RESUMEN
BACKGROUND: Primary ciliary dyskinesia (PCD) is a rare airway disorder caused by defective motile cilia. Only male patients have been reported with pathogenic mutations in X-linked DNAAF6, which result in the absence of ciliary dynein arms, whereas their heterozygous mothers are supposedly healthy. Our objective was to assess the possible clinical and ciliary consequences of X-chromosome inactivation (XCI) in these mothers. METHODS: XCI patterns of six mothers of male patients with DNAAF6-related PCD were determined by DNA-methylation studies and compared with their clinical phenotype (6/6 mothers), as well as their ciliary phenotype (4/6 mothers), as assessed by immunofluorescence and high-speed videomicroscopy analyses. The mutated X chromosome was tracked to assess the percentage of cells with a normal inactivated DNAAF6 allele. RESULTS: The mothers' phenotypes ranged from absence of symptoms to mild/moderate or severe airway phenotypes, closely reflecting their XCI pattern. Analyses of the symptomatic mothers' airway ciliated cells revealed the coexistence of normal cells and cells with immotile cilia lacking dynein arms, whose ratio closely mirrored their XCI pattern. CONCLUSION: This study highlights the importance of searching for heterozygous pathogenic DNAAF6 mutations in all female relatives of male PCD patients with a DNAAF6 defect, as well as in females consulting for mild chronic respiratory symptoms. Our results also demonstrate that about one-third-ranging from 20% to 50%-normal ciliated airway cells sufficed to avoid severe PCD, a result paving the way for gene therapy.
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Cilios , Inactivación del Cromosoma X , Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Masculino , Cilios/patología , Cilios/genética , Trastornos de la Motilidad Ciliar/genética , Trastornos de la Motilidad Ciliar/patología , Metilación de ADN/genética , Dineínas/genética , Síndrome de Kartagener/genética , Síndrome de Kartagener/patología , Mutación , Fenotipo , Inactivación del Cromosoma X/genéticaRESUMEN
Biallelic 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.
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Enfermedades Pulmonares Intersticiales , Fibrosis Pulmonar , Adulto , Niño , Preescolar , Humanos , Recién Nacido , Enfermedades Pulmonares Intersticiales/diagnóstico , Enfermedades Pulmonares Intersticiales/genética , Fibrosis Pulmonar/genética , Proteína B Asociada a Surfactante Pulmonar/genéticaRESUMEN
BACKGROUND: Urticarial lesions are observed in both cutaneous and systemic disorders. Familial forms of urticarial syndromes are rare and can be encountered in systemic autoinflammatory diseases. OBJECTIVE: We sought to investigate a large family with dominantly inherited chronic urticarial lesions associated with hypercytokinemia. METHODS: We performed a genetic linkage analysis in 14 patients from a 5-generation family, as well as whole-exome sequencing, cytokine profiling, and transcriptomic analyses on samples from 2 patients. The identified candidate protein was studied after in vitro expression of the corresponding normal and mutated recombinant proteins. An unsupervised proteomic approach was used to unveil the associated protein network. RESULTS: The disease phenotype of the most affected family members is characterized by chronic urticarial flares associated with extremely high plasma levels of proinflammatory (IL-1ß, IL-6, and TNF-α) and anti-inflammatory (IL-10 and IL-1 receptor antagonist [IL-1RA]) cytokines, with no secondary organ dysfunction, no susceptibility to infections, no fever, and normal C-reactive protein levels. Monocyte transcriptomic analyses identified an immunotolerant profile in the most affected patient. The affected family members carried a loss-of-function mutation in RNF213 that encodes mysterin, a protein with a poorly known physiologic role. We identified the deubiquitinase CYLD, a major regulator of inflammation, as an RNF213 partner and showed that CYLD expression is inhibited by wild-type but not mutant RNF213. CONCLUSION: We identified a new entity characterized by chronic urticarial lesions associated with a clinically blunted hypercytokinemia. This disease, which is due to loss of function of RNF213, reveals mysterin's key role in the complex molecular network of innate immunity.
