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Objectives To analyse the prevalence of CECR1 mutations in patients diagnosed with early onset livedo reticularis and/or haemorrhagic/ischaemic strokes in the context of inflammation or polyarteritis nodosa (PAN). Forty-eight patients from 43 families were included in the study. Methods Direct sequencing of CECR1 was performed by Sanger analysis. Adenosine deaminase 2 (ADA2) enzymatic activity was analysed in monocyte isolated from patients and healthy controls incubated with adenosine and with or without an ADA1 inhibitor. Results Biallelic homozygous or compound heterozygous CECR1 mutations were detected in 15/48 patients. A heterozygous disease-associated mutation (p.G47V) was observed in two affected brothers. The mean age of onset of the genetically positive patients was 24 months (6 months to 7 years). Ten patients displayed one or more cerebral strokes during their disease course. Low immunoglobulin levels were detected in six patients. Thalidomide and anti-TNF (tumour necrosis factor) blockers were the most effective drugs. Patients without CECR1 mutations had a later age at disease onset, a lower prevalence of neurological and skin manifestations; one of these patients displayed all the clinical features of adenosine deaminase 2deficiency (DADA2) and a defective enzymatic activity suggesting the presence of a missed mutation or a synthesis defect. Conclusions DADA2 accounts for paediatric patients diagnosed with PAN-like disease and strokes and might explain an unrecognised condition in patients followed by adult rheumatologist. Timely diagnosis and treatment with anti-TNF agents are crucial for the prevention of severe complications of the disease. Functional assay to measure ADA2 activity should complement genetic testing in patients with non-confirming genotypes.

Background Autoimmune neutropenia is a chronic reduction of absolute neutrophil below the threshold for age, due to peripheral destruction by specific antibodies (AaN). "Primary autoimmune neutropenia" (pAN) usually appears in early infancy and generally resolve within 18-24 months, while "Secondary autoimmune Neutropenia" (sAN) rises later in life and is usually accompanied by autoimmune features. Very few descriptions on the topic are available in the literature. Aim of the study: to describe from a clinical and immunological point of view a cohort of subjects affected with sAN included registered in the Italian Neutropenia Registry (INR) and to compare these data with those from subjects diagnosed with pAN still in the INR . Patient and methods Subjects with neutropenia and positivity of AaN lasting for more than 2 years in subjects older than 5 years of life ( up to 18) or patients affected with neutropenia (plus AaN) associated with autoimmune features registered in the INR from 2005 to 2018 were considered eligible for the present study. Antibodies against neutrophils were always detected throught indirect test . Results: Data from 40 patients affected with sAN (26 females, 65%) were collected. Median age at diagnosis was of 11.6 years (0-21.558mo) with a median follow up of 17 months (0-159mo). The degree of Neutropenia was mild in 12.5%(5/40), moderate in 35% (14/40) and severe in 52.5% (21/40) Neutropenia was diagnosed by chance in half of the cohort and in only 10% (4/40) (10%) definitively resolved . Neutropenia coexisted with leucopenia in almost all the of subjects (36/40, 88%), and moreover, autoimmune haemolytic anemia and thrombocytopenia was concomitant to neutropenia in 12% (5/40) and 32.5% in (13/40) of cases respectively. As for clinical history, 15% (6/40) subjects (15%) were completely asymptomatic , while the remaining 85% (36/40) (85%) had clinical signs of infections and/or autoimmunity features . Infections were documented in 60% (24/40) (60%) of patients. Severe infections namely sepsis , meningitis, pneumonia, osteomyielitis and broad absesses/flemmons were identified in 21% (5/24) of the group. Other type of infections are listed Figure 1A. Apparently there was no correlation between severity of infections and degree of neutropenia ( p=0.2 Fisher' exact test) Autoimmune features and/or autoimmunity markers were present in 30/40 (75% ) of the entire cohort . Thyroiditis and artralgia/arthritis were the most common signs which accounted both for 17,5% of the total episodes. Positivity of ANA and Direct antibodies test ( DAT) were showed in 42% and 29% of cases respectively. (Fig 1B, 1C) As for immunological characteristic 37% (12/32) of the studied subjects were frankly lymphocytopenic, 19% (6/32) had borderline values , while the remaining 44% (14/32) had normal lymphocytes subclasses. In terms of frequency of sub-classes depletion CD19 + and NK were the most frequently decreased subsets. ( Fig 1D) . Dosage of serum immunoglobulin (IgG, IgM and IgA) were shown for all three classes were abnormal (either increased or diminished) in 74% (28/38) of patients (Fig 1D). Mutation analysis performed by Next Generation Sequencing in 16/40 subjects and pathogenic variants of : TACI, TINF 2, CASP 8, CASP 10, PI3K, CARD 11 genes were identified in 4/16 (25%). As for treatment, in 25 % of cases needed Granulocyte-Colony Stimulating Factor to control infections. Rapamicin, micophenolate mofetil, steroids, intravenous immunoglobulin were necessary in 25% (10/40) of patients cases, being (4 of them were already treated on with G-CSF) Comparison with a group of 236 pAN patients of the INR showed that sAN were older at diagnosis (P Conclusions This registry study describes the clinical phenotype of sAN appearing in childhood and by comparative analysis shows that sAN substantially differs from pAN. sAN indeed appears an epiphenomenon of a complex immunological disturbances rather than a disease itself. Occasionally mutations of genes of immunodeficiency/disimmunity can be demonstrated Disclosures No relevant conflicts of interest to declare.

