MEFV Antibody

What is the MEFV gene and what is its significance in research?

The MEFV gene provides instructions for making a protein called pyrin (also known as marenostrin), which plays a critical role in regulating inflammation. While pyrin's function is not fully understood, it likely assists in controlling the inflammatory process by modulating immune signaling pathways .

The MEFV gene is of particular research interest because:

• It is associated with Familial Mediterranean Fever (FMF), an autoinflammatory disorder characterized by recurrent fevers and inflammation

• MEFV mutations have been linked to other inflammatory conditions beyond FMF, including Behçet's disease

• It serves as a model for studying autoinflammatory mechanisms and genetic influences on inflammation

Pyrin is expressed primarily in granulocytes, monocytes, and dendritic cells, making these cell types important models for MEFV research .

What are the recommended applications and protocols for MEFV antibodies?

Based on validated research applications, MEFV antibodies can be used in multiple experimental approaches with specific recommended protocols:

Important methodological consideration: It is recommended that researchers titrate the antibody in each specific testing system to obtain optimal results, as reactivity can be sample-dependent .

How can researchers properly store and handle MEFV antibodies to maintain functionality?

For optimal results with MEFV antibodies, researchers should follow these evidence-based storage and handling protocols:

• Store at -20°C in the storage buffer (PBS with 0.02% sodium azide and 50% glycerol pH 7.3)

• Antibodies remain stable for one year after shipment when properly stored

• Aliquoting is unnecessary for -20°C storage

• Small volume formats (20 μl) typically contain 0.1% BSA as a stabilizer

• Avoid repeated freeze-thaw cycles to prevent antibody degradation

How can researchers distinguish between pathogenic and non-pathogenic MEFV variants?

Differentiating pathogenic from non-pathogenic MEFV variants presents a significant challenge, with many variants historically classified as "variants of uncertain significance" (VUS). Recent methodological approaches have improved classification:

Recommended methodology: Combine the Rare Exome Variant Ensemble Learner (REVEL) metapredictor tool with pyrin domain structural analysis.

This integrated approach has:

• Reduced VUS proportion from 61.6% to 17.6% in recent studies

• Established a gene-specific threshold of 0.298 for pathogenicity prediction

• Demonstrated strong correlation with expert consensus classifications

Additionally, researchers should consider:

• Non-random distribution of pathogenic variants across pyrin's functional domains

• Correlation between specific variants (e.g., M694V) and clinical phenotypes including amyloidosis risk

• Development of functional assays measuring IL-1β and IL-18 secretion ratios, which have shown efficacy in distinguishing FMF patients from controls

What experimental models are most effective for studying MEFV function and mutations?

When designing experiments to investigate MEFV function and mutational effects, researchers should consider these validated cellular and molecular models:

Cellular models:

• THP-1 cells (human monocytic cell line): Validated for MEFV expression studies, IP experiments, and gene silencing approaches

• A431 cells: Suitable for immunofluorescence and flow cytometry applications with MEFV antibodies

• SW982 cells: Effective for transfection studies examining miRNA effects on MEFV expression

Methodological approaches:

• Gene silencing experiments using siRNA to observe downstream inflammatory marker changes

• miRNA transfection studies (e.g., pre-miR-197) to examine effects on inflammatory cytokine expression

• Real-time RT-PCR using TaqMan Gene Expression Assays targeting exon junction 9-10, with B2M as internal control

• Hybridization-based resequencing systems for comprehensive MEFV genomic analysis

How do researchers address discrepancies between genotype and phenotype in MEFV-related conditions?

One of the most challenging aspects of MEFV research is resolving cases where patients have clinical FMF but carry only one identifiable MEFV mutation or show phenotypic variability despite similar mutations.

Recommended investigative approaches:

• Extended genomic analysis: Examine intronic and regulatory regions of MEFV

  • For example, studies identified a novel variant in the regulatory region 1kb upstream (c.-888G>A) in a North American patient when standard coding region screening was insufficient

• Modifier gene evaluation: Assess additional genetic factors that may influence phenotypic expression

  • The SAA1 gene has been shown to modify amyloidosis risk in patients with the M694V MEFV mutation

• Microbiome characterization: Recent findings suggest gut microbiota influences FMF severity

  • FMF patients co-infected with Helicobacter pylori experience more severe and frequent attacks

  • Small Intestinal Bacterial Overgrowth (SIBO) has been linked to more severe symptoms in FMF patients

• miRNA profiling: Analyze differential expression of miRNAs that may regulate MEFV

  • miR-197-3p has demonstrated anti-inflammatory effects by decreasing IL-1β and MEFV expression in experimental models

What methodological considerations are important when using MEFV antibodies for detecting specific mutations?

