Research (Inserm U844)

Physiopathology of auto-inflammatory diseases
FMF
TRAPS
Behçet

Development and transfer of new approaches for diagnosis and evaluation of prognosis
Set up of new tools for diagnosis
New auto-inflammatory genes and candidate genes
Signature of hereditary recurrent fevers
Pharmacogenetics of chronic arthritis

Physiopathology of auto-inflammatory diseases

FMF

Familial Mediterranean fever (FMF) is a recessive auto-inflammatory disease commonly found in Mediterranean populations. Genetic diagnosis has developed since the discovery of the causative gene MEFV in 1997 by our consortium Pub. This gene expressed mainly in leucocytes, encodes the protein Marenostrine/Pyrine (M/P). Colchicine remains the most effective treatment, although, in an unexplained way, approximately 5 to 10 % of the patients do not response to it.
          M/P is thought to play a key role in the regulation of inflammation. Mutations in this protein would trigger a protracted and inappropriate inflammatory response. However, very little is known so far about the nature of the regulating pathways or steps involved in the disease. One of the main characteristics of the inflammatory attacks in FMF patients is the massive chemotaxis of neutrophiles at the inflammatory sites. Moreover, apoptosis is enhanced in monocytes and neutrophiles upon several pathogenic danger signal such as E. coli, whereas phagocytosis is decreased in FMF monocytes.

1. Previous results

Our efforts until 2002 lead to identify the FMF gene (MEFV) Pub, to define phenotype-genotype correlation Pub, to evaluate the risk factors for renal amyloidosis Pub and to highlight MICA as the first modifier locus in FMF Pub. We also showed that MEFV is a factor of susceptibility for Behçet Pub and of severity for Crohn Pub.
               We studied the mRNA expression of the MEFV gene and of cytokines in FMF patients. We showed a reduced mRNA expression Pub and an enhanced cytokine mRNA levels in patients Pub

2. Recent results

Characterisation of the MEFV promoter Pub
               Promoter mapping assays revealed a region important for regulating expression, a putative enhancer element between -571 and -414. As many patients could not be confirmed genetically by routine exon screening, we searched for mutations in the 5’-flanking region of this gene. We found 6 novel sequence variants in a region extending -825bp upstream of the first translated codon. Among them, c.-614C>G resulted in a 70% decrease in promoter activity, while c.-382C>T induced a 100% increase in activity, when compared to the wild type. We observed specific DNA-protein binding to both wild type sites, suggesting that transcription factors may bind to these sequences to modulate MEFV expression.

Post-transcriptional regulation of MEFV expression
               To examine the splicing pattern of MEFV, we screened peripheral blood leucocytes mRNAs from 60 patients and 34 healthy individuals using RT-PCR. Five novel splicing events were observed. Their expression in both patients and controls is not in favour of a severe pathogenic effect. However, these results suggest that MEFV expression is subjected to a complex transcriptional regulation.
               Since alternative splicing and nonsense-mediated decay (NMD) pathways are usually coupled in the post-transcriptional regulation of gene expression, we examined the effect of indirect and direct inhibition of NMD on expression of the MEFV transcripts in THP1, monocyte and neutrophil cells Pub. We showed that MEFV is the first auto-inflammatory gene regulated by NMD in both a cell- and transcript-specific manner. This result suggests the possible translation of alternatively spliced MEFV transcripts into several protein variants according to cell type and inflammatory state. We propose that variation of NMD efficiency could play an important role in FMF pathophysiology as a potent phenotypic modifier.

3. Questions addressed in our laboratory

The aim of our research projects on FMF is to determine the implication of the protein Marenostrin/Pyrin and the effect of colchicin in the steps of inflammation in monocytes/macrophages and neutrophiles.

(1) Are there functional protein variants of the MEFV gene?
               (2) What is the role of M/P in chemotaxis ?
               (3) What is the role of M/P in the resolution of inflammation?

Contact: Isabelle Touitou or Sylvie Grandemange

TRAPS

TRAPS (TNFR-Associated Periodic Syndrome) is a dominantly inherited hereditary recurrent fever. It appears in the early childhood. Glucocorticoïdes remains the most effective treatment in this disease even if some patients respond poorly or badly to the treatment. Pathophysiological studies on the disease made it possible to consider novel biotherapeutics such as anti-TNF or anti-IL1 molecules.
         The causative gene TNFRSF1A encodes the TNF receptor 1 (TNFR1). Binding of TNFa to TNFR1 activates both the NF-kB transcription factor which in turn induces expression of pro-inflammatory cytokines, and apoptosis via caspase 8.
          Several hypotheses have been suggested to account for the TRAPS disease, i.e. defect of the TNFR1 receptor shedding, the binding of the receptor from the endoplasmic reticulum to the cytoplasmic membrane, NF-kB activation, and apoptosis. However, no clear phenotype–genotype correlation has been delineated.
          Our team coordinates a European-supported consortium EuroTRAPS that is devoted to this disease.