OBJECTIVES: Systemic auto-inflammatory disorders (SAIDs) are a heterogeneous group of monogenic diseases sharing a primary dysfunction of the innate immune system. More than 50% of patients with SAID does not show any mutation at gene(s) tested because of lack of precise clinical classification criteria and/or incomplete gene screening. To improve the molecular diagnosis and genotype interpretation of SAIDs, we undertook the development of a next-generation sequencing (NGS)-based protocol designed to simultaneous screening of 10 genes. METHODS: Fifty patients with SAID, already genotyped for the respective causative gene(s), were massively sequenced for the coding portions of MEFV, MVK, TNFRSF1A, NLRP3, NLRP12, NOD2, PSTPIP1, IL1RN, LPIN2 and PSMB8. Three different bioinformatic pipelines (Ion Reporter, CLC Bio Genomics Workbench, GATK-based in-house workflow) were compared. RESULTS: Once resulting variants were compared with the expected mutation list, no workflow turned out to be able to detect all the 79 variants known in the 50 DNAs. Additional variants were also detected, validated by Sanger sequencing and compared to assess true and false positive detection rates of the three workflows. Finally, the overall clinical picture of 34 patients was re-evaluated in the light of the new mutations found. CONCLUSIONS: The present gene panel has resulted suitable for molecular diagnosis of SAIDs. Moreover, genotype-phenotype correlation has confirmed that the interpretation of NGS data in patients with an undefined inflammatory phenotype is remarkably difficult, thus supporting the need of evidence-based and validated clinical criteria to be used concurrently with the genetic analysis for the final diagnosis and classification of patients with SAIDs. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

