Identification of RNF185 as an ubiquitin ligase invovled in the endoplasmic reticulum-assoicated degradation (ERAD) of CFTR

N.cN.

Identification de RNF185 comme une ubiquitine ligase impliquée dans la voie de dégradation associée au réticulum endoplasmique (ERAD) de CFTR

N.cN.cPARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Somatic mosaic mutation in TNFRSF1A as a cause of Tumor necrosis factor receptor-associated periodic syndrome- Impact on genetic counselling

Introduction: Tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS) is a rare monogenic systemic autoinflammatory disease caused mainly by germline heterozygous missense mutations in exons 2-4 of the TNF receptor super family member 1A (TNFRSF1A) gene. TRAPS is known as a familial disorder of autosomal dominant inheritance. Objective: To identify the genetic defect underlying the disease in a patient with a phenotype evocative of TRAPS. Methods: (i) The patient’s DNA was analyzed by Sanger sequencing of TNFRSF1A exons 2-4 followed by a NGS approach based on the targeted sequencing of the main genes involved in systemic autoinflammatory disorders; and (ii) Subpopulations of peripheral blood cells were subsequently analyzed in order to study the cellular distribution of the identified molecular defect.Results: (i) Clinical phenotype: A 36-year-old man presented since the age of 24 with recurrent episodes of fever (38-40C°) lasting for 1-2 weeks. The febrile attacks were accompanied with severe abdominal, thoracic and lower back pain, myalgia, and transitory erythematous eruptions localized at elbows and left lower limb. He also had an acute episode of pericarditis. No lymphadenopathy, ophthalmological or neurological symptoms were observed. Laboratory tests showed elevated inflammatory biological markers: increased serum levels of CRP (171 mg/l) with hyperleukocytosis (15000/l). Serum ferritin levels and kidney function tests were normal. (ii) Molecular bases: No heterozygous mutation was identified after Sanger sequencing of exons 2-4 of TNFRSF1A. The patient’s phenotype, highly evocative of TRAPS, prompted us to deepen the molecular investigations through the use of our autoinflammatory disorders NGS panel. We identified the c.176G>A p.(Cys59Tyr) TNFRSF1A mutation in a mosaic state with 14% of mutated allele in whole blood. This mutation (also known as C30Y) has already been reported, but in the heterozygous state, in three TRAPS families. We further studied the distribution of the mutated alleles in blood cells and observed a higher percentage of the mutated allele in myeloid cells (i.e. 15.8% in monocytes and 14.1% in neutrophils) than in lymphoid cells (i.e., 9.6% in T cells and 8.9% in B cells). Discussion: So far, only 3 other patients have been reported with a somatic mutation in TNFRSF1A. Among them, one displayed a germinal mosaicism. In our patient, the mosaicism predominantly involved the myeloid lineage (involved in the pathophysiology of TRAPS). In the 4 patients now identified with a TNFRSF1A somatic mosaic mutation, the phenotypic severity of TRAPS appears not to be related to the germinal/mosaic status of TNFRSF1A mutations.Conclusion: Somatic mosaic TNFRSF1A mutations should be sought in sporadic cases of TRAPS with no mutation identified by Sanger sequencing. Genetic counseling should take into account the fact that somatic mosaic mutations are not restricted to myeloid cells and may be present in other cell lineages

Mosaic variants in TNFRSF1A : an emerging cause of tumour necrosis factor receptor-associated periodic syndrome

International audienceObjective To identify the molecular basis of a systemic autoinflammatory disorder (SAID) evocative of TNF receptor-associated periodic syndrome (TRAPS). Methods (i) Deep next generation sequencing (NGS) through a SAID gene panel; (ii) variant allele distribution in peripheral blood subpopulations; (iii) in silico analyses of mosaic variants using TNF receptor superfamily 1A (TNFRSF1A) crystal structure; (iv) review of the very rare TNFRSF1A mosaic variants reported previously. Results In a 36-year-old man suffering from recurrent fever for 12 years, high-depth NGS revealed a TNFRSF1A mosaic variant, c.176G>A p.(Cys59Tyr), which Sanger sequencing failed to detect. This mosaic variant displayed a variant allele fraction of 14% in whole blood; it affects both myeloid and lymphoid lineages. p.(Cys59Tyr), a recurrent germline pathogenic variant, affects a crucial cysteine located in the first cysteine-rich domain (CRD1) and involved in a disulphide bridge. Introduction of a tyrosine at this position is expected to disrupt the CRD1 structure. Review of the three previously reported TNFRSF1A mosaic variants revealed that they are all located in a small region of CRD2 and that germinal cells can be affected. Conclusion This study expands the localization of TNFRSF1A mosaic variants to the CRD1 domain. Noticeably, residues involved in germline TNFRSF1A mutational hot spots can also be involved in post-zygotic mutational events. Including our study, only four patients have been thus far reported with TNFRSF1A mosaicism, highlighting the need for a high-depth NGS-based approach to avoid the misdiagnosis of TRAPS. Genetic counselling has to consider the potential occurrence of TNFRSF1A mosaic variants in germinal cells

