A somatic NLRP3 mutation as a cause of a sporadic case of chronic infantile neurologic, cutaneous, articular syndrome/neonatal-onset multisystem inflammatory disease: Novel evidence of the role of low-level mosaicism as the pathophysiologic mechanism underlying mendelian inherited diseases.

OBJECTIVE: Chronic infantile neurologic, cutaneous, articular syndrome (CINCA), also known as neonatal-onset multisystem inflammatory disease (NOMID), is a severe, early-onset autoinflammatory disease characterized by an urticaria-like rash, arthritis/arthropathy, variable neurologic involvement, and dysmorphic features, which usually respond to interleukin-1 blockade. CINCA/NOMID has been associated with dominant Mendelian inherited NLRP3 mutations. However, conventional sequencing analyses detect true disease-causing mutations in only approximately 55-60% of patients, which suggests the presence of genetic heterogeneity. We undertook the current study to assess the presence of somatic, nongermline NLRP3 mutations in a sporadic case of CINCA/NOMID. METHODS: Clinical data, laboratory results, and information on treatment outcomes were gathered through direct interviews. Exhaustive genetic studies, including Sanger method sequencing, subcloning, restriction fragment length polymorphism assay, and pyrosequencing, were performed. RESULTS: The patient's CINCA/NOMID was diagnosed based on clinical features (early onset of the disease, urticaria-like rash, knee arthropathy, and dysmorphic features). The patient has exhibited a successful response to anakinra within the last 28 months. Analysis of NLRP3 identified a novel heterozygous variant (p.D303H) that was detected in approximately 30-38% of circulating leukocytes. The absence of this variant in healthy controls and in the patient's parents suggested a de novo true disease-causing mutation. Additional analyses showed that this novel mutation was present in both leukocyte subpopulations and epithelial cells. CONCLUSION: Our findings identify the novel p.D303H NLRP3 variant in a Spanish patient with CINCA/NOMID as a new disease-causing mutation, which was detected as a somatic, nongermline mutation in hematopoietic and nonhematopoietic cell lineages. Our data provide new insight into the role of low-level mosaicism in NLRP3 as the pathophysiologic mechanism underlying cryopyrin-associated periodic syndrome.

NOD2 gene-associated pediatric granulomatous arthritis: clinical diversity, novel and recurrent mutations, and evidence of clinical improvement with interleukin-1 blockade in a Spanish cohort.

OBJECTIVE: Blau syndrome and early-onset sarcoidosis are NOD2 gene-associated chronic autoinflammatory diseases characterized by skin rash, arthritis, and/or eye involvement, with noncaseating granulomata as their pathologic hallmark. This study was undertaken to describe the expanded clinical phenotype, treatment outcomes, and NOD2 gene mutation analysis in a Spanish cohort with pediatric granulomatous arthritis, a chronic disease resembling Blau syndrome/early-onset sarcoidosis. METHODS: Clinical, laboratory, and treatment data on the 12 patients in the cohort were obtained through direct interviews. NOD2 gene analysis was performed in a central laboratory, by bidirectional sequencing. Cytokine levels were measured using the human Flex-Set cytokine bead array. RESULTS: The classic Blau syndrome/early-onset sarcoidosis triad of skin rash, arthritis, and recurrent uveitis was identified in 5 patients (41.7%), whereas 7 patients (58.3%) presented with fewer than 3 of the classic features. Novel atypical manifestations such as persistent fever and myocardiopathy were also observed. NOD2 analysis revealed 1 heterozygous mutation in each patient, and familial studies confirmed its full penetrance. Of the 12 cases, 58.3% were sporadic, due to de novo mutations. Four different missense mutations on exon 4 were detected. Two of them (R334W and R334Q) were recurrent mutations and were found in 77.8% of the Spanish families, whereas the other 2 (C495Y and R587C) were novel. In the patient who received anakinra treatment, all clinical inflammatory symptoms improved and plasma cytokine levels normalized. CONCLUSION: These findings indicate that the expanding clinical heterogeneity of the disease (that is, the presentation of incomplete forms of the classic triad and atypical manifestations) and the high prevalence of sporadic cases should alert clinicians to the possible genetic basis of the condition and support the inclusion of DNA analysis as a diagnostic test. The positive response to anakinra observed in 1 patient suggests a new potential therapeutic approach that merits further investigation, and suggests that the pathogenesis of pediatric granulomatous arthritis may involve interleukin-1-mediated events.

An unexpectedly high frequency of MEFV mutations in patients with anti-citrullinated protein antibody-negative palindromic rheumatism.