INTRODUCTION: Autoimmune lymphoproliferative syndrome (ALPS) is a rare congenital disorder characterized by an impaired FAS-mediated apoptosis that leads to chronic benign lymphoproliferation and autoimmunity. In most cases mutation on FAS gene are responsible of the disease and the phenotype of individuals carrying the same variants can vary from asymptomatic/mild forms to severe disease, due to incomplete penetrance of pathogenic mutations. More rarely, other genes involved in this pathway, such as CASP10, are mutated. Few clinical and molecular data have been reported on small numbers of patients carrying CASP10 mutation showing that different genetic variations can produce contrasting phenotypic effects. So far, 2 mutations have been recognized as pathogenic (I406L and L258F) and other have been reported with controversial result on their pathogenicity (V410l, Y446C) or are known to be polymorphic variants (L522l) AIMS: The aim of this study is to evaluate the FAS-mediated apoptosis function in patients with ALPS or ALPS-like symptoms carrying mutations on CASP10 gene. METHODS: We evaluated FAS-mediated apoptosis pathway in all patients presenting with an ALPS/ALPS-like phenotype that were found to carry a CASP10 mutation in our Institution. Molecular findings were obtained by NGS analysis of a panel of genes involved in the most common immune-dysregulation syndromes and immune-deficiencies and were confirmed by Sanger sequencing. Functional studies were performed by Western blot analysis of CASP10, CASP8, and PARP proteins after TRIAL-induced apoptosis stimulation. Healthy individuals were used as control. RESULTS: We identified 6 patients with ALPS (2) or ALPS-like (4) phenotype, carrying the following heterozygous CASP10 mutations: I406L (1), V410l (2), Y446C (1) L522l (2). Western blot analysis showed an impaired activation of CASP10, CASP8, and PARP proteins in all cases compared to healthy control (Fig. 1) CONCLUSIONS: In our symptomatic patients, the CASP10 polymorphic variant L522l and other mutations whose pathogenicity is controversial (V410l, Y446C) were associated with impaired CASP10, CASP8, PARP activity -and therefore with apoptosis dysfunction- as in the case of I406L pathogenic mutation. We can speculate that, in addition to the functional impairment of apoptosis, other genetic and epigenetic factors might be crucial for the development of clinical symptoms in CASP10 mutated patients, as already described in FAS mutations, suggesting that the search of other mutations in patients with ALPS/ALPS-like phenotype should be encouraged. Nonetheless, further studies on epigenetic factors potentially implicated in the expression of symptoms are needed to fully understand the heterogeneity of clinical phenotype of this disorder. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Severe Chronic Neutropenia (SChrN) is an heterogeneous group of disorders characterized by persistently low circulating neutrophils ( Thirteen/27 subjects (48%) were phenotypically diagnosed as classical SCN. Of them 10 (77%) and 1 (8%) had mutations in ELANEand JAG1 genes respectively and 2 (16%) had no pathogenic gene detectable. Eight/27(30%) patients were phenotypically diagnosed as SN and the remaining 6/27 ( 22%) had the clinical features of ON. A pathogenic mutation in a Primary Immune Deficiency (PID) gene (RAG1, LRBA, CECR1. TACI, CARD11, CD40 and PI3K plus CASP 10) was found in 7/27 patients (26%). Four of them belonged to the group of 8 patients diagnosed with SN (50%) and 3 to that of the 6 ON subjects (50%). In 5/27(19%) patients no pathogenic gene was found. Table 1 shows clinical hematological characteristic of the 3 categories of patients. Conclusions:In our unselected cohort of SChrNa considerable proportion (26%) of subjects bore a genetic defect that qualifies them as PID. These PID genetic defects are located in the SN and ON patients (50% each) that are clinically different from classical ELANE mutated SCN subjects because of the presence of extra-hematological signs, of markers of autoimmunity, of a diverse marrow maturation block and a frequent involvement of more than one hematopoietic lineage. While the use of NGS still leaves a not negligible proportion of SChrN without pathogenic gene, this extensive genetic diagnostic approach enables to identify a relevant portion of subjects with SChrN who indeed carry a PID genetic defect. This has important clinical implications related to specific treatment and monitoring schedules to apply to these patients. The application of the Whole ExomeSequencing technique might fill the gap of the SCHrN patient who are still gene orphan. Disclosures No relevant conflicts of interest to declare.

Systemic autoinflammatory diseases (SAIDs) are a group of monogenic disorders characterized by inflammation which occurs in the absence of pathogenic auto-antibodies, auto-reactive T lymphocytes or other infective causes. More than 50% of SAID patients recruited to our Unit does not show any mutation at gene(s) tested by direct Sanger sequencing in the routine diagnosis. Clinical misdiagnosis, mutations in untested gene regions and genetic heterogeneity are possible explanations.

