Ancient familial Mediterranean fever mutations in human pyrin and resistance to Yersinia pestis.

Familial Mediterranean fever (FMF) is an autoinflammatory disease caused by homozygous or compound heterozygous gain-of-function mutations in MEFV, which encodes pyrin, an inflammasome protein. Heterozygous carrier frequencies for multiple MEFV mutations are high in several Mediterranean populations, suggesting that they confer selective advantage. Among 2,313 Turkish people, we found extended haplotype homozygosity flanking FMF-associated mutations, indicating evolutionarily recent positive selection of FMF-associated mutations. Two pathogenic pyrin variants independently arose >1,800 years ago. Mutant pyrin interacts less avidly with Yersinia pestis virulence factor YopM than with wild-type human pyrin, thereby attenuating YopM-induced interleukin (IL)-1ß suppression. Relative to healthy controls, leukocytes from patients with FMF harboring homozygous or compound heterozygous mutations and from asymptomatic heterozygous carriers released heightened IL-1ß specifically in response to Y. pestis. Y. pestis-infected MefvM680I/M680I FMF knock-in mice exhibited IL-1-dependent increased survival relative to wild-type knock-in mice. Thus, FMF mutations that were positively selected in Mediterranean populations confer heightened resistance to Y. pestis.

Control of the innate immune response by the mevalonate pathway.

Deficiency in mevalonate kinase (MVK) causes systemic inflammation. However, the molecular mechanisms linking the mevalonate pathway to inflammation remain obscure. Geranylgeranyl pyrophosphate, a non-sterol intermediate of the mevalonate pathway, is the substrate for protein geranylgeranylation, a protein post-translational modification that is catalyzed by protein geranylgeranyl transferase I (GGTase I). Pyrin is an innate immune sensor that forms an active inflammasome in response to bacterial toxins. Mutations in MEFV (encoding human PYRIN) result in autoinflammatory familial Mediterranean fever syndrome. We found that protein geranylgeranylation enabled Toll-like receptor (TLR)-induced activation of phosphatidylinositol-3-OH kinase (PI(3)K) by promoting the interaction between the small GTPase Kras and the PI(3)K catalytic subunit p110δ. Macrophages that were deficient in GGTase I or p110δ exhibited constitutive release of interleukin 1ß that was dependent on MEFV but independent of the NLRP3, AIM2 and NLRC4 inflammasomes. In the absence of protein geranylgeranylation, compromised PI(3)K activity allows an unchecked TLR-induced inflammatory responses and constitutive activation of the Pyrin inflammasome.

Pyrin inflammasome activation and RhoA signaling in the autoinflammatory diseases FMF and HIDS.

Mutations in the genes encoding pyrin and mevalonate kinase (MVK) cause distinct interleukin-1ß (IL-1ß)-mediated autoinflammatory diseases: familial Mediterranean fever (FMF) and hyperimmunoglobulinemia D syndrome (HIDS). Pyrin forms an inflammasome when mutant or in response to bacterial modification of the GTPase RhoA. We found that RhoA activated the serine-threonine kinases PKN1 and PKN2 that bind and phosphorylate pyrin. Phosphorylated pyrin bound to 14-3-3 proteins, regulatory proteins that in turn blocked the pyrin inflammasome. The binding of 14-3-3 and PKN proteins to FMF-associated mutant pyrin was substantially decreased, and the constitutive IL-1ß release from peripheral blood mononuclear cells of patients with FMF or HIDS was attenuated by activation of PKN1 and PKN2. Defects in prenylation, seen in HIDS, led to RhoA inactivation and consequent pyrin inflammasome activation. These data suggest a previously unsuspected fundamental molecular connection between two seemingly distinct autoinflammatory disorders.

Transcriptional licensing is required for Pyrin inflammasome activation in human macrophages and bypassed by mutations causing familial Mediterranean fever.

