Alessandra Gava

Blau syndrome, clinical and genetic aspects

Blau syndrome (BS) is a rare autosomal dominant, autoinflammatory syndrome characterized by the clinical triad of granulomatous recurrent uveitis, dermatitis and symmetric arthritis. The gene responsible for BS has been identified in the caspase recruitment domain gene CARD15/NOD2. In the majority of patients, the disease is characterized by early onset, usually before 3-4years of age. The manifestations at disease onset are usually represented by articular and cutaneous involvement signs, generally followed later by ocular manifestations which are often the most relevant morbidity of BS. In some cases the presence of fever is also observed; atypical cases of BS have been reported with cardiovascular, neurological, renal, intestinal and other organ involvement. The rarity and the variations in the severity and evolution of its expressions do not permit sufficient data about optimal treatment for patients with BS. The first step of therapy is represented by the use of corticosteroids and successively, in case of unsatisfactory response, by additional treatment with immunosuppressive agents. The results with biologic anti-cytokine agents, such as anti-TNFα and anti-IL1β, are different, particularly with regard to ocular morbidity. Clinical and genetic aspects of the familial and the sporadic form of BS will be discussed and focused on. A description of a case study of an Italian family is also included.

Miscellaneous non-inflammatory musculoskeletal conditions. Blau syndrome.

Blau syndrome (BS) is a rare dominantly inherited, inflammatory syndrome characterised by the clinical triad of granulomatous dermatitis, symmetric arthritis and recurrent uveitis. The caspase recruitment domain gene CARD15/NOD2 has been identified as the gene responsible for BS. In the majority of patients, the disease is characterised by early onset, usually before 3-4 years of age. Onset is most often articular and cutaneous. Eye symptoms usually start later; however, eye involvement is the most relevant morbidity of BS. Atypical cases of BS have been reported with involvement of organs other than skin, joint and eyes. Due to its rarity and the variations in the severity and evolution of its expressions, there have been no studies on the optimal treatment for patients with BS. If the therapeutic response to corticosteroids is unsatisfactory, additional treatment with immunosuppressive agents should be tried. The results with biologic anti-cytokine agents, such as infliximab and anakinra, are variable, particularly with regard to ocular morbidity. This review will focus on the clinical and genetics aspects of the familial and the sporadic form of BS. Further, we will describe an Italian family followed by us over the past 25 years.

Ex vivo and in vitro production of pro-inflammatory cytokines in Blau syndrome

The objective was to study both ex vivo and in vitro secretion of pro-inflammatory cytokines in patients affected by Blau syndrome (BS) and carrying p.E383K mutation in the CARD15/NOD2 gene associated with the disease. For ex vivo studies, peripheral blood mononuclear cells (PBMCs), serum from three patients and healthy controls have been collected. PBMCs have been cultured in the presence or absence of inflammatory enhancers, such as lipopolysaccharide (LPS) and muramyl dipeptide (MDP). The levels of interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α and interferon (IFN)-γ were assayed by either immunoassay or array-based system. For in vitro studies, different constructs were created cloning human wild-type and p.E383K-mutated NOD2 cDNA into the expression vector pCMV-Tag2c. HEK293 cell lines were stably transfected, cultured with or without MDP and IL-8 level was assayed in their surnatants. Statistical analysis in both studies was performed using non-parametric tests. Both ex vivo and in vitro studies have not identified a significant increase in secretion of the analyzed proinflammatory cytokines. p.E383K-mutated NOD2 transfected cells express low level of IL-8. The ex vivo basal level results from both serum and PBMCs surnatants present similar levels of IL-1β, IL-6, TNF-α and IFN-γ in patients and controls. The presence of the stimulant agents (LPS and MDP), either individual or paired, does not lead to significant increases in all cytokines concentrations in patients compared to controls. Taken together, the ex vivo and in vitro data suggest that there is not a primary mediation of IL-1β and other pro-inflammatory cytokines in BS patients carrying p.E383K.