Deficiency of the interleukin-36 receptor antagonist (DITRA) is an autosomal recessive disease caused by mutations of IL36RN gene.1 Patients suffer from flares of acute generalised pustular psoriasis and systemic inflammation. We present two paediatric cases of DITRA with a severe clinical course, resistant to multiple therapies in whom the use of high doses of ustekinumab (a monoclonal antibody against the p40 subunit of both IL-12 and IL-23) lead to a persistent control of the disease. A 4-year-old boy, born from unrelated parents, presented at the age of 3 years with inverse psoriasis in the genital area. After some months, he developed diffuse pustular lesions associated with fever, elevation of acute phase reactants and poor general condition, requiring parenteral antibiotics and high-dose steroids (figure 1). Compound heterozygosity for the IL36RN P76L/S113L mutations was detected. Different treatments (acitretin, high-dose ciclosporin, …

The association of pyoderma gangrenosum, acne, and suppurative hidradenitis (PASH) has recently been described and suggested to be a new entity within the spectrum of autoinflammatory syndromes, which are characterized by recurrent episodes of sterile inflammation, without circulating autoantibodies and autoreactive T-cells. We conducted an observational study on 5 patients with PASH syndrome, analyzing their clinical features, genetic profile of 10 genes already known to be involved in autoinflammatory diseases (AIDs), and cytokine expression pattern both in lesional skin and serum. In tissue skin samples, the expressions of interleukin (IL)-1β and its receptors I and II were significantly higher in PASH (P = 0.028, 0.047, and 0.050, respectively) than in controls. In PASH patients, chemokines such as IL-8 (P = 0.004), C-X-C motif ligand (CXCL) 1/2/3 (P = 0.028), CXCL 16 (P = 0.008), and regulated on activation, normal T cell expressed and secreted (RANTES) (P = 0.005) were overexpressed. Fas/Fas ligand and cluster of differentiation (CD)40/CD40 ligand systems were also overexpressed (P = 0.016 for Fas, P = 0.006 for Fas ligand, P = 0.005 for CD40, and P = 0.004 for CD40 ligand), contributing to tissue damage and inflammation. In peripheral blood, serum levels of the main proinflammatory cytokines, that is, IL-1β, tumor necrosis factor-α, and IL-17, were within the normal range, suggesting that in PASH syndrome, the inflammatory process is mainly localized into the skin. Four out of our 5 PASH patients presented genetic alterations typical of well-known AIDs, including inflammatory bowel diseases, and the only patient lacking genetic changes had clinically evident Crohn disease. In conclusion, overexpression of cytokines/chemokines and molecules amplifying the inflammatory network, along with the genetic changes, supports the view that PASH syndrome is autoinflammatory in origin.

Background Mutations of CECR1 have been recently reported as causative of an inflammatory condition characterized by polyarteritis nodosa, cerebral stroke and immunodeficiency; the clinical manifestations of the disease are heterogeneous with a wide range of severity. Objectives To analyze the prevalence of CECR1 mutations in pediatric patients with polyarteritis nodosa, livedo reticularis and/or stroke. Methods Pediatric patients of Caucasian Italian origin with the following diseases/manifestations were included in the study: i) histologically confirmed polyartiritis nodosa (PAN) or cutaneous polyartiritis nodosa (cPAN), ii) persistent livedo reticularis with elevation of acute phase reactants, iii) ischemic or hemorrhagic strokes with systemic inflammation. Direct sequencing of CECR1 gene (exons 1-9) was performed with Sanger analysis. Results Up to January 2015, 27 patients from 25 families were included in the study. Homozygous or compound heterozygous CECR1 mutations with deleterious effects (G47R, G47A, P251L, R312X, E328D, T360A) were detected in 6 patients. A heterozygous causative mutation (G47V) was observed in 2 affected brothers, their father and the unaffected brother. So far a second mutation has not been detected; loss of heterozygosity could not be demonstrated and the mother is being investigated for a possible interstitial deletion. In the remaining patients common polymorphisms (L46L, N53N, H335R, Y453Y) were detected. The mean age of onset of the disease in genetically confirmed patients was 24 months (range 6 months – 5 years); all of them presented fever, elevation of acute phase reactants, livedo reticularis and a skin biopsy suggestive for vasculitis; two of them presented subcutaneous nodules while one of them presented ulcerations at extremities. Hypertension was detected in four patients, while one presented miocarditis. 3 patients presented one or more cerebral stroke during their disease course, while in 3 patients peripheral neuropathy was detected. 4 patients presented intestinal involvement (ranging from recurrent abdominal pain to intestinal perforation) and 2 patients presented growth delay, independent from steroidal treatment. Low immunoglobulin levels were detected in two patients. The clinical characteristics of the two heterozygous patients were similar: fever, livedo reticularis, increased acute phase reactants and hypogammaglobulinemia were detected in both; cerebral stroke occurred in one of them. Conclusions CECR1mutations are present in the Italian population and associated with severe cases of ADA2 deficiency. A clinical heterogeneity has been detected in genetically confirmed patients. In a few patients a typical phenotype was associated to incomplete or negative genotype, thus supporting the hypothesis of a genetic heterogeneity of this condition. Disclosure of Interest None declared