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Síndrome de Liberación de Citoquinas , Proteómica , HumanosRESUMEN
INTRODUCTION: Interstitial lung diseases (ILDs) can be caused by mutations in the SFTPA1 and SFTPA2 genes, which encode the surfactant protein (SP) complex SP-A. Only 11 SFTPA1 or SFTPA2 mutations have so far been reported worldwide, of which five have been functionally assessed. In the framework of ILD molecular diagnosis, we identified 14 independent patients with pathogenic SFTPA1 or SFTPA2 mutations. The present study aimed to functionally assess the 11 different mutations identified and to accurately describe the disease phenotype of the patients and their affected relatives. METHODS: The consequences of the 11 SFTPA1 or SFTPA2 mutations were analysed both in vitro, by studying the production and secretion of the corresponding mutated proteins and ex vivo, by analysing SP-A expression in lung tissue samples. The associated disease phenotypes were documented. RESULTS: For the 11 identified mutations, protein production was preserved but secretion was abolished. The expression pattern of lung SP-A available in six patients was altered and the family history reported ILD and/or lung adenocarcinoma in 13 out of 14 families (93%). Among the 28 SFTPA1 or SFTPA2 mutation carriers, the mean age at ILD onset was 45â years (range 0.6-65â years) and 48% underwent lung transplantation (mean age 51â years). Seven carriers were asymptomatic. DISCUSSION: This study, which expands the molecular and clinical spectrum of SP-A disorders, shows that pathogenic SFTPA1 or SFTPA2 mutations share similar consequences for SP-A secretion in cell models and in lung tissue immunostaining, whereas they are associated with a highly variable phenotypic expression of disease, ranging from severe forms requiring lung transplantation to incomplete penetrance.
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Enfermedades Pulmonares Intersticiales , Neoplasias Pulmonares , Adolescente , Adulto , Anciano , Niño , Preescolar , Humanos , Lactante , Enfermedades Pulmonares Intersticiales/genética , Neoplasias Pulmonares/genética , Persona de Mediana Edad , Mutación , Fenotipo , Proteína A Asociada a Surfactante Pulmonar/genética , Adulto JovenRESUMEN
Cilia and flagella are evolutionarily conserved organelles whose motility relies on the outer and inner dynein arm complexes (ODAs and IDAs). Defects in ODAs and IDAs result in primary ciliary dyskinesia (PCD), a disease characterized by recurrent airway infections and male infertility. PCD mutations in assembly factors have been shown to cause a combined ODA-IDA defect, affecting both cilia and flagella. We identified four loss-of-function mutations in TTC12, which encodes a cytoplasmic protein, in four independent families in which affected individuals displayed a peculiar PCD phenotype characterized by the absence of ODAs and IDAs in sperm flagella, contrasting with the absence of only IDAs in respiratory cilia. Analyses of both primary cells from individuals carrying TTC12 mutations and human differentiated airway cells invalidated for TTC12 by a CRISPR-Cas9 approach revealed an IDA defect restricted to a subset of single-headed IDAs that are different in flagella and cilia, whereas TTC12 depletion in the ciliate Paramecium tetraurelia recapitulated the sperm phenotype. Overall, our study, which identifies TTC12 as a gene involved in PCD, unveils distinct dynein assembly mechanisms in human motile cilia versus flagella.
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Cilios/patología , Trastornos de la Motilidad Ciliar/etiología , Dineínas/metabolismo , Flagelos/patología , Mutación , Proteínas/genética , Cola del Espermatozoide/patología , Adulto , Axonema , Niño , Cilios/metabolismo , Trastornos de la Motilidad Ciliar/patología , Dineínas/genética , Femenino , Flagelos/metabolismo , Homocigoto , Humanos , Infertilidad Masculina/etiología , Infertilidad Masculina/patología , Masculino , Persona de Mediana Edad , Linaje , Fenotipo , Motilidad Espermática , Cola del Espermatozoide/metabolismo , Adulto JovenRESUMEN
BACKGROUND: NLRP3-associated autoinflammatory diseases (NLRP3-AIDs) include conditions of various severities, due to germline or somatic mosaic NLRP3 mutations. OBJECTIVE: To identify mosaic- versus germline-specific NLRP3 mutations' characteristics, we reinterpreted all the mutations reported in NLRP3-AIDs and performed an in-depth study of 3 novel patients. METHODS: The pathogenicity of all reported mosaic/germline mutations was reassessed according to international recommendations and their location on the NLRP3 3-dimensional structure. Deep-targeted sequencing and NLRP3-inflammasome-activation assays were used to identify the disease-causing mutation in 3 patients. RESULTS: We identified, in 3 patients, mosaic mutations affecting the same NLRP3 amino acid (Glu569). This residue belongs to 1 of the 2 mosaic mutational hot spots that face each other in the core of the NLRP3 ATPase domain. The review of the 90 NLRP3 mutations identified in 277 patients revealed that those hot spots account for 68.5% of patients (37 of 54) with mosaic mutations. Glu569 is affected in 22% of the patients (12 of 54) with mosaic mutations and in 0.4% of patients (1 of 223) with germline mutations. Only 8 of 90 mutations were found in mosaic and germinal states. All of the germline mutations were associated with a severe phenotype. These data suggest that mutations found only in mosaic state could be incompatible with life if present in germinal state. None of the 5 most frequent germline mutations was identified in mosaic state. Mutations found only in germinal state could, therefore, be asymptomatic in mosaic state. CONCLUSIONS: The phenotypic spectrum of NLRP3-AIDs appears to be related to the germinal/mosaic status and localization of the underlying mutations.
