, but the results have not been conclusive (8–10). Two recent studies from the same group have shown nominal linkage and positive association of the locus with the disease, by means of family-based association studies and the discovery of the highly polymorphic markers CRHRA1 and CRHRA2 (25 kb and 20 kb downstream of the CRH coding sequences, respectively) (11,12). We performed the first study designed to replicate these results in a different population (121 simplex Spanish families), using the transmission disequilibrium test (TDT), but no significant association was found (13). Family-based association studies are less powerful than traditional case–control studies, but their robustness to population stratification has made them very popular in the last decade (14). However, case–control analysis methods and strategies that allow population stratification have recently been developed (15–17). The aim of the present investigation was to clarify the effect of the CRH locus in RA by performing the first population association study of the markers CRHRA1 and CRHRA2 in a study with a relatively high number of RA cases and the use of the “hypernormal control group” strategy proposed by Morton and Collins (15). We recruited a group of 257 patients (the 121 probands from the original data set plus 136 new patients) who fulfilled the American College of Rheumatology (formerly, the American Rheumatism Association) 1987 criteria for RA (18). There were 198 women (77%) and 59 men. The mean ⫾ SD age of the patients was 69 ⫾ 16.6 years, and the mean ⫾ SD disease duration was 17 ⫾ 9.3 years. Sixty-eight percent of the patients were positive for rheumatoid factor, 76% had erosive lesions, and 22% had nodules. From a well-characterized control group of 409 individuals referred from the HOMFOL group (Hospital Universitari de San Joan, Reus Spain), we selected only those who were ⬎39 years old (age at which risk for RA is increased) at the time of the clinical interview. From these, we subsequently applied the next selection criteria: same geographic area as the cases (Spain), same ethnicity (Mediter2706

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Table 1. Analysis of allele frequency differences between rheumatoid arthritis cases (n ⫽ 257) and “hypernormal” controls (n ⫽ 178)* CRHRA1

CRHRA2

Allele

Cases

Controls

Cases

Controls

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 P†

0 (0.00) 1 (0.19) 0 (0.00) 0 (0.00) 15 (2.92) 52 (10.12) 129 (25.10) 264 (51.36) 35 (6.81) 9 (1.75) 7 (1.36) 1 (0.19) 0 (0.00) 0 (0.00) 0 (0.00) 1 (0.19)

2 (0.56) 2 (0.56) 0 (0.00) 1 (0.28) 10 (2.81) 30 (8.43) 96 (26.97) 173 (48.60) 30 (8.43) 8 (2.25) 3 (0.84) 1 (0.28) 0 (0.00) 0 (0.00) 0 (0.00) 0 (0.00)

19 (3.70) 1 (0.19) 1 (0.19) 2 (0.39) 1 (0.19) 1 (0.19) 9 (1.75) 51 (9.92) 128 (24.90) 80 (15.56) 87 (16.93) 65 (12.65) 46 (8.95) 14 (2.72) 7 (1.36) 2 (0.39)

20 (5.62) 0 (0.00) 2 (0.56) 1 (0.28) 1 (0.28) 2 (0.56) 5 (1.40) 30 (8.43) 87 (24.44) 50 (14.04) 61 (17.13) 49 (13.76) 33 (9.27) 10 (2.81) 3 (0.84) 2 (0.56)

0.8613

0.9302

* Values are the number (%). † Data were analyzed with the CLUMP program (version 2.2). P values are based on the T4 statistic after 100,000 simulations (random number seed ⫽ 2,000) (20).

fied. In addition, a number of strategies that maximize population-based association studies, either by circumventing the stratification problem or by increasing analytic efficiency, have recently been proposed (15–17). In particular, the “extreme discordance” strategy proposed by Morton and Collins (15) should give our study at least a 6-fold efficiency against TDT trios based on the null hypothesis. We therefore started by selecting an age-of-RA-risk group of Caucasian controls who were of Spanish origin, going back at least 2-generations. Discordance between case and control samples was then increased by excluding from the control group all individuals with RA or another autoimmune disease. We further increased the “hypernormality” of the control group by also excluding those individuals who had a first-order relative with RA or another autoimmune disease. Through the use of this selective strategy, our study should have the highest statistical power attainable in this type of investigation. Based on our data, we conclude that the CRH locus is not associated with RA in the Spanish population, although the possibility of a very weak effect cannot be definitively excluded. This is the first population-based association study of the CRH locus and RA using the markers CRHRA1 and CRHRA2. By using controls with the lowest liability for development of RA we maximized the power obtained from standard estimators. Most association studies are performed with small-to-moderate–sized single samples, thus making replication in different populations necessary (25). If the absence of association we report here were to be replicated in other populations, there would be good reason to dismiss the initially reported association (26). Therefore, we encourage groups working with different populations to perform similar studies