Pyrin is a cytosolic immune sensor that nucleates an inflammasome in response to inhibition of RhoA by bacterial virulence factors, triggering the release of inflammatory cytokines, including IL-1ß. Gain-of-function mutations in the MEFV gene encoding Pyrin cause autoinflammatory disorders, such as familial Mediterranean fever (FMF) and Pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND). To precisely define the role of Pyrin in pathogen detection in human immune cells, we compared initiation and regulation of the Pyrin inflammasome response in monocyte-derived macrophages (hMDM). Unlike human monocytes and murine macrophages, we determined that hMDM failed to activate Pyrin in response to known Pyrin activators Clostridioides difficile (C. difficile) toxins A or B (TcdA or TcdB), as well as the bile acid analogue BAA-473. The Pyrin inflammasome response was enabled in hMDM by prolonged priming with either LPS or type I or II interferons and required an increase in Pyrin expression. Notably, FMF mutations lifted the requirement for prolonged priming for Pyrin activation in hMDM, enabling Pyrin activation in the absence of additional inflammatory signals. Unexpectedly, in the absence of a Pyrin response, we found that TcdB activated the NLRP3 inflammasome in hMDM. These data demonstrate that regulation of Pyrin activation in hMDM diverges from monocytes and highlights its dysregulation in FMF.

Pyrin Inflammasome Activation Defines Colchicine-Responsive SURF Patients from FMF and Other Recurrent Fevers.

Syndrome of undifferentiated recurrent fever (SURF) is characterized by recurrent fevers, a lack of confirmed molecular diagnosis, and a complete or partial response to colchicine. Despite the clinical similarities to familial Mediterranean fever (FMF), the underlying inflammatory mechanisms of SURF are not yet understood. We here analyzed the in vitro activation of the pyrin inflammasome in a cohort of SURF patients compared to FMF and PFAPA patients. Peripheral blood mononuclear cells (PBMC) were collected from SURF (both colchicine-treated and untreated), FMF, PFAPA patients, and healthy donors. PBMC were stimulated ex vivo with Clostridium difficile toxin A (TcdA) and a PKC inhibitor (UCN-01), in the presence or absence of colchicine. The assembly of the pyrin inflammasome was evaluated by measuring the presence of apoptosis-associated Speck-like protein containing caspase recruitment domain (ASC) specks in monocytes using flow cytometry. IL-1ß secretion was quantified using an ELISA assay. No differences in TcdA-induced activation of pyrin inflammasome were observed among FMF, PFAPA, and healthy donors. Untreated SURF patients showed a reduced response to TcdA, which was normalized after colchicine treatment. In contrast to FMF, SURF patients, similar to PFAPA patients and healthy donors, did not exhibit pyrin inflammasome activation in response to UCN-01-mediated pyrin dephosphorylation. These data demonstrate that in vitro functional analysis of pyrin inflammasome activation can differentiate SURF from FMF and PFAPA patients, suggesting the involvement of the pyrin inflammasome in the pathophysiology of SURF.

Pyrin variant E148Q potentiates inflammasome activation and the effect of pathogenic mutations in cis.

OBJECTIVE: The p.E148Q variant in pyrin is present in different populations at a frequency of up to 29%, and has been associated with diseases, including vasculitis and FMF. The pathogenicity of p.E148Q in FMF is unclear, even when observed in cis or in trans to a single, typically recessive, pathogenic mutation. We performed functional validation to determine whether p.E148Q increases the ability of pyrin to form an active inflammasome complex in cell lines. METHODS: We interrogated the Australian Autoinflammatory Disease RegistrY (AADRY) to find candidate inheritance patterns for the p.E148Q variant in pyrin. Different pyrin variant combinations were tested in HEK293T cells stably expressing the adaptor protein apoptosis-associated speck-like (ASC), which were analysed by flow cytometry to visualize inflammasome formation, with and without stimulation by Clostridioides difficile toxin B (TcdB). Inflammasome-dependent cytokine secretion was also quantified by ELISA of supernatants from THP-1 cells transduced with lentiviral expression vectors. RESULTS: In AADRY, we observed the p.E148Q allele in individuals with autoinflammatory diseases alone or in conjunction with other pyrin variants. Two FMF families harboured the allele p.E148Q-M694I in cis with dominant heritability. In vitro, p.E148Q pyrin could spontaneously potentiate inflammasome formation, with increased IL-1ß and IL-18 secretion. p.E148Q in cis to classical FMF mutations provided significant potentiation of inflammasome formation. CONCLUSION: The p.E148Q variant in pyrin potentiates inflammasome activation in vitro. In cis, this effect is additive to known pathogenic FMF mutations. In some families, this increased effect could explain why FMF segregates as an apparently dominant disease.