Background Autosomal dominant gain of function mutations in caspase recruitment domain family member 14 (CARD14) were found to cause plaque psoriasis in two families and severe generalized pustular psoriasis as a monogenic form of childhood (CARD14-mediated pustular psoriasis, CAMPS) [1]. CARD14 mutations have also been implicated in plaque-type psoriasis and pityriasis rubra pilaris [2]. Objectives Describing a family with an unusual clinical phenotype characterized by some members with childhood-onset erytrodermic psoriasis first localized and then diffuse over all the skin surface; in some family members is also reported psoriatic arthritis. Methods We assessed for the first time the family in december 2013, because of their skin lesions and family recurrence for erytrodermic psoriasis associated to arthritis in some cases. There are three pairs of twins, five of them presenting psoriasis and two of them presenting psoriatic arthritis. The children presented poor clinical response to topic and systemic therapy with antihistamine, steroid, retinoids, cyclosporine and etanercept. The whole pedigree is depicted in the figure with the grey symbols indicating the affected members and an asterisk pointing out members for whom we got a biological sample (Figure). After exclusion of the most common genes associated to autoinflammatory diseases (IL36RN, IL1RN, MVK, TNFRSF1A, NLRP3, NLRP12, MEFV, IL1RN, NOD2, PSMB8, PSTPIP1, LPIN2). Successively, we approached the new gene search by subjecting to Whole Exome Sequencing (WES) analysis five members of the family, namely those indicated by an arrow. Samples were processed in outsourcing and raw data were transferred to our lab for the bio-informatic analysis. Results Among variants shared by the four affected individuals and not present in the unaffected subject, a missense mutation of the CARD14 gene resulted worth of further investigation. In particular, it was the case of an exon4 heterozygous nucleotide change, c.446T>G, leading to the missense amino acid substitution p.L149R. The presence of this variant was validated by Sanger sequencing not only in the affected members undergone WES, but also assessed in all the rest of the available family members. This allowed us to confirm the expected segregation of the CARD14 mutation with the disease phenotype. Conclusions CARD14 gain of function mutations can give rise to unusual clinical phenotype like diffuse erytrodermic psoriasis and psoriatic arthritis. WES appears to be a powerful, suitable and proper approach to identify new SAID genes, thus also disclosing new molecular pathogenic mechanisms. References Jordan CT, Cao L, Roberson ED, Pierson KC, Yang CF, Joyce CE, et al. PSORS2 is due to mutations in CARD14. American journal of human genetics. 2012; 90(5):784–95. Epub 2012/04/24. Fuchs-Telem D, Sarig O, van Steensel MA, Isakov O, Israeli S, Nousbeck J, et al. Familial pityriasis rubra pilaris is caused by mutations in CARD14. American journal of human genetics.n 2012; 91(1):163–70. Epub 2012/06/19. Disclosure of Interest None declared

Methods Pediatric patients of Caucasian Italian origin with the following diseases/manifestations were included in the study: i) histologically confirmed polyartiritis nodosa (PAN) or cutaneous polyartiritis nodosa (cPAN), ii) persistent livedo reticularis with elevation of acute phase reactants, iii) ischemic or hemorrhagic strokes with systemic inflammation. Direct sequencing of CECR1 gene (exons 1-9) was performed with Sanger analysis.

ADA2 deficiency, a recently described disease, is characterized by systemic vasculopathy and episodes of strokes. The defect is due to a loss of function mutation of CECR1 gene, codifying for Adenosine Deaminase 2 protein. This protein regulates the catabolism of extracellular adenosine, which we have recently shown is an important regulator of Class Switch Recombination in B lymphocytes. Accordingly DADA2 patients can present hypogammaglobulinemia.