Blood-based test for diagnosis and functional subtyping of familial Mediterranean fever.

BACKGROUND AND OBJECTIVE: Familial Mediterranean fever (FMF) is the most common monogenic autoinflammatory disease (AID) worldwide. The disease is caused by mutations in the MEFV gene encoding the inflammasome sensor Pyrin. Clinical diagnosis of FMF is complicated by overlap in symptoms with other diseases, and interpretation of genetic testing is confounded by the lack of a clear genotype-phenotype association for most of the 340 reported MEFV variants. In this study, the authors designed a functional assay and evaluated its potential in supporting FMF diagnosis. METHODS: Peripheral blood mononuclear cells (PBMCs) were obtained from patients with Pyrin-associated autoinflammation with an FMF phenotype (n=43) or with autoinflammatory features not compatible with FMF (n=8), 10 asymptomatic carriers and 48 healthy donors. Sera were obtained from patients with distinct AIDs (n=10), and whole blood from a subset of patients and controls. The clinical, demographic, molecular genetic factors and other characteristics of the patient population were assessed for their impact on the diagnostic test read-out. Interleukin (IL)-1ß and IL-18 levels were measured by Luminex assay. RESULTS: The ex vivo colchicine assay may be performed on whole blood or PBMC. The functional assay robustly segregated patients with FMF from healthy controls and patients with related clinical disorders. The diagnostic test distinguished patients with classical FMF mutations (M694V, M694I, M680I, R761H) from patients with other MEFV mutations and variants (K695R, P369S, R202Q, E148Q) that are considered benign or of uncertain clinical significance. CONCLUSION: The ex vivo colchicine assay may support diagnosis of FMF and functional subtyping of Pyrin-associated autoinflammation.

NACHT, LRR, and PYD domains-containing Protein 3 and LL-37: prognostic value of new biomarkers in community-acquired pneumonia. / Proteína 3 contendo um domínio NACHT, porção C-terminal rica em repetições de leucina e de domínio pirina e LL-37: valor prognóstico de novos biomarcadores em pneumonia adquirida na comunidade.

OBJECTIVE: This study aimed to determine the serum levels of NACHT, Leucine-rich repeat (LRR), and Pyrin (PYD) domains-containing Protein 3 (NLRP3) and cathelicidin LL-37, and investigate their prognostic significance in community-acquired pneumonia (CAP). METHODS: The sample of this prospective study was composed of 76 consecutive patients with CAP. Demographic data and clinical characteristics were collected. Serum levels of NLRP3 and LL-37 were determined by ELISA. Spearman's analysis was used to evaluate the correlation between NLRP3 and LL-37. Association of NLRP3 and LL-37 with 30-day survival and mortality rates was assessed using the Kaplan-Meier curve and logistic regression analysis. RESULTS: Serum NLRP3 significantly increased whereas serum LL-37 significantly decreased in patients with severe CAP. Significant correlation was observed between serum NLRP3 and LL-37 in CAP patients. Patients with higher levels of NLRP3 and lower levels of LL-37 showed lower 30-day survival rate and higher mortality compared with those with lower NLRP3 and higher LL-37 levels. CONCLUSION: Severe CAP patients tend to present higher serum NLRP3 and lower serum LL-37, which might serve as potential biomarkers for CAP prognosis.

OBJETIVO: Este estudo teve como objetivo determinar os níveis séricos de proteína 3 contendo um domínio NACHT, porção C-terminal rica em repetições de leucina e de domínio pirina (NLRP3) e catelicidina LL-37, bem como investigar sua importância prognóstica em pneumonia adquirida na comunidade (PAC). MÉTODOS: Este estudo prospectivo incluiu 76 pacientes com PAC. Foram obtidos dados demográficos e características clínicas. Os níveis séricos de NLRP3 e LL-37 foram determinados por meio do teste ELISA. A correlação entre NLRP3 e LL-37 foi estimada por intermédio da análise de Spearman. A associação entre NLRP3 e LL-37 com 30 dias de taxa de sobrevida e de mortalidade foi avaliada pela curva de Kaplan-Meier e análise de regressão logística. RESULTADOS: Os níveis séricos de NLRP3 estavam elevados, enquanto os níveis de LL-37 apresentaram redução significativa em pacientes com PAC grave. Observou-se correlação significativa entre os níveis séricos de NLRP3 e LL-37 em pacientes com PAC. Pacientes com níveis elevados de NLRP3 e níveis reduzidos de LL-37 exibiram maior taxa de sobrevida em 30 dias e de mortalidade quando comparados com aqueles com níveis inferiores de NLRP3 e LL-37. CONCLUSÕES: Pacientes com PAC grave tendem a apresentar níveis séricos elevados de NLRP3 e níveis reduzidos de LL-37, o que pode ser utilizado como um potencial biomarcador prognóstico.