Characteristics and course of patients with AA amyloidosis: single centre experience with 174 patients from Turkey.

OBJECTIVES: This study aimed to evaluate the clinical, laboratory and genetic characteristics and outcomes of patients with AA amyloidosis. METHODS: Patients followed up in a tertiary referral centre in Turkey with the diagnosis of inflammatory rheumatic diseases and immunohistologically proven AA amyloidosis were included in the study and retrospectively analysed. RESULTS: Among 184 patients with the diagnosis of AA amyloidosis, 174 (83 female, 91 male) were included in the analysis. The most common cause of AA amyloidosis was FMF (78.7%), and 91% of FMF-AA amyloidosis patients were carrying the p.M694V variant (74.1% homozygous). AA amyloidosis was identified earlier in patients with homozygous or compound heterozygous MEFV exon 10 variants compared with the heterozygous patients (27, 30 and 41 years, respectively). Patients with an estimated glomerular filtration rate <60 ml/min at admission had a higher frequency of progression to end-stage renal disease (P < 0.001). The overall mortality rate was 15.3% and it increased gradually in association with the amyloid burden (10% in patients with renal, 15% in renal + gastrointestinal and 43% in those with additional cardiac involvement). Renal findings responded completely to treatment in 31% of the patients, a partial response was observed in 4%, a stable course in 23.6% and progression in 38.5%. Amyloid storm was identified in nine patients and was found to be associated with increased mortality within 1 year. CONCLUSION: FMF patients still constitute the majority of AA amyloidosis patients in Turkey. The MEFV genotype and associated inflammatory load may affect the age of onset of AA amyloidosis, and earlier diagnosis and stricter follow-up and treatment may delay progression of the disease.

Gut microbiota alterations are associated with phenotype and genotype in familial Mediterranean fever.

OBJECTIVE: FMF is the most common monogenic autoinflammatory disease associated with MEFV mutations. Disease phenotype and response to treatment vary from one patient to another, despite similar genotype, suggesting the role of environmental factors. The objective of this study was to analyse the gut microbiota of a large cohort of FMF patients in relation to disease characteristics. METHODS: The gut microbiotas of 119 FMF patients and 61 healthy controls were analysed using 16 s rRNA gene sequencing. Associations between bacterial taxa, clinical characteristics, and genotypes were evaluated using multivariable association with linear models (MaAslin2), adjusting on age, sex, genotype, presence of AA amyloidosis (n = 17), hepatopathy (n = 5), colchicine intake, colchicine resistance (n = 27), use of biotherapy (n = 10), CRP levels, and number of daily faeces. Bacterial network structures were also analysed. RESULTS: The gut microbiotas of FMF patients differ from those of controls in having increased pro-inflammatory bacteria, such as the Enterobacter, Klebsiella and Ruminococcus gnavus group. Disease characteristics and resistance to colchicine correlated with homozygous mutations and were associated with specific microbiota alteration. Colchicine treatment was associated with the expansion of anti-inflammatory taxa such as Faecalibacterium and Roseburia, while FMF severity was associated with expansion of the Ruminococcus gnavus group and Paracoccus. Colchicine-resistant patients exhibited an alteration of the bacterial network structure, with decreased intertaxa connectivity. CONCLUSION: The gut microbiota of FMF patients correlates with disease characteristics and severity, with an increase in pro-inflammatory taxa in the most severe patients. This suggests a specific role for the gut microbiota in shaping FMF outcomes and response to treatment.

Old paradigms and new concepts in familial Mediterranean fever (FMF): an update 2023.

Familial Mediterranean fever (FMF) is a hereditary autoinflammatory disease characterized by recurrent attacks of fever and polyserositis. Its first description as a new entity was published by Siegal in 1945. Colchicine has been the treatment of choice for this disease since 1972. Significant progress has been made over the years in understanding FMF's clinical features, diagnosis, mode of inheritance, pathogenesis and therapeutic approach. However, many old paradigms related to FMF have proven inaccurate, leading to the emergence of new concepts that provide more precise insights. The term 'FMF' is no longer appropriate as the disease is found beyond the Mediterranean basin. The concept of diagnosis based only upon clinical ground proved to be wrong. The paradigm that MEFV mutations in FMF lead to loss of function of the encoded peptide pyrin turned out to be a gain of function mutation. Finally, the concept that as a genetic disease FMF should be treated for life was found to be inaccurate for the subpopulation of the heterozygote patients. Thus, the breakthroughs of identifying the gene associated with the disease (MEFV) and the deciphering of its pathogenesis revolutionized our old paradigms and replaced them with new and more precise insights.