1. Recent results
            We investigated the transcript pattern of the TNFRSF1A gene from individuals’ controls and patients and identified a new splicing isoform lacking exon 2, that we named TNFR1-d2. Its expression is tissue-specific in contrast to that of the full-length transcript which is ubiquitous.

2. Questions addressed in our laboratory
            The aim of our research projects on TRAPS is to determine the function of TNFR1-d2 and its implication in the pathophysiology of TRAPS.

            (1) How is the expression of TNFR1-d2 regulated?
            (2) What is the function of TNFR1-d2 ?

Contact: Isabelle Touitou or Sylvie Grandemange

Behçet’s disease

Behçet’s disease (BD) is a multifactorial chronic and relapsing auto-inflammatory disorder. It is a multisystemic vasculitis of unknown origin. It has been mainly described in young adults from Eastern Asia and Mediterranean countries. Because of this population specificity, the existence of familial cases, and the strong association with HLAB51, it is considered to have a strong genetic component. BD is difficult to recognize and its diagnosis only relies on the clinical criteria defined in 1990. Its prognosis is serious, at the functional level (blindness, encephalopathy) but also at the vital level (thromboses, aneurysm).

1. Previous results
            We and others showed that MEFV and TNFRSF1 mutations are more frequent in BD Pub. We screened 3 more auto-inflammatory genes (MVK, PSTPIP1, and LPIN2) in 97 BD patients and 51 matched healthy controls to test them as possible additional susceptibility factors for BD. This study could not evidence a significant increase of mutations in BD patients as compared to controls Pub.
            We have shown an increase of familial aggregation in pediatric compared with non-pediatric patients Pub. We then provided the first evidence of genetic heterogeneity in BD, and of the existence of a Mendelian entity in the paediatric BD subgroup Pub

2. Questions addressed in our laboratory
            The aim of our research projects on BD is to identify the putative gene in early onset Behçet patients

Contact: Isabelle Touitou or Mouna Barat

Development and transfer of new approaches for diagnosis and evaluation of prognosis

Set up of new tools for diagnosis

Connections between all auto-inflammatory diseases from clinical but also genetic being increasingly narrow, it appears essential to us to improve the screening techniques of sequences variants.

We have set up high resolution melting curves (HRM) and quantitative analysis for CNVs (copy number variants) identification. We screened 10 hereditary AID genes and found several new punctual mutations but no large gene rearrangements Pub.

A wide number of methods have been developed for genotyping. We searched for a quick reliable and affordable approach for SNP screening in our laboratories. We have set up the following methods: TPAP (tetra primer amplification refractory mutation system PCR), UPMA (unlabeled probe melting analysis) and TaqMan SNP genotyping assay.

Contact: Isabelle Touitou or Laurent Philibert or Nathalie Pallares-Ruiz

New auto-inflammatory genes and candidate gene

1. Recent results
            NLRP7 (Nucleotide-binding domain, leucine-rich repeat family, pyrin domain containing 7) is related to NLRP3 and NLRP12, two genes responsible of hereditary recurrent fevers. Unexpectedly, NLRP7 was recently associated with recurrent hydatidiform moles, a rare cause of reproductive wastage. We implemented this gene in our diagnosis facilities, and could find several new mutations Pub

2. Questions addressed in our laboratory
            (1) Which candidate genes are responsible for hereditary reproductive wastage in NLRP7 negative women?
            (2) Which candidate inflammatory genes are responsible for hereditary auto-inflammatory diseases in patients without genetically confirmed diagnosis?

Contact: Isabelle Touitou or Laurent Philibert or Cécile Rittore

Signature of hereditary recurrent fevers

Although the 4 principal recurring fevers (FMF, TRAPS, CAPES and MKD) are genetically different, in clinical these 4 entities have common points. Each one showing a phenotypic and genetic heterogeneity (absence of mutations in spite of specific clinical signs), it is sometimes difficult to pose a precise diagnosis on which therapeutic effective depends. It thus appeared significant to us to analyze other ways enabling us to help the classification of these diseases.

The aim of the research projects is to define a specific molecular signature of the hereditary recurring fevers

Contact: Isabelle Touitou or Sylvie Grandemange

Pharmacogenetics of chronic arthritis

The discovery of the target defective signalling pathway in auto-inflammatory diseases prompted clinicians to test new anti-TNF and anti-IL-1 biotherapeutics. These were effective in most patients, but some of them remained refractory or even develop side effects.

The aim of our research projects on chronic arthritis is to identify a genetic signature of the response to treatment in rheumatoid arthritis, juvenile arthritis, and TRAPS by the combined impact of a set of 40 candidate polymorphisms already reported in the literature.

Contact: Isabelle Touitou or Cécile Rittore