68 research outputs found
Loss-of-Function Mutations in LRRC6, a Gene Essential for Proper Axonemal Assembly of Inner and Outer Dynein Arms, Cause Primary Ciliary Dyskinesia
Primary ciliary dyskinesia (PCD) is a group of autosomal-recessive disorders resulting from cilia and sperm-flagella defects, which lead to respiratory infections and male infertility. Most implicated genes encode structural proteins that participate in the composition of axonemal components, such as dynein arms (DAs), that are essential for ciliary and flagellar movements; they explain the pathology in fewer than half of the affected individuals. We undertook this study to further understand the pathogenesis of PCD due to the absence of both DAs. We identified, via homozygosity mapping, an early frameshift in LRRC6, a gene that encodes a leucine-rich-repeat (LRR)-containing protein. Subsequent analyses of this gene mainly expressed in testis and respiratory cells identified biallelic mutations in several independent individuals. The situs inversus observed in two of them supports a key role for LRRC6 in embryonic nodal cilia. Study of native LRRC6 in airway epithelial cells revealed that it localizes to the cytoplasm and within cilia, whereas it is absent from cells with loss-of-function mutations, in which DA protein markers are also missing. These results are consistent with the transmission-electron-microscopy data showing the absence of both DAs in cilia or flagella from individuals with LRRC6 mutations. In spite of structural and functional similarities between LRRC6 and DNAAF1, another LRR-containing protein involved in the same PCD phenotype, the two proteins are not redundant. The evolutionarily conserved LRRC6, therefore, emerges as an additional player in DA assembly, a process that is essential for proper axoneme building and that appears to be much more complex than was previously thought
Mowat-Wilson syndrome in a Moroccan consanguineous family
Mowat-Wilson syndrome is a mental retardation-multiple congenital
anomaly syndrome characterized by a typical facies, developmental
delay, epilepsy, and variable congenital malformations, including
Hirschsprung disease, urogenital anomalies, congenital heart disease,
and agenesis of the corpus callosum. This disorder is sporadic and is
caused by heterozygous mutations or deletions of the ZFHX1B gene
located in the 2q22 region. We report here the first Moroccan patient,
born to consanguineous parents, with Mowat-Wilson syndrome, due to a de
novo, unreported mutation of the ZFHX1B gene
Mowat-Wilson syndrome in a Moroccan consanguineous family
Mowat-Wilson syndrome is a mental retardation-multiple congenital
anomaly syndrome characterized by a typical facies, developmental
delay, epilepsy, and variable congenital malformations, including
Hirschsprung disease, urogenital anomalies, congenital heart disease,
and agenesis of the corpus callosum. This disorder is sporadic and is
caused by heterozygous mutations or deletions of the ZFHX1B gene
located in the 2q22 region. We report here the first Moroccan patient,
born to consanguineous parents, with Mowat-Wilson syndrome, due to a de
novo, unreported mutation of the ZFHX1B gene
Mowat-Wilson syndrome in a Moroccan consanguineous family