The significance of carrying MEFV variants in symptomatic and asymptomatic individuals.

Familial Mediterranean fever (FMF) is an autoinflammatory disease characterized by recurrent attacks of fever, serositis (peritonitis, pleuritis, or synovitis), and erysipelas-like erythema. Genetic variants in the MEFV gene are associated with this disease. Familial Mediterranean fever is considered an autosomal recessive disease. However, in Middle Eastern countries, a third of the patients expressing FMF manifestations, carry a single mutation only. Moreover, some cases of pure dominant inheritance linked to specific single MEFV variants have also been described. This complex inheritance of MEFV-associated inflammatory diseases poses a serious challenge when interpreting the results of genetic testing in patients having recurrent fever syndromes. In addition, in certain situations, asymptomatic individuals may be incidentally found to carry MEFV variants. These cases pose the question of their exact diagnosis and whether they should be treated. Previous studies have focused on genetic results interpretations among symptomatic patients. In the current article, we would like to elaborate on the genetic interpretation in cases of symptomatic individuals suspected to have FMF and on asymptomatic individuals carrying MEFV variants. We aim to assist physicians unfamiliar with FMF to cope with genetic results interpretation when facing symptomatic and asymptomatic individuals carrying MEFV variants and suggest a management plan accordingly.

Experimental models in Familial Mediterranean Fever (FMF): Insights into pathophysiology and therapeutic strategies.

Familial Mediterranean Fever (FMF) is a recurrent polyserositis characterized by self-limiting episodes or attacks of fever along with serosal inflammation. It mainly impacts people of the Mediterranean and Middle Eastern basin. FMF is a recessive autoinflammatory condition caused by mutation in the MEFV gene located on chromosome 16p13. MEFV mutations lead to the activation of the pyrin inflammasome resulting in an uncontrolled release of IL-1ß. Various in vitro, in vivo and ex vivo experimental models have been developed to further comprehend the etiology and pathogenesis of FMF. These models have been proven to be clinically relevant to human FMF and can provide significant information about biological systems with respect to this condition. Additionally, these models have provided pertinent contributions to the development of potent therapeutic strategies against FMF. In this review, we describe the different experimental models utilized in FMF and we focus primarily on the most widely used models that have produced prominent insights into the pathophysiology of the disease.

Molecular analyses of MEFV gene mutation variants in Turkish population.

BACKGROUND: Familial Mediterranean fever (FMF) is an autosomal recessive autoinflammatory disease primarily affecting individuals of Turkish, Armenian, Arab, and non-Ashkenazi Jewish descent, caused by mutations in the MEFV gene. The aim of this study was to review the common genotype distributions of MEFV variants and mutations in the Turkish population and evaluate rare mutations. METHODS AND RESULTS: The study included 2984 patients who applied to Ankara University Ibni Sina Hospital Immunology Laboratory with clinical suspicion of FMF between 2004 and 2014. The data of patients from different regions of the country who were followed up in the immunology-rheumatology clinic with clinical suspicion and presumptive diagnosis of FMF were evaluated retrospectively. Patients were tested for all mutations in Exon 2 and Exon 10, including M694V, M680I, M694I, V726A, E148Q and R202Q. There were 2504 patients with FMF variant. According to genotyping, R202Q (n = 1567, 39.2%) was the most common mutation. The most common co-variant was the R202Q/M694V genotype (n = 507, 16.98%). Allele frequencies for MEFV mutations were as follows: R202Q (n = 1567, 39.2%), M694V (n = 1004, 25.1%), E148Q (n = 463, 11.5%), M680I (n = 354, 8.8%), V726A (n = 319, 7.9%), A744S (n = 51, 1.2%), R761H (N = 41, 1.0%), P706P (N = 25, 0.6%), E167D (N = 23, 0.5%), M694I (N = 23, 0.5%), and K695R (N = 20, 0.5%). CONCLUSION: This research revealed the prevalence of both common and rare MEFV gene mutations in Turkish FMF patients in various age groups. R202Q was the most prevalent mutation.