in order to help clarify the conflicting results, or even to conduct meta-analyses of the existing data. Supported by the Societat Catalana de Reumatologia. Antonio Julia` Hospital General i Universita Vall d’Hebron Dominique Gallardo, PhD Francisco Vidal, PhD Centre de Transfusions i Banc de Teixits Carles Toma`s, MD Pere Barcelo ´, MD Miquel Vilardell, MD, PhD Sara Marsal, MD, PhD Hospital General i Universitari Vall d’Hebron Barcelona, Spain 1. Carmona L, Villaverde V, Hernandez-Garcia C, Ballina J, Gabriel R, Laffon A. The prevalence of rheumatoid arthritis in the general population of Spain. Rheumatology (Oxford) 2002;41:88–95. 2. Shiozawa S, Hayashi S, Tsukamoto Y, Goko H, Kawasaki H, Wada T, et al. Identification of the gene loci that predispose to rheumatoid arthritis. Int Immunol 1998;10:1891–5. 3. Cornelis F, Faure S, Martinez M, Prudhomme JF, Fritz P, Dib C, et al. New susceptibility locus for rheumatoid arthritis suggested by a genome-wide linkage study. Proc Natl Acad Sci U S A 1998;95: 10746–50. 4. Jawaheer D, Seldin ME, Amos CI, Chen WV, Shigeta R, Monteiro J, et al. A genomewide screen in multiplex rheumatoid arthritis families suggests genetic overlap with other autoimmune diseases. Am J Hum Genet 2001;68:927–36. 5. MacKay K, Eyre S, Myerscough A, Milicic A, Barton A, Laval S, et al. Whole-genome linkage analysis of rheumatoid arthritis susceptibility loci in 252 affected sibling pairs in the United Kingdom. Arthritis Rheum 2002;46:632–9.

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6. Sternberg EM, Young WS III, Bernardini R, Calogero AE, Chrousos GP, Gold PW, et al. A central nervous system defect in biosynthesis of corticotropin-releasing hormone is associated with susceptibility to streptococcal cell wall-induced arthritis in Lewis rats. Proc Natl Acad Sci U S A 1989;86:4771–5. 7. Chrousos GP. The hypothalamic-pituitary-adrenal axis and immune-mediated inflammation. N Engl J Med 1995;332:1351–62. 8. Baerwald CG, Panayi GS, Lanchbury JS. Corticotropin releasing hormone promoter region polymorphisms in rheumatoid arthritis. J Rheumatol 1997;24:215–6. 9. Baerwald CG, Mok CC, Tickly M, Lau CS, Wordsworth BP, Ollier B, et al. Corticotropin releasing hormone (CRH) promoter polymorphisms in various ethnic groups of patients with rheumatoid arthritis. Z Rheumatol 2000;59:29–34. 10. Gonzalez-Gay MA, Hajeer AH, Garcia-Porrua C, Dababneh A, Amoli MM, Botana MA, et al. Corticotropin-releasing hormone promoter polymorphisms in patients with rheumatoid arthritis from northwest Spain. J Rheumatol 2003;30:913–7. 11. Fife MS, Fisher SA, John S, Worthington J, Shah CJ, Ollier WE, et al. Multipoint linkage analysis of a candidate gene locus in rheumatoid arthritis demonstrates significant evidence of linkage and association with the corticotropin-releasing hormone genomic region. Arthritis Rheum 2000;43:1673–8. 12. Fife M, Steer S, Fisher S, Newton J, McKay K, Worthington J, et al. Association of familial and sporadic rheumatoid arthritis with a single corticotropin-releasing hormone genomic region (8q12.3) haplotype. Arthritis Rheum 2002;46:75–82. 13. Julia A, Gallardo D, Vidal F, de Agustin JJ, Barcelo P, Villardell M, et al. Association study between corticotrophin-releasing hormone genomic region (8q13) and rheumatoid arthritis in the Spanish population. Rheumatology (Oxford) 2003;42:1534–8. 14. Spielman RS, McGinnis RE, Ewens WJ. Transmission test for linkage disequilibrium: the insulin gene region and insulin-dependent diabetes mellitus (IDDM). Am J Hum Genet 1993;52: 506–16. 15. Morton NE, Collins A. Tests and estimates of allelic association in complex inheritance. Proc Natl Acad Sci U S A 1998;95:11389–93. 16. Pritchard JK, Rosenberg NA. Use of unlinked genetic markers to detect population stratification in association studies. Am J Hum Genet 1999;65:220–8. 17. Devlin B, Roeder K. Genomic control for association studies. Biometrics 1999;55:997–1004. 18. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988;31:315–24. 19. Schneider S, Roessli D, Excoffier L. ARLEQUIN, software for population genetics data analysis: version 2.000. Geneva; 2000. URL: http://lgb.unige.ch/arlequin/software/2.000/doc/faq/faqlist.htm. 20. Sham PC, Curtis D. Monte Carlo tests for associations between disease and alleles at highly polymorphic loci. Ann Hum Genet 1995;59:97–105. 21. Zhao JH, Sham PC. Faster haplotype frequency estimation using unrelated subjects. Hum Hered 2002;53:36–41. 22. Xie X, Ott J. Testing linkage disequilibrium between a disease gene and marker loci. Am J Hum Genet 1993;53:1107. 23. Wacholder S, Rothman N, Caporaso N. Counterpoint: bias from population stratification is not a major threat to the validity of conclusions from epidemiological studies of common polymorphisms and cancer. Cancer Epidemiol Biomarkers Prev 2002;11: 513–20. 24. Ardlie KG, Lunetta KL, Seielstad M. Testing for population subdivision and association in four case-control studies. Am J Hum Genet 2002;71:304–11. 25. Cardon LR, Bell JI. Association study designs for complex diseases. Nat Rev Genet 2001;2:91–9. 26. Freely associating [editorial]. Nat Genet 1999;22:1–2.

