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.

Updates on the role of epigenetics in familial mediterranean fever (FMF).

Familial Mediterranean Fever (FMF) is an autosomal recessive autoinflammatory disease caused by mutations in the MEFV (MEditerranean FeVer) gene that affects people originating from the Mediterranean Sea. The high variability in severity and clinical manifestations observed not only between ethnic groups but also between and within families is mainly related to MEFV allelic heterogeneity and to some modifying genes. In addition to the genetic factors underlying FMF, the environment plays a significant role in the development and manifestation of this disease through various epigenetic mechanisms, including DNA methylation, histone modification, and noncoding RNAs. Indeed, epigenetic events have been identified as an important pathophysiological determinant of FMF and co-factors shaping the clinical picture and outcome of the disease. Therefore, it is essential to better understand the contribution of epigenetic factors to autoinflammatory diseases, namely, FMF, to improve disease prognosis and potentially develop effective targeted therapies. In this review, we highlight the latest updates on the role of epigenetics in FMF.

Epigenetic insights into Familial Mediterranean Fever: Increased RGS10 expression and histone modifications accompanies inflammation in familial Mediterranean fever disease.

BACKGROUND: Familial Mediterranean fever (FMF) is an autosomal recessive autoinflammatory disease characterized by recurring fever, erythema, joint pain, and abdominal discomfort during acute episodes. While FMF patients typically share MEFV gene mutations, they display varying clinical manifestations, suggesting the involvement of modifying genes, epigenetic mechanisms, or environmental factors. G protein regulator signal 10 (RGS10), a member of the RGS protein family, exhibits anti-inflammatory effects in autoinflammatory diseases. There are no studies on the role of plays in FMF pathogenesis or histone modification in FMF. AIMS: This study aimed to shed light on the epigenetic regulation of FMF from several perspectives. The relationship between RGS10 DNA hypermethylation in FMF clinical parameters and the regulation of 22 histone modifications were examined in FMF attack patients and the control group. METHODS: Sixty FMF (remission/attack) and thirty healthy individuals were included in the study. First, RNA was isolated from the blood of patients/controls, and the expression of RGS10 was examined. Then, DNA was isolated from the patients, and gene-specific hypermethylation was investigated using the bisulfite conversion method. Finally, histone extraction was performed for FMF patients and controls and 22 histone H3 modifications were determined. In addition, using ADEX bioinformatics tools, RGS10 expression and methylation profiles were detected in different autoinflammatory diseases. RESULTS: This study indicate that RGS10 expression decreased in attack-free/attack patients than control, attributed to DNA methylation. In addition, there were a positive correlation between FMF patients and attack, WBC, neutrophil, MCHC and MPV. Moreover, higher H3K4 me3, H3K9 me2, and H3K14ac levels were observed in patients with FMF attacks. This research also showed a consistent decrease in RGS10 expression in patients with SjS, SSc, and T1D compared with controls. I also obtained five prognosis-related CpGs (cg17527393, cg19653161, cg20445950, cg18938673 and cg13975098) of RGS10 in patients with SjS, RA, SSc, SLE and T1D. CONCLUSION: The present study provides insights into the complex relationship between RGS10, epigenetic modifications, and immune responses in FMF. While RGS10 may initially enhance immune responses, genetic mutations and epigenetic changes associated with FMF acute episode may override this regulatory effect, resulting in increased inflammation and clinical symptoms. Moreover, our study revealed elevated levels of specific histone modifications in the context of FMF, suggesting significant epigenetic changes that could contribute to the disease pathogenesis. Understanding these associations opens new avenues for research and potential therapeutic interventions, potentially involving epigenetic therapies targeting histone modifications.

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.

Unraveling the genome: Familial Mediterranean fever.