Molecular investigation of MEFV gene polymorphisms among patients with familial mediterranean fever-like symptoms.

The diagnosis of familial Mediterranean fever (FMF) is primarily based on clinical standards. The purpose of this study was to investigate the relevance of Mediterranean fever (MEFV) genetic testing in the diagnosis of FMF as well as to identify the most frequent variant alleles and their relationship to clinical symptoms in Egyptian patients. Egyptian patients with a clinical suspicion of having FMF were studied in order to determine MEFV genotypes. Each patient was meticulously evaluated through an extensive collection of their medical history, a thorough clinical examination, and a series of laboratory tests, encompassing CBC, ESR, and CRP measurements. The MEFV variant screening procedure included the use of reverse dot blot hybridization. The average age of our patients when they were given a diagnosis was 22.8 ± 1.404 years old. The predominant clinical manifestations identified were abdominal pain, fever, and arthralgia.  Molecular interrogation of the MEFV gene unveiled that a significant proportion of the cohort, constituting 72 individuals (60%), displayed heterozygosity, whereas a smaller fraction, comprising 12 subjects (10%), demonstrated homozygosity and an equivalent number (10%) exhibited compound heterozygosity. Pertaining to the distribution of allele variants, E148Q emerged as the most prevalent, succeeded by M694I, accounting for 12.5% of the cases, and M680I (G/A), representing 10.41%. This notable prevalence of heterozygous genotypes among the Egyptian demographic, preliminarily identified as potential FMF cases, underscores the imperative for molecular diagnostics to enhance the precision of FMF identification.

The Impact of Different MEFV Genotypes on Clinical Phenotype of Patients with Familial Mediterranean Fever: Special Emphasis on Joint Involvement.

Familial Mediterranean Fever (FMF) is the most common monogenic autoinflammatory disease worldwide. In this retrospective cohort study, we aimed to assess the effects of various MEFV genotypes on the clinical characteristics of the patients, with a special focus on the joint involvement. In total, 782 patients with FMF were categorized into 3 groups according to the MEFV mutation; Group 1: Patients homozygous for M694V; Group 2: Patients carrying other pathogenic MEFV variants in exon 10 in homozygous or compound heterozygous states; and Group 3: FMF patients with other variants or without mutations. Clinical and demographic findings were compared between groups. Among the 782 FMF patients, total frequency of arthritis was 237 (30.3%): 207 (26.4%) were acute monoarthritis and 67 (8.5%) were chronic arthritis. Both the frequency of arthritis (acute and/or chronic) (40.4% vs. 24.8% vs. 26.7%; p:0.001) and acute monoarthritis (35.4% vs. 20% vs. 23.7%; p:0.001) were significantly higher in Group 1 than in the other groups. FMF patients with chronic arthritis showed a distinct juvenile idiopathic arthritis (JIA) distribution pattern with a more frequent enthesitis-related arthritis (ERA) subtype (43.2%). HLA-B27 was positive in 24% of the ERA patients.Conclusion: Homozygous M694V mutation is associated with a more frequent and longer acute monoarthritis comparing to other MEFV genotypes. In addition, the risk of chronic arthritis seems not related to the MEFV mutations. However, FMF patients with chronic arthritis show a distinct ILAR JIA distribution pattern with a more frequent ERA and undifferentiated arthritis subtype. What is known: • Homozygous M694V mutation is associated with a more frequent and longer acute monoarthritis What is new: • FMF patients with chronic arthritis show a distinct ILAR JIA distribution pattern with a more frequent ERA subtype • ERA patients with negative HLA-B27 antigen should also be assessed for polyserositis episodes of FMF, especially in countries with high FMF carrier frequency.

E148Q variant: a familial Mediterranean fever-causing mutation or a sequence variant?