Mowat-Wilson syndrome is a mental retardation-multiple congenital anomaly syndrome characterized by a typical facies, developmental delay, epilepsy, and variable congenital malformations, including Hirschsprung disease, urogenital anomalies, congenital heart disease, and agenesis of the corpus callosum. This disorder is sporadic and is caused by heterozygous mutations or deletions of the ZFHX1B gene located in the 2q22 region. We report here the first Moroccan patient, born to consanguineous parents, with Mowat-Wilson syndrome, due to a de novo, unreported mutation of the ZFHX1B gene
Isolation and Expression of the Human hPF20 Gene Orthologous to Chlamydomonas pf20
International audiencePrimary ciliary dyskinesia (PCD) is a heterogeneous congenital disorder characterized by bronchiectasis and chronic sinusitis, sometimes associated with situs inversus (i.e., Kartagener's syndrome) and male infertility. At the cell level, the disease phenotype includes various axonemal abnormalities of respiratory cilia and sperm flagella. We have previously isolated DNAI1, the first gene involved in these diseases in patients lacking outer dynein arms. In this study, designed to find additional genes for other axonemal defects, we report the isolation of a novel human gene, hPF20, which is orthologous to Chlamydomonas pf20. The hPF20 gene is expressed as two major transcripts: one is expressed in testis only, whereas the second is weakly expressed in many other tissues. As flagella of Chlamydomonas strains carrying pf20 mutations lack the axonemal central complexes, we tested the involvement of the hPF20 gene in the disease phenotype of five patients in whom cilia or flagella display abnormal central complexes. Five intragenic polymorphisms were identified and used to exclude hPF20 in two consanguineous patients, while no mutation was found in the remaining patients. However, given the genetic heterogeneity of PCD, we consider that this gene remains a good candidate to be investigated in patients with abnormal central complexes
SP-A restaure lâoligomĂ©risation et la sĂ©crĂ©tion de mutants de SFTPA1 et SFTPA2
National audienceIntroductionLa protĂ©ine A du surfactant (SP)-A est composĂ©e de SP-A1 et SP-A2 (codĂ©s respectivement par SFTPA1 et SFTPA2) oligomĂ©risĂ©es en hexamĂšres de SP-A1 et SP-A2. Les variants hĂ©tĂ©rozygotes de SFTPA1 et SFTPA2 sont associĂ©s Ă des fibroses et des adĂ©nocarcinomes pulmonaires. Cette Ă©tude a pour but dâanalyser lâeffet des variants de SFTPA1 et SFTPA2 sur la localisation subcellulaire, lâoligomĂ©risation et la sĂ©crĂ©tion de SP-A.MĂ©thodesDes cellules HEK293T ont Ă©tĂ© co-transfectĂ©es de façon transitoire avec des vecteurs dâexpression des ADNc de SFTPA1 ou SFTPA2 normaux (wt), mutĂ©s (m) ou vecteur vide (vv) selon les combinaisons suivantes : [SFTPA1_wt + vv], [SFTPA2_wt + vv], [SFTPA1_m + vv], [SFTPA2_m + vv], [SFTPA1_wt + SFTPA2_wt], [SFTPA1_wt + SFTPA1_m], [SFTPA1_wt + SFTPA2_m], [SFTPA2_wt + SFTPA2_m] ou [SFTPA2_wt + SFTPA1_m]. Plusieurs variants ont Ă©tĂ© testĂ©s. La localisation subcellulaire des protĂ©ines exprimĂ©es a Ă©tĂ© analysĂ©e par immunofluorescence (IF) aprĂšs transfection dans des cellules HEK293. Le profil dâoligomĂ©risation et la sĂ©crĂ©tion ont Ă©tĂ© Ă©valuĂ©s par western blot (WB) des protĂ©ines du lysat cellulaire et du surnageant. La spĂ©cificitĂ© des interactions entre SP-A1 et SP-A2 a Ă©tĂ© vĂ©rifiĂ©e par co-immunoprĂ©cipitation (co-IP). Pour chaque expĂ©rience, des Tags diffĂ©rents ont Ă©tĂ© introduits dans les protĂ©ines recombinantes pour identifier les isoformes en jeu. RĂ©sultatsEn IF, les protĂ©ines SP-A2_wt sont dĂ©celables dans des vĂ©sicules de sĂ©crĂ©tion alors que le marquage cellulaire associĂ© aux