ABSTRACT: Familial Mediterranean fever (FMF) is an inherited, autoinflammatory disease with a high prevalence in Middle Eastern and Mediterranean populations including Turks, Iranian, Spanish, Sephardic Jews, Arabs, and other Mediterranean ethnic groups. Autoinflammatory diseases are genetically predetermined disorders with multisystem effects primarily caused by defects in innate immunity. Although primarily known for an autosomal recessive mode of inheritance, there are increasing case reports associated with single Mediterranean fever (MEFV) mutation or dominant transmission. There have been over 300 variants identified in the MEFV gene; however, roughly 9-11 variants are responsible for the phenotypical expression seen with FMF. Symptoms include recurrent episodes of fever of unknown origin, abdominal, chest, or joint pain because of serosal inflammation. Persistent elevations in serum amyloid A can lead to complications like renal amyloidosis, kidney dysfunction, and end-stage kidney disease. Familial Mediterranean fever is diagnosed clinically using the Tel-Hashomer criteria and confirmed through genetic testing. Treatment includes initiation of colchicine with the goal of stopping attacks and preventing renal dysfunction and end-stage kidney disease. Genetic testing helps to identify the specific mutation allowing the provider to create a patient-specific treatment plan, monitor for complications such as renal amyloidosis, and enhance knowledge on the genetic heterogeneity and possible epigenetic factors.

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.

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.

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.

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.

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.

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.

Inflammatory comorbidities in the largest pediatric Familial Mediterranean fever cohort: a multicenter retrospective study of Pediatric Rheumatology Academy (PeRA)-Research Group (RG).

AIM: The aim of this study was to investigate the frequency and type of FMF-associated inflammatory diseases in a large FMF pediatric patients and to compare them to those FMF patients without concomitant inflammatory diseases. MATERIALS AND METHODS: Familial Mediterranean fever patients enrolled in the Pediatric Rheumatology Academy (PeRA)-Research Group (RG) were included. The patients were divided into two groups according to concomitant inflammatory disease as FMF patients who had a concomitant inflammatory disease (group 1) and FMF patients who did not have a concomitant inflammatory disease (group 1). The clinical findings and treatments were compared between the two groups. RESULTS: The study group comprised 3475 patients with FMF. There were 294 patients (8.5%) in group 1 and 3181 patients (91.5%) in group 2. Juvenile idiopathic arthritis (n = 136) was the most common accompanying inflammatory disease. Arthritis, M694V homozygosity, and the need for biological therapy were more frequently observed in Group 1 (p < 0.05). Fever and abdominal pain were more frequently detected in Group 2 (p < 0.05). FMF patients with concomitant inflammatory diseas more frequently demonstrated colchicine resistance. There were no significant differences in the median attack frequency, chest pain, amyloidosis, erysipelas-like erythema, or family history of FMF between the two patient groups. CONCLUSION: To the best of our knowledge, this is the largest pediatric cohort reviewed to date. FMF patients may have different clinical profiles and colchicine responses if they have with concomitant inflammatory diseases. Key points • FMF is associated with some inflammatory comorbidities diseases. • To the best of our knowledge, this is the largest cohort evlauated pediatric FMF associated inflammatory comorbidities diseases reviewed to date.

Recent Insights in Pyrin Inflammasome Activation: Identifying Potential Novel Therapeutic Approaches in Pyrin-Associated Autoinflammatory Syndromes.

Pyrin is a cytosolic protein encoded by the MEFV gene, predominantly expressed in innate immune cells. Upon activation, it forms an inflammasome, a multimolecular complex that enables the activation and secretion of IL-1ß and IL-18. In addition, the Pyrin inflammasome activates Gasdermin D leading to pyroptosis, a highly pro-inflammatory cell death. Four autoinflammatory syndromes are associated with Pyrin inflammasome dysregulation: familial Mediterranean fever, hyper IgD syndrome/mevalonate kinase deficiency, pyrin-associated autoinflammation with neutrophilic dermatosis, and pyogenic arthritis, pyoderma gangrenosum, and acne syndrome. In this review, we discuss recent advances in understanding the molecular mechanisms regulating the two-step model of Pyrin inflammasome activation. Based on these insights, we discuss current pharmacological options and identify a series of existing molecules with therapeutic potential for the treatment of pyrin-associated autoinflammatory syndromes.