Identification of a new exon 2-skipped TNFR1 transcript: regulation by three functional polymorphisms of the TNFR-associated periodic syndrome (TRAPS) gene.

BACKGROUND: Mutations in the TNFRSF1A gene encoding the tumour necrosis factor α cell surface receptor, TNFR1, cause TNFR-associated periodic syndrome (TRAPS) and polymorphisms in TNFRSF1A, including rs4149570, rs767455 and rs1800692, are associated with inflammatory diseases. OBJECTIVES: To describe a new exon 2-spliced transcript-TNFR1-d2-and the impact of these three single nucleotide polymorphisms on exon 2 splicing, transcriptional activity of TNFRSF1A and TRAPS phenotype. METHODS: Expression of TNFRSF1A transcripts was performed by reverse-transcription-PCR in a range of human cells and tissues. Exon 2 splicing and transcriptional activity were analysed in HEK293T and SW480 cells by in vitro alternative splicing and luciferase assays, respectively. We constructed haplotypes containing rs4149570, rs767455 and rs1800692 in controls (n=72), patients with TRAPS (n=111) and in TRAPS-like patients (n=450) to compare their distribution and association with clinical features of TRAPS. RESULTS: TNFR1-d2 was expressed in a tissue-specific manner, whereas TNFR1 expression was ubiquitous. Alternative splicing assays showed that the T-A-T haplotype at rs4149570-rs767455-rs1800692 had a significantly higher expression of exon 2-skipping product (p=0.02) compared with the G-G-C haplotype. Transcriptional activity from the T-T haplotype at rs4149570-rs1800692 was increased compared with the G-C haplotype (p=0.03). In patients with TRAPS, rs1800692 T/T homozygotes were excessively rare (p<10(-4)) and TRAPS-like patients with this genotype experienced less fever. CONCLUSIONS: Our study provides a new mechanism of TNFRSF1A regulation whereby three polymorphisms in the promoter, exon 1 and intron 4 have a functional and combined effect on exon 2 splicing, via a coupling mechanism between transcription and splicing. These polymorphisms may affect the phenotype of TRAPS and TRAPS-like patients.

Expression of the familial Mediterranean fever gene is regulated by nonsense-mediated decay.

Mutations in the MEditerranean FeVer (MEFV) gene are responsible for familial Mediterranean fever (FMF), a recessively inherited auto-inflammatory disease. Cases of dominant inheritance and phenotype-genotype heterogeneity have been reported; however, the underlying molecular mechanism is not currently understood. The FMF protein named pyrin or marenostrin (P/M) is thought to be involved in regulating innate immunity but its function remains subject to controversy. Recent studies postulate that a defect in MEFV expression regulation may play a role in FMF physiopathology. Our group, along with others, has identified several alternatively spliced MEFV transcripts in leukocytes. Since alternative splicing and nonsense-mediated decay (NMD) pathways are usually coupled in the post-transcriptional regulation of gene expression, we hypothesized that NMD could contribute to the regulation of the MEFV gene. To address this issue, we examined the effect of indirect and direct inhibition of NMD on expression of the MEFV transcripts in THP1, monocyte and neutrophil cells. We showed that MEFV is the first auto-inflammatory gene regulated by NMD in both a cell- and transcript-specific manner. These results and preliminary western-blot analyses suggest the possible translation of alternatively spliced MEFV transcripts into several P/M variants according to cell type and inflammatory state. Our results introduce the novel hypothesis that variation of NMD efficiency could play an important role in FMF physiopathology as a potent phenotypic modifier.

Impact of cannabis use on outcomes of patients admitted to an involuntary psychiatric unit: A retrospective cohort study.