When designing experiments to detect MEFV mutations using antibody-based approaches, researchers should consider:

• Epitope location relative to mutations: Ensure the antibody's epitope does not overlap with the mutation site of interest, which could affect binding

• Validation across mutation types: Commercial MEFV antibodies (such as 24280-1-AP) have been tested for reactivity with human samples but may require additional validation when studying specific mutations

• Complementary techniques: Combine antibody-based detection with:

  • Fluorescent sequencing of coding regions and splice junctions

  • Hybridization-based resequencing systems that can detect single nucleotide variations and small insertions/deletions

  • PCR amplification with overlapping fragments to rule out genomic deletions

• Controls for non-specific binding: Use appropriate negative controls, particularly when working with less common mutations

How can researchers investigate the role of MEFV in diseases beyond FMF?

Studies have revealed MEFV's potential involvement in other inflammatory conditions, creating opportunities for broader research applications:

Methodological approaches for investigating MEFV in non-FMF conditions:

• For Behçet's Disease (BD):

  • Compare endoscopic findings between patients with and without MEFV mutations

  • Assess refractoriness to treatment based on mutation status

  • Analyze cytokine expression patterns, particularly in intestinal manifestations

• For Palindromic Rheumatism (PR):

  • Stratify patients based on anti-citrullinated protein antibody (ACPA) status

  • Investigate the unexpectedly high frequency (22.2%) of MEFV mutations in ACPA-negative PR patients

  • Compare demographic and clinical characteristics between mutation carriers and non-carriers

• Recommended experimental design:

  • Include appropriate disease controls without MEFV mutations

  • Match subjects for age, sex, and ethnicity to control for population-specific variant frequencies

  • Perform comprehensive genotyping beyond common mutations

What are the latest techniques for functional characterization of MEFV variants?

Recent advances have expanded the toolbox for functional characterization of MEFV variants:

• Inflammasome activation assays: Measure ASC speck formation and IL-1β/IL-18 processing in response to different MEFV variants

• Functional IL-1β/IL-18 secretion ratio analysis: This approach has emerged as an effective diagnostic tool and can be applied to variant characterization

  • Whole blood tests measuring IL-1β have shown particular utility in improving diagnosis of FMF and related conditions

• Structural biology approaches: Analyze how variants affect the structure and interaction surfaces of pyrin domains

  • This is particularly important as pyrin normally works with ASC to create an inflammasome complex

• In silico classification tools: Integrate multiple prediction algorithms through metapredictors like REVEL, which have shown good correlation with expert classifications

How can researchers accurately assess the impact of MEFV variants on treatment response?

When investigating how MEFV variants influence treatment efficacy, consider these methodological approaches:

• Stratification by mutation type: Different mutations may respond differently to therapies

  • For example, in Behçet's Disease patients, MEFV mutation carriers showed different treatment requirements compared to non-carriers

• Combined genomic and clinical response analysis: Document treatment responses across:

  • Colchicine (standard therapy)

  • Biological agents (IL-1 antagonists, TNF inhibitors)

  • Novel therapeutic approaches

• Diet-genotype interaction studies: Recent findings suggest diet may modify treatment response

  • Lower salt and fat intake might enhance colchicine response

  • Anti-inflammatory diets with supplements have shown promise in improving symptoms in patients on colchicine therapy

• Longitudinal monitoring protocols: Implement standardized monitoring of:

  • Attack frequency and severity

  • Inflammatory markers

  • Development of complications (particularly amyloidosis)

This research approach has significant implications for personalized medicine in FMF and related disorders.

What considerations are important when interpreting contradictory MEFV mutation data?