Familial Mediterranean fever (FMF) is an autosomal recessive autoinflammatory disease, linked to mutations in the MEFV gene. The p.E148Q variant, found on exon 2, has an uncertain role in FMF, with debates on whether it is a benign polymorphism or a pathogenic mutation. This study aimed to assess the clinical characteristics and severity of FMF in patients homozygous for the p.E148Q variant and to evaluate the impact of the p.V726A variant in these patients. This retrospective cohort study analyzed data from electronic medical records at Carmel Medical Center, Israel. Patients who underwent genetic testing for FMF from November 2004 to December 2019 and had p.E148Q/p.E148Q or p.E148Q/p.E148Q + p.V726A variants were included. Disease severity was assessed using the Tel Hashomer Key to Severity Score. Statistical analyses compared clinical characteristics and severity between genotype groups. The study included 61 FMF patients, with 24 (39%) having p.E148Q/p.E148Q and 37 (61%) having p.E148Q/p.E148Q + p.V726A variants. The majority (72%) were Druze. Most patients (65.5%) exhibited mild disease, while 31.1% had moderate disease, with no cases of severe disease. Colchicine treatment significantly reduced CRP levels in all patients. CONCLUSION: These findings suggest that the p.E148Q variant, whether alone or with p.V726A, generally results in mild to moderate FMF severity, supporting its pathogenic role in particular ethnicity. These results contribute to understanding the clinical significance of the p.E148Q variant and considering the patient's need for Colchicine treatment. WHAT IS KNOWN: • The role of the p.E148Q variant in FMF is debated, with questions about whether it is a benign polymorphism or a pathogenic mutation. • The prevalence of MEFV variants can vary significantly among different ethnic groups. WHAT IS NEW: • The p.E148Q variant has clinical significance in particular ethnicities, as supported by a significant reduction in CRP levels following colchicine treatment. • The p.E148Q variant, whether alone or with p.V726A, generally results in mild to moderate FMF severity.

Exploring factors for predicting colchicine responsiveness in children with PFAPA.

Periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis syndrome (PFAPA) are the most common autoinflammatory syndromes in children. This study aimed to evaluate the clinical and laboratory parameters that may predict colchicine responsiveness.This retrospective, multicenter, cross-sectional study involved nine pediatric rheumatology centers from our country., The patients diagnosed with PFAPA were compared on the basis of their responses to colchicine. In the 806 (42.3% female 57.7% male) patients, the most common clinical findings were fever (100%), exudative tonsillitis (86.5%), pharyngitis (80.9%), and aphthous stomatitis (50.5%). The mean attack frequency was 13.5 ± 6.8 attacks per year lasting for a mean of 3.9 ± 1.1 days. Colchicine treatment was attempted in 519 (64.4%) patients, with 419 (80.7%) showing a favorable response. In patients who underwent MEFV gene analysis (70.8%), the most common variant was M694V heterozygous (16.8%). The presence of pharyngitis (p = 0.03, 95% CI 0.885 to 0.994), the presence of arthralgia (p = 0.04, 95% CI 0.169 to 0.958), and having more frequent attacks (p = 0.001, 95% CI 0.028 to 0.748) were found to be associated with colchicine unresponsiveness, whereas the carriage of the M694V variant (p = 0.001, 95% CI 0.065 to 0.242) was associated with colchicine responsiveness. CONCLUSION: This study identified the presence of pharyngitis, arthralgia, and increased attack frequency in patients with PFAPA as factors predicting colchicine unresponsiveness, whereas the carriage of the M694V variant emerged as a predictor of colchicine responsiveness. Predicting colchicine response at disease onset may facilitate a more effective management of PFAPA. WHAT IS KNOWN: • Colchicine treatment can be used in the prophylaxis of PFAPA disease. • Having the MEFV variant is the most commonly known factor in predicting response to colchicine. WHAT IS NEW: • The presence of pharyngitis or arthralgia, and more frequent attacks in PFAPA disease were found to be independently associated with colchicine unresponsiveness. • Carrying the M694V variant was identified as the sole factor predicting colchicine responsiveness.

Plasminogen activator inhibitor-1 genotype 4G/5G associates with skin involvement in Armenian familial Mediterranean fever patients.