OBJECTIVE: To investigate whether the MEFV gene, which is involved in the regulation of the inflammatory response and has been associated with familial Mediterranean fever (FMF) and intermittent hydrarthrosis, is implicated in the pathogenesis of palindromic rheumatism (PR) and to examine its clinical presentation and its evolution in a Spanish cohort of PR patients. METHODS: Family histories, demographic clinical data, and laboratory characteristics of 75 patients diagnosed as having PR were collected from medical records and personal interviews. The healthy control group included 325 blood bank donors. The FMF control group was made up of 84 Spanish FMF patients. Genomic DNA was isolated, and MEFV gene mutation analysis was performed by polymerase chain reaction amplification and sequence analysis. RESULTS: Sixty-five unrelated PR patients were finally included in the study. MEFV gene mutation analysis identified 8 of these 65 patients (12.3%) as carriers of at least 1 mutated MEFV allele. Patients with MEFV mutations had higher mean age and age at disease onset, but lower mean serum levels of anti-citrullinated protein antibodies (ACPAs). No other significant differences were observed between patients with and those without mutations. The frequency of MEFV mutations in ACPA-negative PR patients was 22.2%, compared with 5.3% in ACPA-positive PR patients (P = 0.058). CONCLUSION: This study shows a previously unreported high prevalence of mutations of the MEFV gene in patients with ACPA-negative PR. This supports the hypothesis that it might be a susceptibility gene. Our findings also support the hypothesis that the MEFV gene might participate in the pathogenesis of other undifferentiated relapsing inflammatory rheumatic disorders.

Effect of NOD2/CARD15 variants in T-cell depleted allogeneic stem cell transplantation.

BACKGROUND AND OBJECTIVES: Three single nucleotide polymorphisms (SNP) in the NOD2/CARD15 gene have been associated with the incidence and the severity of acute graft-versus-host disease (GVHD) following allogeneic stem cell transplantation (SCT). We hypothesized that the clinical effect of SNP in NOD2/CARD15 might be different in patients submitted to T-cell-depleted allogeneic SCT, in which donor T cells, the main effectors of GVHD, are eliminated. DESIGN AND METHODS: SNP 8, 12 and 13 in NOD2/CARD15 were studied using a Taqman protocol in 85 patients undergoing HLA-identical, T-cell-depleted SCT and in 71 of their sibling donors. RESULTS: NOD2/CARD15 variants were present in nine (11%) patients and six (8%) donors. The incidences of acute GVHD and chronic GVHD were not associated with either the donors' or recipients' NOD2/CARD15 variants. In contrast, these genetic variants were associated with a lower disease-free survival (17% vs. 48%, p=0.03). Death due to pulmonary infection was more frequent in the group of patients with NOD2/CARD15 variants. In the multivariate analysis, only NOD2/CARD15 variants (RR 2.3, p=0.04) and older age (RR 2.2; p=0.04) were independent prognostic factors for disease-free survival. INTERPRETATION AND CONCLUSIONS: NOD2/CARD15 variants have a deleterious effect on clinical outcome in T-cell-depleted allogeneic SCT, which is independent of GVHD. These results supports the hypothesis that the detrimental effect of NOD2/CARD15 variants in such a transplant setting might be produced by an alteration of the innate immune system more than by activation of the adaptive immune system.

Etanercept plus colchicine treatment in a child with tumour necrosis factor receptor-associated periodic syndrome abolishes auto-inflammatory episodes without normalising the subclinical acute phase response.

UNLABELLED: We investigated the cause of hereditary periodic fever syndrome in a Spanish child with recurrent long episodes of fever, migratory skin rash, myalgia, arthralgia, conjunctivitis and abdominal pain. Infectious and autoimmune causes were ruled out. No familial history was reported. Analysis of the tumour necrosis factor receptor superfamily 1A (TNFRSF1A) gene identified a missense mutation (G36E) on exon 3. The absence of this variant in the patient's parents and in controls identified it as a de novo disease-associated mutation. Clinical symptoms disappeared with administration of etanercept; however, levels of acute-phase reactants remained increased and could not be stabilised by the addition of colchicine. We believe that this patient gained some symptomatic relief with etanercept therapy, although not enough to completely avoid the risk of amyloidosis. Thus it is debatable whether etanercept alone or combined with other drugs, is the treatment of choice for patients with tumour necrosis factor receptor-associated periodic syndrome. CONCLUSION: Since there is variability in treatment responses among different patients with tumour necrosis factor receptor-associated periodic syndrome, we suggest that a systematic evaluation of acute-phase reactants, especially SAA-1, could be useful in maintaining or modifying a given therapeutic approach in these patients.

Clinical and genetic heterogeneity among Spanish patients with recurrent autoinflammatory syndromes associated with the CIAS1/PYPAF1/NALP3 gene.