protĂ©ines SP-A2_m est diffus dans le cytoplasme sans marquage vĂ©siculaire. Cette observation confirme lâabsence de sĂ©crĂ©tion de SP-A2_m que nous avions prĂ©cĂ©demment rapportĂ©e. En WB, le profil dâoligomĂ©risation de SP-A2_m est anormal avec la formation de tĂ©tramĂšres sans hexamĂšres dĂ©celables, et la sĂ©crĂ©tion de SP-A2_m est absente. La co-transfection [SFTPA2_wt + SFTPA2_m] conduit Ă lâaugmentation de lâexpression de SP-A2_m, Ă la restauration des hexamĂšres et dâune sĂ©crĂ©tion pour deux des trois variants testĂ©s. Les mĂȘmes rĂ©sultats ont Ă©tĂ© retrouvĂ©s avec la co-transfection [SFTPA2_wt + SFTPA1_m]. La co-transfection [SFTPA1_wt + SFTPA2_m] conduit aussi Ă lâaugmentation de lâexpression de SP-A2_m, sans toutefois restaurer la formation des hexamĂšres ni une sĂ©crĂ©tion significative de SP-A2_m. Les mĂȘmes rĂ©sultats ont Ă©tĂ© retrouvĂ©s avec la co-transfection [SFTPA1_wt + SFTPA1_m]. Les expĂ©riences de co-IP ont par ailleurs confirmĂ© la spĂ©cificitĂ© des interactions SP-A1/SP-A2. ConclusionCette Ă©tude rĂ©vĂšle une interaction spĂ©cifique de SP-A1 et SP-A2 associĂ©e, lors de la co-expression avec SP-A2_wt, Ă une augmentation de lâexpression de SP-A1_m ou SP-A2_m, mais aussi Ă la restauration dâun profil dâoligomĂ©risation en hexamĂšres et Ă une sĂ©crĂ©tion de protĂ©ines du surfactant. Dans une perspective thĂ©rapeutique, ces rĂ©sultats permettent dâenvisager lâadjonction de SP-A2 exogĂšne dans des modĂšles cellulaires surexprimant SP-A1_m ou SP-A2_m
WT SP-A restores abnormal oligomerization and secretion of mutant SFTPA1 and SFTPA2
International audienc
Hypomorphic pathogenic variant in SFTPB leads to adult pulmonary fibrosis
International audienceBiallelic pathogenic variants in the surfactant protein (SP)-B gene (SFTPB) have been associated with fatal forms of interstitial lung diseases (ILD) in newborns and exceptional survival in young children. We herein report the cases of two related adults with pulmonary fibrosis due to a new homozygous SFTPB pathogenic variant, c.582G>A p.(Gln194=). In vitro transcript studies showed that this SFTPB synonymous pathogenic variant induces aberrant splicing leading to three abnormal transcripts with the preservation of the expression of a small proportion of normal SFTPB transcripts. Immunostainings on lung biopsies of the proband showed an almost complete loss of SP-B expression. This hypomorphic splice variant has thus probably allowed the patients' survival to adulthood while inducing an epithelial cell dysfunction leading to ILD. Altogether, this report shows that SFTPB pathogenic variants should be considered in atypical presentations and/or early-onset forms of ILD particularly when a family history is identified
uORF-creating mutations in Van der Woude syndrome: why it is important to study 5âUTRs
International audienceBackground/Objectives:Van der Woude syndrome (VWS, MIM 119300) is an autosomal dominant cleft lip and/or palate with typical lower lip pits. Most patients carry loss-of-function mutations in IRF6. Upstream open reading frame (uORF)-creating mutations have been reported in four VWS patients. Pathogenic uORFcreating mutations are mostly out-of-frame upstream start codons (uAUG) in the 5âUTR. We searched for IRF6 uORF mutations and assessed the ability to predict the pathogenicity of uORFcreating variations of 5 prediction tools.Methods:We analyzed IRF6 UTR and coding regions in 68 VWS probands. By using a set of 44 reference genes, we assessed 5 in silico tools predicting the probability of ATGs to initiate translation: NetStart, ATGpr, TIS Miner, AltORFev, TIS Predictor. We then assessed the potential pathogenicity of all the theoretical uORFs in IRF6 5âUTR.Results:We have identified two novel uORF-creating mutations (c.