There is little and conflicting data on the role of the plasminogen activator inhibitor-1 (PAI-1, SERPINE1) 4G/5G polymorphism in familial Mediterranean fever (FMF). Therefore this study aimed at evaluating the impact of this polymorphism on the disease course in a cohort of 303 Armenian FMF patients. Genotyping for 12 Mediterranean fever (MEFV) gene mutations and the PAI-1 4G/5G (rs1799762) polymorphism were performed by PCR/reverse-hybridization (StripAssay) and real-time PCR, respectively. PAI-1 genotypes 4G/4G, 4G/5G, and 5G/5G could be identified in 4 (5.88%), 30 (18.63%) and 9 (12.16%) patients with erysipelas-like erythema (ELE), while this was the case for 64 (94.12%), 131 (81.37%), and 65 (87.84%) patients without ELE, respectively (P < 0.033). We have identified a significant relationship between the PAI-1 4G/5G genotype and the occurence of ELE in a relatively large cohort of Armenian FMF patients. Because of conflicting results concerning the impact of this polymorphism on the clinical course of FMF in different populations, further studies are desirable to substantiate the findings reported here.

Clinical presentation and genetic variants in patients with autoinflammatory diseases: results from the German GARROD registry.

To investigate clinical symptoms and genetic variants in patients from the German anti-IL-1 registry for autoinflammatory orphan diseases (GARROD) between 2013 and 2022. Multicentre, retrospective analysis of demographic, clinical and genetic data of patients with autoinflammatory diseases (AID) who received anti-IL-1 targeted therapy. The cohort comprised 152 patients with familial Mediterranean fever (FMF; n = 71), cryopyrin-associated periodic syndromes (CAPS; n = 43), TNF-receptor associated periodic syndrome (TRAPS; n = 19), mevalonate kinase deficiency (MKD; n = 3) and unclassified AID (uAID; n = 16). Inflammatory attacks started in 61.2% of the patients before the age of 18 years. The delay between the first AID attack and anti-IL-1 therapy was 17.8 years. Monogenetic AIDs were diagnosed by clinical symptoms. Genetic analyses confirmed the diagnosis in 87.3% of patients with FMF, 65.2% with CAPS and 94.8% with TRAPS. Among this group, heterozygous MEFV variants and variants of unknown significance (VUS) were detected in 22.5% of patients with FMF, 51.2% with CAPS and 47.4% with TRAPS. Patients with VUS were older at disease onset which is consistent with a milder phenotype. Twenty-four patients had secondary AA amyloidosis (AA) at initiation of anti-IL-1 therapy. The mean age of these patients was 16.4 years at their first attack and 44.9 years at the time of AA diagnosis. Turkish-Armenian ancestry correlated with MEFV variants and higher FMF disease activity compared to German ancestry. Molecular genetic analyses should substantiate the clinical diagnosis of a monogenetic AID. Our data support the concept of variable penetrance of VUS which can be associated with late-onset AID.

Next-generation sequencing based newborn screening and comparative analysis with MS/MS.

BACKGROUND: Newborn screening (NBS), such as tandem mass spectrometry (MS/MS), may yield false positive/negative results. Next-generation sequencing (NGS) has the potential to provide increased data output, efficiencies, and applications. This study aimed to analyze the types and distribution of pathogenic gene mutations in newborns in Huzhou, Zhejiang province, China and explore the applicability of NGS and MS/MS in NBS. METHODS: Blood spot samples from 1263 newborns were collected. NGS was employed to screen for pathogenic variants in 542 disease-causing genes, and detected variants were validated using Sanger sequencing. Simultaneously, 26 inherited metabolic diseases (IMD) were screened using MS/MS. Positive or suspicious samples identified through MS/MS were cross-referenced with the results of NGS. RESULTS: Among all newborns, 328 had no gene mutations detected. NGS revealed at least one gene mutation in 935 newborns, with a mutation rate of 74.0%. The top 5 genes were FLG, GJB2, UGT1A1, USH2A, and DUOX2. According to American College of Medical Genetics guidelines, gene mutations in 260 cases were classified as pathogenic or likely pathogenic mutation, with a positive rate of 20.6%. The top 5 genes were UGT1A1, FLG, GJB2, MEFV, and G6PD. MS/MS identified 18 positive or suspicious samples for IMD and 1245 negative samples. Verification of these cases by NGS results showed no pathogenic mutations, resulting in a false positive rate of 1.4% (18/1263). CONCLUSION: NBS using NGS technology broadened the range of diseases screened, and enhanced the accuracy of diagnoses in comparison to MS/MS for screening IMD. Combining NGS and biochemical screening would improve the efficiency of current NBS.