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DOI 10.1002/art.20357

Response to anakinra in a de novo case of neonatal-onset multisystem inflammatory disease Neonatal-onset multisystem inflammatory disease (NOMID), which in Europe is called chronic infantile neurologic, cutaneous, articular syndrome (CINCA syndrome), is a severe and hitherto poorly treatable autoinflammatory disease. It is caused by mutations in the gene known as NALP3 or CIAS1, which encodes a member of the pyrin superfamily of death domain fold proteins that are involved in inflammation and apoptosis (1–3). Manifestations of NOMID/CINCA syndrome include arthropathy, conjunctivitis, an urticariaappearing skin rash, chronic sterile meningitis, progressive visual and severe auditory defects, and abnormal growth and neurologic development (1). Patients typically have an intense acute-phase response, and some develop AA amyloidosis. The disease responds only modestly to high-dose corticosteroids and other antiinflammatory and immunosuppressive therapies. Herein we describe a patient with NOMID/CINCA syndrome associated with G571R, a de novo variant of NALP3, whose inflammatory disease remitted rapidly and completely following treatment with recombinant interleukin-1 receptor antagonist (rIL-1Ra) (anakinra). The patient was a 32-year-old British woman who had developed a generalized erythematous/urticarial rash and conjunctivitis at birth. These symptoms persisted virtually constantly except during brief periods when she received high doses of corticosteroids. She had marked arthralgia most of her life, affecting her ankles, knees, wrists, and elbows, and had mild frontal bossing of the skull and flattening of the nasal bridge characteristic of NOMID/CINCA syndrome. Her growth was restricted, and exogenous estrogen treatment was needed to stimulate secondary sexual development. Hearing difficulties had been evident since she was pre–school age, and she had become profoundly deaf over the last 5 years. Longstanding neurologic features included irritability, headaches, papilledema, and progressive loss of vision consistent with chronic elevated intracranial pressure. She reported severe fatigue and the need for an aftenoon nap most days. Numerous blood tests throughout her life had revealed persistent massive neutrophilia, normocyctic anemia, and an intense acute-phase response. Renal and liver function were preserved, and AA amyloidosis was excluded by serum amyloid P component scintigraphy. Prior to the institution of anakinra treatment, she was being treated with prednisone 5 mg on alternate days and azathioprine 50 mg daily, with little evidence of benefit. Sequencing of the NALP3 gene revealed that the patient was heterozygous for a point mutation encoding the NALP3 variant G571R, which was not present in either parent, consistent with the reported high frequency of de novo mutations underlying NOMID/CINCA syndrome (1). Mutations in this gene were originally found to be responsible for the ostensibly distinct inherited periodic fever syndromes familial cold urticaria/familial cold autoinflammatory syndrome and Muckle-Wells syndrome (4), and we have shown that the latter is highly responsive to treatment with anakinra (5,6). The patient consented to undergo a therapeutic trial of anakinra (Kineret; Amgen) at 100 mg daily (the dosage licensed for treatment of rheumatoid arthritis).