-141C>T and c.-162C>T), representing 3% (2/68) of the probands. The 7 carriers of the two families had typical VWS signs. Our in silico analyses revealed a higher accuracy for AI-based tools over those based on Kozak consensus scoring. There are 28 theoretical uAUG-creating SNVs in IRF6 5âUTR. With AI-based tools, the six uAUG identified in VWS patients have high translation initiation site scores; 3 to 4 of the theoretical uAUG-creating SNVs had high scores and could correspond to pathogenic mutations. For the dozen of theoretical SNVs with intermediate scores, predicting pathogenicity remains challenging.Conclusion:As untranslated regions are frequently understudied in NGS strategies, uORF-creating mutations may be underdiagnosed in VWS and in human pathology in general
SPA restaure lâoligomĂ©risation et la sĂ©crĂ©tion de mutants de SFTPA1 et SFTPA2
National audienceIntroductionLa protĂ©ine du surfactant (SP)-A mature, sĂ©crĂ©tĂ©e dans lâespace alvĂ©olaire, est une combinaison de SP-A1 et SP-A2 principalement rapportĂ©e sous forme dâoctadĂ©camĂšres. Les variations hĂ©tĂ©rozygotes dans les gĂšnes codant pour SP-A (SFTPA1 et SFTPA2) sont associĂ©es Ă des pneumopathies interstitielles diffuses (PID) fibrosantes et des adĂ©nocarcinomes pulmonaires. Ă ce jour aucun traitement spĂ©cifique nâexiste. Cette Ă©tude a pour but dâanalyser lâeffet des mutants SP-A1 et SP-A2 sur lâoligomĂ©risation et la sĂ©crĂ©tion ainsi que le potentiel thĂ©rapeutique de SP-A.MĂ©thodesĂtude in vitro par expression ou co-expression transitoire de SP-A WT et/ou mutĂ©e (mutation faux sens) dans la lignĂ©e cellulaire HEK293T. Les protĂ©ines extraites du lysat cellulaire et du surnageant (sĂ©crĂ©tion) sont analysĂ©es par western blot et co-immunoprĂ©cipitation (co-IP). Les protĂ©ines sont Ă©galement Ă©tudiĂ©es par immunofluorescence.RĂ©sultatsLes protĂ©ines SP-A mutĂ©es prĂ©sentent un profil dâoligomĂ©risation anormal dans le lysat soluble avec une absence dâhexamĂšre en comparaison au WT. De façon intĂ©ressante, la co-expression avec SP-A WT augmente la quantitĂ© de SP-A mutĂ©e et restaure la formation dâhexamĂšres. ParallĂšlement, dans le lysat insoluble, SP-A mutĂ©e seule est plus prĂ©sente quâen Ă©tant co-exprimĂ©e avec du WT. Cette observation est en faveur dâune solubilisation de SP-A mutĂ©e en prĂ©sence de WT. Nous avons prĂ©cĂ©demment montrĂ© que SP-A mutĂ©e nâĂ©tait pas sĂ©crĂ©tĂ©e. Cette Ă©tude confirme ces observations Ă©galement par immunofluorescence mettant en Ă©vidence des vĂ©sicules de sĂ©crĂ©tion seulement pour SP-A WT. LĂ encore, la co-expression avec SP-A WT permet Ă nouveau dâobserver une sĂ©crĂ©tion partielle des protĂ©ines mutĂ©es. Les analyses par co-IP du lysat et du surnageant ont permis de confirmer lâinteraction entre SP-A WT et SP-A mutĂ©e.ConclusionNous rapportons pour la premiĂšre fois Ă travers cette Ă©tude le bĂ©nĂ©fice de la protĂ©ine SP-A WT dans la restauration du profil dâoligomĂ©risation et de la sĂ©crĂ©tion des protĂ©ines SP-A mutĂ©es. Cette Ă©tude pilote permet dâenvisager lâĂ©tude de SP-A comme traitement des patients prĂ©sentant une fibrose pulmonaire associĂ©e Ă des variations de SFTPA1 ou SFTPA2
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