Proteína 3 contendo um domínio NACHT, porção C-terminal rica em repetições de leucina e de domínio pirina e LL-37: valor prognóstico de novos biomarcadores em pneumonia adquirida na comunidade / NACHT, LRR, and PYD domains-containing Protein 3 and LL-37: prognostic value of new biomarkers in community-acquired pneumonia

RESUMO Objetivo Este estudo teve como objetivo determinar os níveis séricos de proteína 3 contendo um domínio NACHT, porção C-terminal rica em repetições de leucina e de domínio pirina (NLRP3) e catelicidina LL-37, bem como investigar sua importância prognóstica em pneumonia adquirida na comunidade (PAC). Métodos Este estudo prospectivo incluiu 76 pacientes com PAC. Foram obtidos dados demográficos e características clínicas. Os níveis séricos de NLRP3 e LL-37 foram determinados por meio do teste ELISA. A correlação entre NLRP3 e LL-37 foi estimada por intermédio da análise de Spearman. A associação entre NLRP3 e LL-37 com 30 dias de taxa de sobrevida e de mortalidade foi avaliada pela curva de Kaplan-Meier e análise de regressão logística. Resultados Os níveis séricos de NLRP3 estavam elevados, enquanto os níveis de LL-37 apresentaram redução significativa em pacientes com PAC grave. Observou-se correlação significativa entre os níveis séricos de NLRP3 e LL-37 em pacientes com PAC. Pacientes com níveis elevados de NLRP3 e níveis reduzidos de LL-37 exibiram maior taxa de sobrevida em 30 dias e de mortalidade quando comparados com aqueles com níveis inferiores de NLRP3 e LL-37. Conclusões Pacientes com PAC grave tendem a apresentar níveis séricos elevados de NLRP3 e níveis reduzidos de LL-37, o que pode ser utilizado como um potencial biomarcador prognóstico.

ABSTRACT Objective This study aimed to determine the serum levels of NACHT, Leucine-rich repeat (LRR), and Pyrin (PYD) domains-containing Protein 3 (NLRP3) and cathelicidin LL-37, and investigate their prognostic significance in community-acquired pneumonia (CAP). Methods The sample of this prospective study was composed of 76 consecutive patients with CAP. Demographic data and clinical characteristics were collected. Serum levels of NLRP3 and LL-37 were determined by ELISA. Spearman's analysis was used to evaluate the correlation between NLRP3 and LL-37. Association of NLRP3 and LL-37 with 30-day survival and mortality rates was assessed using the Kaplan-Meier curve and logistic regression analysis. Results Serum NLRP3 significantly increased whereas serum LL-37 significantly decreased in patients with severe CAP. Significant correlation was observed between serum NLRP3 and LL-37 in CAP patients. Patients with higher levels of NLRP3 and lower levels of LL-37 showed lower 30-day survival rate and higher mortality compared with those with lower NLRP3 and higher LL-37 levels. Conclusion Severe CAP patients tend to present higher serum NLRP3 and lower serum LL-37, which might serve as potential biomarkers for CAP prognosis.

Investigation of MEFV gene polymorphisms (G138G and A165A) in adult patients with familial Mediterranean fever / Investigação de polimorfismos no gene MEFV (G138G e A165A) em pacientes adultos com febre mediterrânica familiar

Abstract Aim: Various mutations have been identified in the Mediterranean fever (MEFV) gene which is reported to be responsible from Familial Mediterranean fever (FMF). In our study, we aimed to determine the frequency of the MEFV mutations in our region and to investigate the impact of G138G (rs224224, c.414A>G) and A165A (rs224223, c.495C>A) gene polymorphisms on the clinical findings of the disease. Methods: One hundred and sixteen patients diagnosed with FMF and 95 control subjects were included in this study. We used the DNA sequence analysis method to identify the most prevailing 10 mutations located in exon 2 and 10 of MEFV gene. Results: As a result of the MEFV mutation analysis, the most common mutation was the M694V mutation allele with a frequency rate of 41.8%. When the patients group and control group were compared in terms of frequency of both polymorphic alleles (G polymorphic allele, observed in G138G and the A polymorphic allele, observed in A165A), the variation was observed to be statistically significant (p < 0.001). It was found that the MEFV mutation types have no relation with clinical findings and amyloidosis (p > 0.05). Conclusions: To our knowledge, our study is the first study in the Southern Marmara region that reports the frequency of MEFV mutations. Our findings imply that the polymorphisms of G138G and A165A may have an impact on progress of the disease. We think that more studies, having higher number of cases and investigating the polymorphisms of MEFV gene, are needed.