AB0012 Functional Characterization of P.E383k Blau Syndrome-Related Mutation

Background The Blau syndrome (BS) is a rare autosomal dominant autoinflammatory disease clinically characterized by symmetrical arthritis, granulomatous dermatitis and recurrent uveitis. The disease is caused by single mutations in CARD15/NOD2, encoding the NOD2 protein that is known to regulate the defense against pathogens by activating the NF-κB signalling pathway (1). Objectives In vitro and ex vivo functional analysis aimed at characterizing p.E383K mutation found in an Italian family affected by Blau syndrome. Methods For ex vivo studies, peripheral blood mononuclear cells (PBMC) are collected from patients carrying p.E383K mutation, then lysed and analyzed by a protein microarray technique (RPPA) to determine the NF-κB activity. IL1β, IL6, IL8, TNFα, IFNγ, IL12, IL17, IL22, IL23 releases are quantified by ELISA and antibody microarray techniques in PBMC cultured for 7 hours in presence or absence of lipopolysaccharide (LPS) and muramyldipeptide (MDP). In vitro analysis started from the creation and stably transfection of constructs containing human NOD2 cDNA wild-type and mutated p.E383K into HEK293 cells. The cells were cultured for 7 and 24 hours with or without MDP. NF-κB activity was determined by Western blot, evaluating the expression of IKBα and its phosphorylated form in cellular lysates, and also by RPPA. IL8 was assayed in the supernatants of the reported cell cultures through antibody microarray technique. The significance of all the data was tested using non-parametric analysis. Results Confocal microscopy revealed that p.E383K NOD2 resides in cytoplasm of transfected HEK293 cells, as well as wild-type. The expression of p.E383K NOD2, assessed by Western blot, did not present any variations after MDP stimulation. Regarding the NF-κB activity, both in vitro RPPA and Western blot studies showed an inactivation of this pathway, presenting lower expression of phosphorylated IKBα than IKBα in presence or absence of MDP stimulation (p<0.05). However, all the pathway components (NF-κB, IKBα and IKKα/β and their phosphorylated forms) were found upregulated ex vivo compared with controls (p<0.05). analysis Concerning the cytokines, both in vitro and ex vivo studies have not identified a significant increase secretion of most of the proinflammatory cytokines analyzed. IL8 showed both in vitro and ex vivo a significant decrease (p<0.05) compared to controls. IL17, IL22 and IL23 release ex vivo was higher in patients than in controls (p<0.05 p<0.001 and p<0.005 respectively), while IL12 is reduced in patients (p<0.05). After stimulation (LPS, MDP or both), IL1β, IL6, IL8, TNFα and IFNγ production was not augmented in patients compared to controls. Conclusions The contrasting results presented by our in vitro and ex vivo data on NF-κB pathway may indicate that the activation do not depend only on NOD2 in carriers of p.E383K mutation. Further experiments may clarify these results, using also different in vitro models, such as THP1 cells. Regarding the cytokines profile, it seems that there is not a primary mediation of IL1β and other pro-inflammatory cytokines in patients presenting p.E383K mutation, as reported in literature for p.R334W/Q carriers (2), with the exception of IL17/22/23 whose role has to be deeply investigated. References Punzi L et al. Best Pract Res Clin Rheumatol 2011;25:703-14 Martin TM et al. Arthritis Rheum 2009;60:611-8 Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.2906

A Novel Mutation (Ser59Pro) in the TNFRSF1A Gene Associated with Adult- Onset Sporadic Traps

Background: TRAPS is an autosomal dominant autoinflammatory disease, caused by mutations in the TNFRSF1A gene. To date 68 variants have been identified. Attacks are associated with fever, abdominal pain, myalgias and skin rash. Long term inflammatory response can lead to AA amyloidosis. Genetic testing confirms the diagnosis. We herein report an apparently sporadic case of TRAPS manifesting after the age of 40 years and associated with a novel mutation in exon 3 of the TNFRSF1A gene. Methods: A 69 ys old man was evaluated for a chronic inflammatory characterized by fever, leukocytosis, refractory iron-deficiency anaemia, recurrent bronchopneumonias, palpebral ptosis, episodes of arthralgia, myalgia, cutaneous erythematosus lesions migrant and intermittent to limbs and trunk, and one episode of pericarditis. Serum IgD, IgA and SAA, along with the main acute phase protein response, were elevated. Since the clinical aspects were suggestive for an autoinflammatory syndrome, we decided to investigate mutations involved in TRAPS, MKD and cryopyrin-associated periodic syndromes (CAPS). Genomic DNA was isolated from peripheral blood lymphocytes by standard methods. Sequencing of MVK, TNFRSF1A and NLRP3 genes was performed by means of an automatic sequencer. Congo Red Dye Test was made for amyloid deposits research. The technique involves sterilizing and anesthetizing a small area of the anterior abdominal wall. Subcutaneous fat is then aspirated into a syringe. The small bits of fat obtained are crushed under a glass slide and stained with Congo red. Positive staining with the Congo red stain is indicated by apple-green birefringence utilizing a polarized microscope. Results: Genetic testing was negative for MVK and NLRP3 mutations. Sequencing of the TNFRSF1A gene showed the presence of an heterozygous single-base mutation (c.262T>C) in exon 3, resulting in a Pro for Ser amino acid substitution at residue 59 of the mature protein (S59P), which has not been reported before in any TRAPS patient. Moreover, this mutation was not identified in 200 control chromosomes. Subcutaneous fat aspirate was negative for amyloid deposits. Conclusion: The new S59P variant is located in a region of the receptor that is crucial for its function and is predicted to result in a protein conformational change: we are developing functional tests in order to assess this aspect. Overall, this mutation is likely to be responsible for our patient’s disease. More studies should be made in order to investigate also the concentration of the soluble receptor of TNF. At present, the disease is controlled with steroids.