INTRODUCTION: Cannabis is associated with an increased risk of mental disorders, including the onset or adverse evolution of schizophrenia, bipolar disorder and depression. The aim of our study was to examine how cannabis use influences length of stay and readmissions in a unit of involuntary care. METHOD: All patients admitted to one secure adult psychiatry unit in France in 2016 were included (n = 370). Patients were assigned to one of two groups according to detection of urinary tetrahydrocannabinol (THC + or -). Clinical outcomes, such as length of stay and readmissions, were compared between the groups in multivariate analyses. RESULTS: We identified 130 THC + patients and 240 THC- patients. THC + patients were often young men. In adjusted analyses, THC + status was significantly associated with one-year readmissions (OR = 2.29, p = .0082) and more prescriptions of benzodiazepines (OR = 1.93, p = .02), but not antipsychotics, at discharge. CONCLUSIONS: Cannabis users seem to have a particular profile in secure units, and are associated with specific diagnoses and treatments, and a higher risk of readmissions. Adapted management strategies might be warranted for these patients.

TNFR1-d2 carrying the p.(Thr79Met) pathogenic variant is a potential novel actor of TNFα/TNFR1 signalling regulation in the pathophysiology of TRAPS.

Binding of tumour necrosis factor α (TNFα) to its receptor (TNFR1) is critical for both survival and death cellular pathways. TNFα/TNFR1 signalling is complex and tightly regulated at different levels to control cell fate decisions. Previously, we identified TNFR1-d2, an exon 2-spliced transcript of TNFRSF1A gene encoding TNFR1, whose splicing may be modulated by polymorphisms associated with inflammatory disorders. Here, we investigated the impact of TNFRSF1A variants involved in TNFR-associated periodic syndrome (TRAPS) on TNFR1-d2 protein expression and activity. We found that TNFR1-d2 could be translated by using an internal translation initiation codon and a de novo internal ribosome entry site (IRES), which resulted in a putative TNFR1 isoform lacking its N-terminal region. The kinetic of assembly of TNFR1-d2 clusters at the cell surface was reduced as compared with full-length TNFR1. Although co-localized with the full-length TNFR1, TNFR1-d2 neither activated nuclear factor (NF)-κB signalling, nor interfered with TNFR1-induced NF-κB activation. Translation of TNFR1-d2 carrying the severe p.(Thr79Met) pathogenic variant (also known as T50M) was initiated at the mutated codon, resulting in an elongated extracellular domain, increased speed to form preassembled clusters in absence of TNFα, and constitutive NF-κB activation. Overall, TNFR1-d2 might reflect the complexity of the TNFR1 signalling pathways and could be involved in TRAPS pathophysiology of patients carrying the p.(Thr79Met) disease-causing variant.

Search for copy number alterations in the MEFV gene using multiplex ligation probe amplification, experience from three diagnostic centres.

Familial mediterranean fever (FMF) is a hereditary autoinflammatory autosomal recessive disease caused by mutations in the MEFV gene. Despite the identification of many disease associated MEFV mutations, often the clinical diagnosis cannot be genetically confirmed. The currently used diagnostic sequencing techniques only allow the detection of point mutations, small deletions or duplications. The question as to whether larger genetic alterations are also involved in the pathophysiology of FMF remains to be answered. To address this question, we used multiplex ligation-dependent probe amplification (MLPA) on a total of 216 patients with FMF symptoms. This careful analysis revealed that not a single deletion/duplication could be detected in this large cohort of patients. This result suggests that single or multiexon MEFV gene copy number changes do not contribute substantially, if at all, to the MEFV mutation spectrum.

TGF beta1 polymorphisms are candidate predictors of the clinical response to rituximab in rheumatoid arthritis.

OBJECTIVE: To evaluate the association between several candidate single-nucleotide polymorphisms (SNPs) and responsiveness to rituximab in patients with rheumatoid arthritis (RA). METHODS: Sixty-three RA patients were included. Nine genes (13 SNPs) were subsequently analyzed, including those coding for cytokines involved in synovitis (IL10, LTA, TGFß1, TNF-α, TNF receptor II) and genes associated with RA susceptibility (-C5 TRAF1, STAT4, TNFAIP3 and PTPN22). RESULTS: Forty-four patients were defined as responders and 19 as nonresponders. TGFß1 Codon 10 and TGFß1 Codon 25 SNPs were both associated with clinical response (probability to respond to treatment with the Codon 10C/T genotype: OR = 1.6; P = 0.002, and with the Codon 25 G/C genotype: OR = 1.6; P = 0.025). The probability to be a responder when the TGFß Codon10 C/T and TGFß Codon 25 G/C genotypes were co-inherited, doubled (OR = 2.6; P = 0.008). CONCLUSION: The TGFß1 SNPs are associated with a good response to rituximab therapy and as such could be useful genetic biomarkers in predicting therapy outcome.