Resumo Objetivo: Identificaram-se mutações no gene da febre mediterrânica (MEFV) relatadas como responsáveis pela febre mediterrânica familiar (FMF). Este estudo teve como objetivo determinar a frequência de mutações no MEFV na região sul do mar de Mármara e investigar o impacto dos polimorfismos genéticos G138G (rs224224, c.414A > G) e A165A (rs224223, c.495C > A) nos achados clínicos da doença. Métodos: Foram incluídos neste estudo 116 pacientes com diagnóstico de FMF e 95 indivíduos no grupo controle. Usou-se o método de análise da sequência de DNA para identificar as 10 mutações mais prevalentes localizadas nos éxons 2 e 10 do gene MEFV. Resultados: Como resultado da análise da mutação MEFV, a mutação mais comum foi a mutação alélica M694 V, com uma taxa de frequência de 41,8%. Quando os grupos de pacientes e controles foram comparados em termos de frequência de ambos os alelos polimórficos (alelo polimórfico G, observado no G138G e o alelo polimórfico A, observado no A165A), a variação observada foi estatisticamente significativa (p < 0,001). Verificou-se que os tipos de mutação no MEFV não tinham relação com os achados clínicos nem com a amiloidose (p > 0,05). Conclusões: Que se tem conhecimento, este estudo é o primeiro feito na região sul do mar de Mármara que relata a frequência de mutações no MEFV. Os achados indicam que os polimorfismos G138G e A165A podem ter um impacto sobre o progresso da doença. Acredita-se que são necessários mais estudos que abranjam um maior número de casos e investiguem os polimorfismos do gene MEFV.

Investigation of MEFV gene polymorphisms (G138G and A165A) in adult patients with familial Mediterranean fever.

AIM: Various mutations have been identified in the Mediterranean fever (MEFV) gene which is reported to be responsible from Familial Mediterranean fever (FMF). In our study, we aimed to determine the frequency of the MEFV mutations in our region and to investigate the impact of G138G (rs224224, c.414A>G) and A165A (rs224223, c.495C>A) gene polymorphisms on the clinical findings of the disease. METHODS: One hundred and sixteen patients diagnosed with FMF and 95 control subjects were included in this study. We used the DNA sequence analysis method to identify the most prevailing 10 mutations located in exon 2 and 10 of MEFV gene. RESULTS: As a result of the MEFV mutation analysis, the most common mutation was the M694V mutation allele with a frequency rate of 41.8%. When the patients group and control group were compared in terms of frequency of both polymorphic alleles (G polymorphic allele, observed in G138G and the A polymorphic allele, observed in A165A), the variation was observed to be statistically significant (p<0.001). It was found that the MEFV mutation types have no relation with clinical findings and amyloidosis (p>0.05). CONCLUSIONS: To our knowledge, our study is the first study in the Southern Marmara region that reports the frequency of MEFV mutations. Our findings imply that the polymorphisms of G138G and A165A may have an impact on progress of the disease. We think that more studies, having higher number of cases and investigating the polymorphisms of MEFV gene, are needed.

A novel Pyrin-Associated Autoinflammation with Neutrophilic Dermatosis mutation further defines 14-3-3 binding of pyrin and distinction to Familial Mediterranean Fever.

OBJECTIVE: Pyrin-Associated Autoinflammation with Neutrophilic Dermatosis (PAAND) is a recently described monogenic autoinflammatory disease. The causal p.S242R MEFV mutation disrupts a binding motif of the regulatory 14-3-3 proteins within pyrin. Here, we investigate a family with clinical features consistent with PAAND in whom the novel p.E244K MEFV mutation, located in the +2 site of the 14-3-3 binding motif in pyrin, has been found. METHODS: Multiplex cytokine analyses were performed on p.E244K patient and control serum. Peripheral blood mononuclear cells were stimulated ex vivo with lipopolysaccharide (LPS). In vitro, inflammasome complex formation was evaluated by flow cytometry of Apoptosis-associated Speck-like protein containing a Caspase recruitment domain (ASC) specks. Interleukin-1ß (IL-1ß) and IL-18 production was quantified by ELISA. The ability of the p.E244K pyrin mutation to interact with 14-3-3 was assessed by immunoprecipitation. RESULTS: PAAND p.E244K patient serum displayed a different cytokine profile compared with patients with Familial Mediterranean Fever (FMF). In overexpression models, p.E244K pyrin was associated with decreased 14-3-3 binding and increased ASC speck formation. THP-1 monocytes expressing PAAND pyrin mutations demonstrated spontaneous caspase-1-dependent IL-1ß and IL-18 secretion, as well as cell death, which were significantly greater than those of wild-type and the FMF-associated mutation p.M694V. CONCLUSION: In PAAND, disruption of the +2 position of a 14-3-3 binding motif in pyrin results in its constitutive activation, with spontaneous production of IL-1ß and IL-18, associated with inflammatory cell death. The altered serum cytokine profile may explain the different clinical features exhibited by PAAND patients compared with those with FMF.