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Investigations performed immediately before anakinra was instituted revealed a hemoglobin level of 10.1 gm/dl, a white blood cell (WBC) count of 29 ⫻ 109/liter (mainly neutrophils), a platelet count of 899 ⫻ 109/liter, reactive polyclonal hyperglobulinemia, and plasma serum amyloid A protein (SAA) and C-reactive protein (CRP) concentrations of 284 mg/liter and 109 mg/liter, respectively (normal ⬍5 for both). Within 24 hours of the initiation of this treatment the rash, conjunctivitis, and arthralgia had disappeared, and the patient reported a steady and dramatic improvement in general well-being thereafter. Her headaches and fatigue ceased, and the intense acute-phase response resolved completely within 5 days of the first injection of anakinra. At review 1 month later, her hemoglobin level was 12.4 gm/dl, the WBC count was 10.7 ⫻ 109/liter, and the platelet count was 366 ⫻ 109/liter. During the next 6 months, the dosage of anakinra was tapered to 33 mg daily, and her other antiinflammatory therapy was discontinued without any recrudescence of the inflammatory disease activity. The median plasma SAA and CRP concentrations, measured every 2 weeks, decreased from 281 mg/liter and 121 mg/liter, respectively, during the 2 months before institution of anakinra treatment to 3.7 mg/liter and 5.5 mg/liter during the 7 months of therapy. Her deafness and visual acuity have not changed. NALP3 is a member of the recently characterized pyrin superfamily of death domain fold proteins which contain a characteristic 6–alpha helix death domain–related structure; now known as the pyrin domain, originally identified as the product of the gene responsible for familial Mediterranean fever (7). The NALP3 gene is expressed in peripheral blood leukocytes and chondrocytes, and the pyrin domain of NALP3 is thought to interact with ASC, leading to signaling of NF-␬B and increased IL-1␤ production (1,3). Up-regulation of IL-1␤ has been observed in unstimulated monocytes obtained from a patient with NOMID/CINCA syndrome (3), and the rapid and complete clinical and serologic responses to rIL-1Ra in the patient described herein and in Muckle-Wells syndrome patients we have treated (5,6) confirm that IL-1␤ has a fundamental role in the pathogenesis of the inflammatory disorders associated with mutations in the NALP3 gene. Interestingly, the dramatic rate of decline in this patient’s plasma SAA concentration following anakinra therapy indicates that SAA must have a circulating half-life of ⬍13 hours, reinforcing its status as the most sensitive and dynamic marker of the acute-phase response. It is notable that as a child, this patient had been thought to have a severe sporadic form of Muckle-Wells

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syndrome, long before the genetic basis of the disease or its relationship with NOMID/CINCA syndrome were known. It has recently become clear that NOMID/CINCA syndrome, Muckle-Wells syndrome, and familial cold urticaria represent a spectrum of overlapping phenotypes that correlate inconsistently with particular mutations among the ⬃20 allelic variants of NALP3 thus far identified. Our findings strongly indicate that trials of anakinra therapy are warranted in patients with all of these disorders and offer hope that early and prolonged treatment with rIL-1Ra might prevent the deafness and abnormal neurologic and skeletal development that occur in children with NOMID/CINCA syndrome. Philip N. Hawkins, PhD, FRCP, FMedSci Alison Bybee, PhD Royal Free and University College Medical School Ebun Aganna, PhD Michael F. McDermott, MRCPI St. Bartholomew’s and the Royal London School of Medicine London, UK 1. Feldmann J, Prieur AM, Quartier P, Berquin P, Certain S, Cortis E, et al. Chronic infantile neurological cutaneous and articular syndrome is caused by mutations in CIAS1, a gene highly expressed in polymorphonuclear cells and chondrocytes. Am J Hum Genet 2002;71:198–203. 2. Dode C, Le Du N, Cuisset L, Letourneur F, Berthelot JM, Vaudour G, et al. New mutations of CIAS1 that are responsible for MuckleWells syndrome and familial cold urticaria: a novel mutation underlies both syndromes. Am J Hum Genet 2002;70:1498–506. 3. 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 neonatalonset multisystem inflammatory disease (NOMID): a new member of the expanding family of pyrin-associated autoinflammatory diseases. Arthritis Rheum 2002;46:3340–8. 4. Hoffman HM, Mueller JL, Broide DH, Wanderer AA, Kolodner RD. Mutation of a new gene encoding a putative pyrin-like protein causes familial cold autoinflammatory syndrome and Muckle-Wells syndrome. Nat Genet 2001;29:301–5. 5. Hawkins PN, Lachmann HJ, McDermott MF. Efficacy of interleukin-1 receptor antagonist in Muckle-Wells syndrome. N Engl J Med 2003;348:2583–4. 6. Hawkins PN, Lachmann HJ, Aganna E, McDermott MF. Spectrum of clinical features in Muckle-Wells syndrome and response to anakinra. Arthritis Rheum 2004;50:607–12. 7. Tschopp J, Martinon F, Burns K. NALPs: a novel protein family involved in inflammation. Nat Rev Mol Cell Biol 2003;4:95–104.