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Enfermedades Autoinmunes/genética , Inflamasomas/metabolismo , Inflamación/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Preescolar , Cristalografía por Rayos X , Femenino , Mutación de Línea Germinal/genética , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inflamasomas/genética , Masculino , Persona de Mediana Edad , Proteína con Dominio Pirina 3 de la Familia NLR/química , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Fenotipo , Conformación Proteica , Índice de Severidad de la Enfermedad , Células THP-1RESUMEN
Chronic urticaria is a common skin disorder with heterogeneous causes. In the absence of physical triggers, chronic urticarial rash is called idiopathic or spontaneous. The objective of this study was to identify the molecular and cellular bases of a disease condition displayed by two unrelated patients aged over 60 years who presented for two decades with a chronic urticaria resistant to standard therapy that occurred in the context of systemic inflammation not triggered by cold. In both patients, a targeted sequencing approach using a next generation technology identified somatic mosaic mutations in NLRP3, a gene encoding a key inflammasome component. The study of several of both patients' cell types showed that, despite the late onset of the disease, NLRP3 mutations were not found to be restricted to myelomonocytic cells. Rather, the data obtained strongly suggested that the mutational event occurred very early, during embryonic development. As shown by functional studies, the identified mutations-an in-frame deletion and a recurrent NLRP3 missense mutation-have a gain-of-function effect on NLRP3-inflammasome activation. Consistently, a complete remission was obtained in both patients with anti-IL-1 receptor antagonists. This study unveils that in late-onset chronic urticaria, the search for autoinflammatory markers and somatic mosaic NLRP3 mutations may have important diagnostic and therapeutic consequences.
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Urticaria Crónica/genética , ADN/genética , Inflamasomas/genética , Mutación , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Anciano , Urticaria Crónica/metabolismo , Análisis Mutacional de ADN , Femenino , Humanos , Inflamasomas/metabolismo , Masculino , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismoRESUMEN
OBJECTIVE: To determine the molecular and cellular bases of autoinflammatory syndromes in a multigenerational French family with Muckle-Wells syndrome and in a patient originating from Portugal with familial cold autoinflammatory syndrome. METHODS: Sequencing of NLRP3 exon 3 was performed in all accessible patients. Microsatellite and whole-genome single nucleotide polymorphism genotyping was used i) to test the intrafamilial segregation of the identified variant and ii) to look for a founder effect. Functional analyses included the study of i) apoptosis-associated speck-like protein containing a CARD (ASC) speck formation in HEK293T cells (stably expressing ASC-green fluorescent protein and pro-caspase 1-FLAG) transiently expressing the wild-type or mutated NLRP3 protein, ii) levels of IL-1ß secreted from transfected THP-1 cells, and iii) inflammasome-related gene expression and cytokine secretion from monocytes isolated from patients in crisis (probands from the two families), related patients out of crisis, and from controls. RESULTS: The same heterozygous mutation (c.1322C>T, p.A441V) located in the NACHT domain, segregating with the disease within the first family, was identified in the two families. This mutation was found to be associated with different core haplotypes. NLRP3-A441V led to increased ASC speck formation and high levels of secreted IL-1ß. Monocyte inflammasome-related gene expression and cytokine secretion, which were within the normal range in patients out of crisis, were found to be differentially regulated between the two probands, correlating with their phenotypic status. CONCLUSION: These molecular and cellular findings, which indicate a recurrent mutational event, clearly demonstrate the pathogenicity of the p.A441V missense mutation in NLRP3-associated autoinflammatory disease and point to the interest of studying patients' primary cells to assess disease activity.