Chronic hepatic involvement in the clinical spectrum of A20 haploinsufficiency

International audienceBackground & aims: Secondary to tumour necrosis factor-alpha induced protein 3 (TNFAIP3) mutations, A20 haploinsufficiency (HA20) is a recently described autoinflammatory disease with clinical features similar to those of Behçet's and Crohn's diseases but with a constantly expanding clinical spectrum. Here, we describe HA20 liver involvement in three new patients from the same family.Methods: We retrospectively assessed clinical, biological and/or histological findings for eight patients over three generations of the same family with heterozygous mutations in the TNFAIP3 gene (c.259C > T, p.Arg87*).Results: The eight patients exhibited the following: aphthous ulcers (8/8, bipolar in 7), autoimmune features (6/8, including 5 with definitive autoimmune disease diagnoses, ie, type I diabetes, Hashimoto thyroiditis, pernicious anaemia, and/or 5 with antinuclear antibodies ≥320), pustulosis/folliculitis (5/8), abdominal pain (4/8), arthralgia (3/8), enlarged cervical lymph nodes (3/8) and pericarditis (1/8). In addition, three patients (twin sisters and their grandmother aged 23 and 70 years, respectively) exhibited persistent mild hepatic cytolysis associated with splenomegaly (n = 3), hepatomegaly (n = 1) and/or liver atrophy (n = 1) on echography. We could not detect any other causes of chronic liver diseases. Liver biopsies from three patients displayed hepatic fibrosis, hepatocyte injury and/or CD4+ /CD8+ T lymphocyte infiltration, and patterns of inflammatory cells and NLRP3 or NF-κB immunostaining differed from the predominant neutrophil infiltration observed in skin or some digestive tract biopsies.Conclusions: This study reinforces the dual involvement of innate and adaptive immunity in HA20 according to both acute and chronic injury and the organ involved and widens its clinical spectrum to include chronic hepatic involvement

CCDC65 , encoding a component of the axonemal Nexin‐Dynein regulatory complex, is required for sperm flagellum structure in humans

Abstract Sperm flagella share an evolutionary conserved microtubule‐based structure with motile cilia expressed at the surface of several cell types, such as the airways epithelial cells. As a result, male infertility can be observed as an isolated condition or a syndromic trait, illustrated by Primary Cilia Dyskinesia (PCD). We report two unrelated patients showing multiple morphological abnormalities of the sperm flagella (MMAF) and carrying distinct homozygous truncating variants in the PCD‐associated gene CCDC65 . We characterized one of the identified variants ( c.1208del ; p.Asn403Ilefs*9), which induces the near absence of CCDC65 protein in patient sperm. In Chlamydomonas , CCDC65 ortholog (DRC2, FAP250) is a component of the Nexin‐Dynein Regulatory complex (N‐DRC), which interconnects microtubule doublets and coordinates dynein arms activity. In sperm cells from the patient, we also show the loss of GAS8, another component of the N‐DRC, supporting a structural/functional link between the two proteins. Our work indicates that, similarly to ciliary axoneme, CCDC65 is required for sperm flagellum structure. Importantly, our work provides first evidence that mutations in the PCD‐associated gene CCDC65 also cause asthenozoospermia

Autoinflammation liée à l’haploinsuffisance A20 : identification et caractérisation fonctionnelle de nouveaux variants A20

A20 est une enzyme d'édition de l'ubiquitine codée par le gène A20 (ou TNFAIP3, tumor necrosis factor a-induced portien 3). A travers son activité E3 ligase et déubiquitinase, A20 régule plusieurs substrats de la voie NF-kappaB, tels que RIP1, NEMO et TRAF6. La protéine A20 joue un rôle central dans la régulation de l'inflammation et de l'immunité en inhibant cette voie de signalisation. Les premières mutations d’A20 décrites en 2016 conduisaient à une haploinsuffisance, donnant ainsi le nom haploinsuffisance A20 (HA20) à cette maladie auto-inflammatoire (MAI) de transmission autosomique dominante. Le phénotype de HA20 est partiellement chevauchant avec celui de la maladie de Behçet mais se caractérise par un âge de début de maladie précoce ; il associe des accès récurrents de fièvre, des ulcères buccaux et génitaux, une atteinte gastro-intestinale avec diarrhée, des arthralgies ou polyarthrites et des uvéites antérieures bilatérales. Avec plus de 25 mutations tronquantes de A20 rapportées à ce jour, HA20 est une MAI monogénique fréquemment diagnostiquée. Cependant, la signification pathogénique des variations faux-sens de A20 a été peu étudiée est reste difficile à établir. Dans cette étude, nous avons identifié chez six patients indépendants présentant une MAI, cinq nouvelles variations de A20 dont le retentissement fonctionnel a été évalué par des tests cellulaires in vitro : deux variations responsables d’un décalage de cadre de lecture et trois faux-sens. Les deux variations tronquantes c.716dup (p.Ala240Cysfs*14) et c.1504del (p.Arg502Glyfs*195), entraînent une haploinsuffisance due à une dégradation des transcrits A20 par non-sens mRNA decay. La variation faux-sens c.707T>C (p.Leu236Pro) (de classe 3 selon les recommandations de l’American College of Medical Genetics), est responsable d’une dégradation accrue de la protéine mutée (suivi de la stabilité de la protéine et de la dégradation par le protéasome). La baisse d’expression de la protéine A20-Leu236Pro a été par ailleurs confirmée dans les PBMCs provenant des patients. En plus du défaut d’expression, la protéine A20-Leu236Pro présente un défaut fonctionnel avec une moindre suppression de l'activité NF-kappaB que la protéine A20 normale. Quant aux deux autres variations faux-sens identifiées : c.464C>T (p.Thr155Met) et c.237C>G (p.Ser79Arg), de classe 3 également, elles n’étaient responsables d’aucune anomalie dans les tests in vitro utilisés (étude de l’expression de la protéine et de l’activité NF-kappaB). Au total, cette étude souligne l’importance d’évaluer le retentissement fonctionnel des variations faux-sens identifiées dans le gène A20 chez les patients chez qui un diagnostic de HA20 est suspecté : les résultats de ces analyses conditionnent la prise en charge thérapeutique des patients et le conseil génétique