OBJECTIVE: To investigate the involvement of the CIAS1/PYPAF1/NALP3 gene in 7 unrelated Spanish families with recurrent autoinflammatory diseases characterized by early onset, recurrent fever, and a chronic urticarial rash, in whom a clinical diagnosis of cryopyrin-associated periodic syndromes (CAPS) is suspected. METHODS: Clinical symptoms, results of laboratory analyses, and data on previous treatments in members of the 7 families were recorded on a questionnaire specific for hereditary autoinflammatory diseases. All coding regions and intronic flanking boundaries of the CIAS1/PYPAF1/NALP3 gene were amplified by polymerase chain reaction and sequenced. RESULTS: Five different missense mutations, including 2 de novo and 1 previously unreported mutation (R488K), were identified in exon 3 of the CIAS1/PYPAF1/NALP3 gene in 5 of the 7 affected families. Expanded genetic analysis among the healthy individuals identified incomplete penetrance in 2 families. No mutations were found in 2 of the 3 patients with chronic infantile neurologic, cutaneous, articular (CINCA) syndrome/neonatal-onset multisystem inflammatory disease (NOMID). CONCLUSION: The clinical data suggested a diagnosis of familial cold-induced autoinflammatory syndrome in 3 families, CINCA/NOMID syndrome in 3 others, and a possible Muckle-Wells syndrome, whereas mutational analysis showed different CIAS1/PYPAF1/NALP3 missense mutations in 5 families. These data are consistent with a common molecular basis of these diseases and highlights the phenotypic heterogeneity among CIAS1/PYPAF1/NALP3 gene-associated syndromes. The previously unreported mutation and the incomplete penetrance found in 2 families expand the genetic basis underlying these autoinflammatory syndromes. These findings should alert clinicians to the possible genetic basis of these conditions, even in the absence of a family history, in their attempts to establish an accurate diagnosis and the optimal therapeutic approach.

The west side story: MEFV haplotype in Spanish FMF patients and controls, and evidence of high LD and a recombination "hot-spot" at the MEFV locus.

Mutations at the MEFV gene cause, with various degrees of penetrance, familial Mediterranean fever (FMF). This disease is more prevalent in the Middle East than elsewhere, and most studies have focused on those populations. However, FMF occurs also in the Western Mediterranean and these populations should be taken into account for a complete view of FMF. We have analyzed intragenic MEFV SNPs in Spanish and Chueta (descendants of converted Jews) FMF patients and controls, and this constitutes the first systematic survey of normal MEFV SNP haplotype structure and variability. Our findings have allowed us to systematize the nomenclature of MEFV haplotypes and show that there is strong linkage disequilibrium (LD) at the MEFV locus and an intragenic recombination hot spot. The high local LD, regardless the recombination hot spot, is responsible for the limited diversity of the MEFV control haplotypes found in the Spanish population and it suggests that it may be a common feature to all Mediterranean populations. The MEFV mutation spectrum in Spain is quite diverse, and similar to those of France and Italy. On the contrary, the Chueta spectrum was poorer and closer to that of North African Jews, suggesting a direct connection with the Jewish diaspora.

A severe autosomal-dominant periodic inflammatory disorder with renal AA amyloidosis and colchicine resistance associated to the MEFV H478Y variant in a Spanish kindred: an unusual familial Mediterranean fever phenotype or another MEFV-associated periodic inflammatory disorder?

Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by recurring short attacks of fever and serositis. Secondary AA amyloidosis is the worst complication of the disease and often determines the prognosis. The MEFV gene, on chromosome 16p13.3, is responsible for the disease and around 30 mutations have been reported to date. Colchicine is the standard FMF treatment today, and prevents both attacks and amyloid deposition in 95% of patients. Here we describe a three-generation Spanish kindred with five family members affected by a severe periodic inflammatory disorder associated with renal AA amyloidosis and colchicine unresponsiveness. Clinical diagnosis of definite FMF disease was made based on the Tel-Hashomer criteria set. Genetic analyses revealed that all subjects were heterozygous for the new H478Y MEFV variant, segregating with the disease. In addition, mutations in the TNFRSF1A and CIAS1/PYPAF1/NALP3 genes, related to the dominantly inherited autoinflammatory periodic syndromes, were ruled out. However, the dominant inheritance of the disease, the long fever episodes with a predominant joint involvement, and the resistance to colchicine in these patients raise the question of whether the periodic syndrome seen in this kindred is a true FMF disease with unusual manifestations or rather another MEFV-associated periodic syndrome. We conclude that the new H478Y MEFV mutation is the dominant pathological variant causing the inflammatory periodic syndrome in this kindred and that full-length analyses of the MEFV gene are needed to obtain an adequate diagnosis of patients with clinical suspicion of a hereditary periodic fever syndrome, especially those from non-ancestral populations.