RESUMEN
Isolated growth hormone deficiency (IGHD) is a rare condition mainly caused by mutations in GH1. The aim of this study was to assess the contribution of GHRHR mutations to IGHD in an unusually large group of patients. All GHRHR coding exons and flanking intronic regions were sequenced in 312 unrelated patients with nonsyndromic IGHD. Functional consequences of all newly identified missense variants were assessed in vitro (i.e., study of the expression of recombinant GHRHRs and their ability to activate the cyclic adenosine monophosphate (cAMP) signaling pathway). Genotype-phenotype correlation analyses were performed according to the nature of the identified mutation. We identified 20 different disease-causing GHRHR mutations (truncating and missense loss-of-function mutations), among which 15 are novel, in 24 unrelated patients. Of note, about half (13/24) of those patients represent sporadic cases. The clinical phenotype of patients with at least one missense GHRHR mutation was found to be indistinguishable from that of patients with bi-allelic truncating mutations. This study, which unveils disease-causing GHRHR mutations in 8% (24/312) of IGHD cases, identifies GHRHR as the second IGHD gene most frequently involved after GH1. The finding that 8% of IGHD cases without GH1 mutations are explained by GHRHR molecular defects (including missense mutations), together with the high proportion of sporadic cases among those patients, has important implications for genetic counseling.
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Enanismo Hipofisario/genética , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Mutación , Receptores de Neuropéptido/genética , Receptores de Hormona Reguladora de Hormona Hipofisaria/genética , Alelos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , AMP Cíclico , Análisis Mutacional de ADN , Enanismo Hipofisario/diagnóstico , Femenino , Genotipo , Hormona de Crecimiento Humana/genética , Humanos , Masculino , Linaje , Receptores de Neuropéptido/química , Receptores de Hormona Reguladora de Hormona Hipofisaria/químicaRESUMEN
Motile cilia and sperm flagella share an evolutionarily conserved axonemal structure. Their structural and/or functional defects are associated with primary ciliary dyskinesia (PCD), a genetic disease characterized by chronic respiratory-tract infections and in which most males are infertile due to asthenozoospermia. Among the well-characterized axonemal protein complexes, the outer dynein arms (ODAs), through ATPase activity of their heavy chains (HCs), play a major role for cilia and flagella beating. However, the contribution of the different HCs (γ-type: DNAH5 and DNAH8 and ß-type: DNAH9, DNAH11, and DNAH17) in ODAs from both organelles is unknown. By analyzing five male individuals who consulted for isolated infertility and displayed a loss of ODAs in their sperm cells but not in their respiratory cells, we identified bi-allelic mutations in DNAH17. The isolated infertility phenotype prompted us to compare the protein composition of ODAs in the sperm and ciliary axonemes from control individuals. We show that DNAH17 and DNAH8, but not DNAH5, DNAH9, or DNAH11, colocalize with α-tubulin along the sperm axoneme, whereas the reverse picture is observed in respiratory cilia, thus explaining the phenotype restricted to sperm cells. We also demonstrate the loss of function associated with DNAH17 mutations in two unrelated individuals by performing immunoblot and immunofluorescence analyses on sperm cells; these analyses indicated the absence of DNAH17 and DNAH8, whereas DNAH2 and DNALI, two inner dynein arm components, were present. Overall, this study demonstrates that mutations in DNAH17 are responsible for isolated male infertility and provides information regarding ODA composition in human spermatozoa.
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Astenozoospermia/complicaciones , Dineínas Axonemales/genética , Infertilidad Masculina/etiología , Mutación , Espermatozoides/patología , Adulto , Humanos , Infertilidad Masculina/metabolismo , Infertilidad Masculina/patología , Masculino , Linaje , Fenotipo , Espermatozoides/metabolismoRESUMEN
Circulating serum amyloid A (SAA) is increased in various inflammatory conditions. The human SAA protein family comprises the acute phase SAA1/SAA2, known to activate a large set of innate and adaptive immune cells, and the constitutive SAA4. The liver synthesis of SAA1/SAA2 is well-established but there is still an open debate on extrahepatic SAA expression especially in macrophages. We aimed to investigate the ability of human primary monocytes and monocyte-derived macrophages to express SAA1, SAA2 and SAA4 at both the transcriptional and protein levels, as previous studies almost exclusively dealt with monocytic cell lines. Monocytes and derived macrophages from healthy donors were stimulated under various conditions. In parallel with SAA, pro-inflammatory IL1A, IL1B and IL6 cytokine expression was assessed. While LPS alone was non-effective, a combined LPS/dexamethasone treatment induced SAA1 and to a lesser extent SAA2 transcription in human monocytes and macrophages. In contrast, as expected, pro-inflammatory cytokine expression was strongly induced following stimulation with LPS, an effect which was dampened in the presence of dexamethasone. Furthermore, in monocytes polarized towards a pro-inflammatory M1 phenotype, SAA expression in response to LPS/dexamethasone was potentiated; a result mainly seen for SAA1. However, a major discrepancy was observed between SAA mRNA and intracellular protein levels under the experimental conditions used. Our results demonstrate that human monocytes and macrophages can express SAA genes, mainly SAA1 in response to an inflammatory environment. While SAA is considered as a member of a large cytokine network, its expression in the monocytes-macrophages in response to LPS-dexamethasone is strikingly different from that observed for classic pro-inflammatory cytokines. As monocytes-macrophages are major players in chronic inflammatory diseases, it may be hypothesized that SAA production from macrophages may contribute to the local inflammatory microenvironment, especially when macrophages are compactly organized in granulomas as in sarcoidosis.