A comparison of restriction fragment length polymorphism, tetra primer amplification refractory mutation system PCR and unlabeled probe melting analysis for LTA+252 C>T SNP genotyping.

BACKGROUND: From the wide range of methods currently available for genotyping, we wished to identify a quick, reliable and affordable approach for routine use in our laboratory for LTA+252 C>T SNP screening. METHODS: We set up and compared three genotyping methods for SNP detection: restriction fragment length polymorphism (RFLP), tetra primer amplification refractory mutation system PCR (TPAP) and unlabeled probe melting analysis (UPMA). The SNP model used was LTA+252 C>T, a cytokine gene polymorphism that has been associated with response to treatment in rheumatoid arthritis. The study was performed using 46 samples from healthy Caucasian volunteers. RESULTS: Allele and genotype distribution was similar to that previously described in the same population. All three genotyping methods showed good reproducibility and are suitable for a medium scale throughput molecular platform. UPMA was the most cost effective, reliable and safe method since it required the shortest technician time, could be performed in a single closed tube and involved automatic data analysis. CONCLUSION: This work is the first to compare these three genotyping techniques and provides evidence for UPMA being the method of choice for LTA+252 C>T SNP genotyping.

Combined mutation and rearrangement screening by quantitative PCR high-resolution melting: is it relevant for hereditary recurrent Fever genes?

The recent identification of genes implicated in hereditary recurrent fevers has allowed their specific diagnosis. So far however, only punctual mutations have been identified and a significant number of patients remain with no genetic confirmation of their disease after routine molecular approaches such as sequencing. The possible involvement of sequence rearrangements in these patients has only been examined in familial Mediterranean fever and was found to be unlikely. To assess the existence of larger genetic alterations in 3 other concerned genes, MVK (Mevalonate kinase), NLRP3 (Nod like receptor family, pyrin domain containing 3) and TNFRSF1A (TNF receptor superfamily 1A), we adapted the qPCR-HRM method to study possible intragenic deletions and duplications. This single-tube approach, combining both qualitative (mutations) and quantitative (rearrangement) screening, has proven effective in Lynch syndrome diagnosis. Using this approach, we studied 113 unselected (prospective group) and 88 selected (retrospective group) patients and identified no intragenic rearrangements in the 3 genes. Only qualitative alterations were found with a sensitivity similar to that obtained using classical molecular techniques for screening punctual mutations. Our results support that deleterious copy number alterations in MVK, NLRP3 and TNFRSF1A are rare or absent from the mutational spectrum of hereditary recurrent fevers, and demonstrate that a routine combined method such as qPCR-HRM provides no further help in genetic diagnosis. However, quantitative approaches such as qPCR or SQF-PCR did prove to be quick and effective and could still be useful after non contributory punctual mutation screening in the presence of clinically evocative signs.

Activation of a dimeric metabotropic glutamate receptor by intersubunit rearrangement.

Although many G protein-coupled receptors (GPCRs) can form dimers, a possible role of this phenomenon in their activation remains elusive. A recent and exciting proposal is that a dynamic intersubunit interplay may contribute to GPCR activation. Here, we examined this possibility using dimeric metabotropic glutamate receptors (mGluRs). We first developed a system to perfectly control their subunit composition and show that mGluR dimers do not form larger oligomers. We then examined an mGluR dimer containing one subunit in which the extracellular agonist-binding domain was uncoupled from the G protein-activating transmembrane domain. Despite this uncoupling in one protomer, agonist stimulation resulted in symmetric activation of either transmembrane domain in the dimer with the same efficiency. This, plus other data, can only be explained by an intersubunit rearrangement as the activation mechanism. Although well established for other types of receptors such as tyrosine kinase and guanylate cyclase receptors, this is the first clear demonstration that such a mechanism may also apply to GPCRs.