Researchers frequently encounter contradictory data when studying MEFV mutations, requiring careful methodological approaches:

• Population-specific variant interpretation: MEFV mutation frequencies vary significantly across different ethnic groups

  • FMF primarily affects people from the Eastern Mediterranean (Turks, Jews, Arabs, and Armenians)

  • The novel variant c.-888G>A has a 4% frequency in Caucasian controls and 3% in Ashkenazi Jewish controls

• Incomplete penetrance analysis: Many individuals with MEFV mutations never develop FMF symptoms

  • Design experiments to investigate molecular differences between symptomatic and asymptomatic carriers

• Complex inheritance patterns: While FMF is traditionally considered recessive, clinical disease can occur with a single mutation

  • 46 FMF patients with only one high-penetrance mutation were extensively studied to find second mutations, suggesting other genetic or environmental factors may be involved

• Bioinformatic reclassification approaches: Apply current classification algorithms to historical variants of uncertain significance for consistent interpretation

By addressing these aspects systematically, researchers can better interpret contradictory data and advance understanding of MEFV pathophysiology.

How should researchers optimize protocols for Western blot applications of MEFV antibodies?

When using MEFV antibodies for Western blot applications, researchers should consider these technical optimizations:

• Lysate preparation: THP-1 cells and human plasma have been validated as positive controls

  • Sample processing should maintain protein integrity to preserve the 86 kDa molecular weight observed for MEFV

• Dilution optimization: Start with the recommended 1:1000-1:5000 range and adjust based on signal strength

  • Antibody titration is essential as optimal concentration may vary between different experimental systems

• Detection considerations: The expected molecular weight of MEFV protein is 86 kDa (based on its 781 amino acids)

  • Both calculated and observed molecular weights align at 86 kDa, simplifying band identification

• Appropriate controls: Include positive controls (THP-1 cells) and negative controls based on experimental design

What methodological approaches are recommended for studying the interaction between MEFV and other inflammatory pathways?

To investigate interactions between MEFV/pyrin and other inflammatory pathways, researchers should consider:

• Co-immunoprecipitation studies: Use MEFV antibodies at 0.5-4.0 μg per 1.0-3.0 mg of total protein lysate to pull down protein complexes

  • THP-1 cells have been validated for IP applications with MEFV antibodies

• Inflammasome complex analysis: Investigate pyrin's interaction with ASC to form inflammasomes

  • This interaction initiates processes leading to IL-1β and IL-18 release

• miRNA regulatory network analysis:

  • Studies show miR-197-3p reduces expression of both IL-1β and MEFV genes when transfected into cells

  • TGF-β and TNF-α expression levels were also decreased (though non-significantly) in pre-miR-197 transfected cells

• Gene silencing approaches: MEFV silencing in THP-1 cells has been shown to significantly increase expression of miR-4520a, which targets RHEB, an activator in the mTOR pathway

These approaches provide complementary insights into MEFV's role in inflammatory regulation and pathway interactions.

How can researchers address antibody specificity issues when studying MEFV?

When encountering specificity challenges with MEFV antibodies, implement these systematic approaches:

• Validation across multiple applications: If specificity issues arise in one application, verify performance in alternative applications

  • For example, if WB results are ambiguous, confirm with IF/ICC or flow cytometry

• Positive control selection: Use validated positive controls for your specific application:

  • WB: THP-1 cells, human plasma

  • IP: THP-1 cells

  • IF/ICC: A431 cells

  • Flow cytometry: A431 cells (intracellular staining)

• Blocking optimization: Adjust blocking conditions to minimize non-specific binding

  • The antibody's storage buffer (PBS with 0.02% sodium azide and 50% glycerol pH 7.3) may influence optimal blocking parameters

• Multiple antibody comparison: When critical results are obtained, confirm with alternative MEFV antibodies targeting different epitopes

What approaches can resolve discrepancies between genetic findings and functional outcomes in MEFV research?

When genetic data and functional outcomes appear contradictory, consider these methodological approaches:

• Comprehensive variant analysis: Extend beyond common mutations to identify rare or novel variants

  • In one study, extensive genomic analysis of the MEFV region identified a novel variant (c.-888G>A) in the regulatory region of the gene

• Consider gene expression variation: Examine whether MEFV expression levels differ between samples with similar genetic profiles

  • Real-time RT-PCR using exon junction 9-10 primers with B2M as internal control can quantify relative expression

• Epigenetic and regulatory analysis: Investigate whether epigenetic modifications or regulatory elements affect gene expression

  • Include analysis of microRNAs like miR-197-3p that have been shown to influence MEFV expression

• Environmental and microbiome factors: Consider environmental triggers and microbiome composition

  • The gut microbiome has been linked to FMF severity, with specific pathogens like H. pylori associated with attack frequency and severity