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Inflamación/sangre , Proteína Amiloide A Sérica/genética , Dexametasona/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Voluntarios Sanos , Humanos , Inflamación/inducido químicamente , Inflamación/genética , Inflamación/patología , Interleucina-1alfa/sangre , Interleucina-1beta/sangre , Interleucina-6/sangre , Lipopolisacáridos/toxicidad , Hígado/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Monocitos/efectos de los fármacos , Monocitos/metabolismoRESUMEN
Inflammasomes are multiprotein complexes nucleating around an NLR (Nucleotide-binding domain and Leucine-rich Repeat containing protein), which regulate the secretion of the pro-inflammatory interleukin (IL)-1ß and IL-18 cytokines. Monocytes and macrophages, the main cells expressing the inflammasome genes, adapt to their surrounding microenvironment by a phenotypic polarization towards a pro-inflammatory M1 phenotype that promotes inflammation or an anti-inflammatory M2 phenotype important for resolution of inflammation. Despite the importance of inflammasomes in health and disease, little is known about inflammasome gene expression in relevant human cells and the impact of monocyte and macrophage polarization in inflammasome gene expression. We examined the expression of several members of the NLR, caspase and cytokine family, and we studied the activation of the well-described NLRP3 inflammasome in an experimental model of polarized human primary monocytes and monocyte-derived macrophages (M1/M2 phenotypes) before and after activation with LPS, a well-characterized microbial pattern used in inflammasome activation studies. Our results show that the differentiation of monocytes to macrophages alters NLR expression. Polarization using IFN-γ (M1 phenotype), induces among the NLRs studied, only the expression of NOD2. One of the key results of our study is that the induction of NLRP3 expression by LPS is inhibited in the presence of IL-4+IL-13 (M2 phenotype) at both mRNA and protein level in monocytes and macrophages. Unlike caspase-3, the expression of inflammasome-related CASP1 (encodes caspase-1) and CASP4 (encodes caspase-4) is up-regulated in M1 but not in M2 cells. Interestingly, the presence of LPS marginally influenced IL18 mRNA expression and secretion, unlike its impact on IL1B. Our data provide the basis for a better understanding of the role of different inflammasomes within a given environment (M1 and M2) in human cells and their impact in the pathophysiology of several important inflammatory disorders.
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Inflamasomas/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Proteínas NLR/inmunología , Proteína Adaptadora de Señalización NOD2/inmunología , Caspasas/genética , Caspasas/inmunología , Polaridad Celular , Células Cultivadas , Citocinas/genética , Citocinas/inmunología , Regulación de la Expresión Génica , Humanos , Inflamasomas/genética , Lipopolisacáridos/inmunología , Macrófagos/citología , Macrófagos/metabolismo , Monocitos/citología , Monocitos/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteínas NLR/genética , Proteína Adaptadora de Señalización NOD2/genéticaRESUMEN
By moving essential body fluids and molecules, motile cilia and flagella govern respiratory mucociliary clearance, laterality determination and the transport of gametes and cerebrospinal fluid. Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder frequently caused by non-assembly of dynein arm motors into cilia and flagella axonemes. Before their import into cilia and flagella, multi-subunit axonemal dynein arms are thought to be stabilized and pre-assembled in the cytoplasm through a DNAAF2-DNAAF4-HSP90 complex akin to the HSP90 co-chaperone R2TP complex. Here, we demonstrate that large genomic deletions as well as point mutations involving PIH1D3 are responsible for an X-linked form of PCD causing disruption of early axonemal dynein assembly. We propose that PIH1D3, a protein that emerges as a new player of the cytoplasmic pre-assembly pathway, is part of a complementary conserved R2TP-like HSP90 co-chaperone complex, the loss of which affects assembly of a subset of inner arm dyneins.