Identification of IQCH as a calmodulin-associated protein required for sperm motility in humans

Summary: Sperm fertilization ability mainly relies on proper sperm progression through the female genital tract and capacitation, which involves phosphorylation signaling pathways triggered by calcium and bicarbonate. We performed exome sequencing of an infertile asthenozoospermic patient and identified truncating variants in MAP7D3, encoding a microtubule-associated protein, and IQCH, encoding a protein of unknown function with enzymatic and signaling features. We demonstrate the deleterious impact of both variants on sperm transcripts and proteins from the patient. We show that, in vitro, patient spermatozoa could not induce the phosphorylation cascades associated with capacitation. We also provide evidence for IQCH association with calmodulin, a well-established calcium-binding protein that regulates the calmodulin kinase. Notably, we describe IQCH spatial distribution around the sperm axoneme, supporting its function within flagella. Overall, our work highlights the cumulative pathological impact of gene mutations and identifies IQCH as a key protein required for sperm motility and capacitation

Autoinflammatory Diseases: Germline vs. Somatic Mosaic Variations

National audienceDuring the period of 2018-2023, we have identified 10 pathogenic variations in the NLRP3 gene (Figure 1) including:-8 different mosaic variations in 11 independent patients-2 different germline variations in 3 independent patients. A total of 34 mosaic and 88 germline NLRP3 variations have been reported in the literature to date (Figure 1). Clinically, NLRP3 mosaic variations are associated with various NLRP3-AID phenotypes ranging from late-onset urticaria in the context of MWS to CINCA. Conclusions This study emphasizes the high frequency of somatic mosaic variations in NLRP3-AIDs. The phenotypic spectrum of NLRP3-AIDs appears to be related to both the germinal/mosaic status and the localization in cryopyrin of the underlying variations. Mosaic variations in the NLRP3, TNFRSF1A, and NLRC4 genes can cause symptoms even when present at very low allele frequencies (below 5%). Furthermore, misdiagnosis may occur due to the low allele frequency of mosaic variations

A critical region of A20 unveiled by missense TNFAIP3 variations that lead to autoinflammation

International audienceA20 haploinsufficiency (HA20) is an autoinflammatory disease caused by heterozygous loss-of-function variations in TNFAIP3 , the gene encoding the A20 protein. Diagnosis of HA20 is challenging due to its heterogeneous clinical presentation and the lack of pathognomonic symptoms. While the pathogenic effect of TNFAIP3 truncating variations is clearly established, that of missense variations is difficult to determine. Herein, we identified a novel TNFAIP3 variation, p.(Leu236Pro), located in the A20 ovarian tumor (OTU) domain and demonstrated its pathogenicity. In the patients’ primary cells, we observed reduced A20 levels. Protein destabilization was predicted in silico for A20_Leu236Pro and enhanced proteasomal degradation was confirmed in vitro through a flow cytometry-based functional assay. By applying this approach to the study of another missense variant, A20_Leu275Pro, for which no functional characterization has been performed to date, we showed that this variant also undergoes enhanced proteasomal degradation. Moreover, we showed a disrupted ability of A20_Leu236Pro to inhibit the NF-κB pathway and to deubiquitinate its substrate TRAF6. Structural modeling revealed that two residues involved in OTU pathogenic missense variations (i.e. Glu192Lys and Cys243Tyr) establish common interactions with Leu236. Interpretation of newly identified missense variations is challenging, requiring, as illustrated here, functional demonstration of their pathogenicity. Together with functional studies, in silico structure analysis is a valuable approach that allowed us (i) to provide a mechanistic explanation for the haploinsufficiency resulting from missense variations and (ii) to unveil a region within the OTU domain critical for A20 function