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Proteínas Reguladoras de la Apoptosis/genética , Dineínas Axonemales/metabolismo , Genes Ligados a X/genética , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Síndrome de Kartagener/genética , Proteínas de Microtúbulos/genética , Chaperonas Moleculares/genética , Adolescente , Adulto , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Axonema/patología , Niño , Preescolar , Cilios/patología , Cilios/ultraestructura , Citoplasma/patología , Modelos Animales de Enfermedad , Femenino , Enfermedades Genéticas Ligadas al Cromosoma X/patología , Células HEK293 , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Recién Nacido , Péptidos y Proteínas de Señalización Intracelular , Síndrome de Kartagener/patología , Masculino , Microscopía Electrónica de Transmisión , Linaje , Filogenia , Mutación Puntual , Pliegue de Proteína , Alineación de Secuencia , Eliminación de Secuencia , Motilidad Espermática/genética , Secuenciación del Exoma , Pez CebraRESUMEN
Context: LHX4 encodes a LIM-homeodomain transcription factor that is implicated in early pituitary development. In humans, only 13 heterozygous LHX4 mutations have been associated with congenital hypopituitarism. Objective: The aims of this study were to evaluate the prevalence of LHX4 mutations in patients with hypopituitarism, to define the associated phenotypes, and to characterize the functional impact of the identified variants and the respective role of the 2 LIM domains of LHX4. Design and Patients: We screened 417 unrelated patients with isolated growth hormone deficiency or combined pituitary hormone deficiency associated with ectopic posterior pituitary and/or sella turcica anomalies for LHX4 mutations (Sanger sequencing). In vitro studies were performed to assess the functional consequences of the identified variants. Results: We identified 7 heterozygous variations, including p.(Tyr131*), p.(Arg48Thrfs*104), c.606+1G>T, p.Arg65Val, p.Thr163Pro, p.Arg221Gln, and p.Arg235Gln), that were associated with variable expressivity; 5 of the 7 were also associated with incomplete penetrance. The p.(Tyr131*), p.(Arg48Thrfs*104), p.Ala65Val, p.Thr163Pro, and p.Arg221Gln LHX4 variants are unable to transactivate the POU1F1 and GH promoters. As suggested by transactivation, subcellular localization, and protein-protein interaction studies, p.Arg235Gln is probably a rare polymorphism. Coimmunoprecipitation studies identified LHX3 as a potential protein partner of LHX4. As revealed by functional studies of LIM-defective recombinant LHX4 proteins, the LIM1 and LIM2 domains are not redundant. Conclusion: This study, performed in the largest cohort of patients screened so far for LHX4 mutations, describes 6 disease-causing mutations that are responsible for congenital hypopituitarism. LHX4 mutations were found to be associated with variable expressivity, and most of them with incomplete penetrance; their contribution to pituitary deficits that are associated with an ectopic posterior pituitary and/or a sella turcica defect is â¼1.4% in the 417 probands tested.
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Hipopituitarismo/genética , Proteínas con Homeodominio LIM/genética , Mutación/genética , Factores de Transcripción/genética , Adolescente , Secuencia de Aminoácidos , Biomarcadores/metabolismo , Western Blotting , Niño , Preescolar , Estudios de Cohortes , Femenino , Estudios de Seguimiento , Humanos , Inmunoprecipitación , Lactante , Recién Nacido , Masculino , Linaje , Pronóstico , Homología de Secuencia de AminoácidoRESUMEN
The respiratory tract is continually exposed to various insults that are a permanent threat to the maintenance of lung homeostasis. Repair of the parenchyma structure, particularly of the alveolar epithelium, requires complex cellular strategies. Among the molecular components that play an important role in these processes are the surfactant proteins (SPs), particularly SP-A. The present review examines current evidence regarding the role of SP-A in lung host defence mechanisms through its implication in innate/adaptive immunity of the lung and epithelium integrity and repair. New information on SP-A deficiency in various forms of pulmonary diseases could help define therapeutic strategies aimed at restoring functional SP-A within the alveolar structure.
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Homeostasis , Pulmón/metabolismo , Proteína A Asociada a Surfactante Pulmonar/metabolismo , Animales , Homeostasis/inmunología , Humanos , Pulmón/inmunología , Enfermedades Pulmonares/inmunología , Enfermedades Pulmonares/metabolismo , Enfermedades Pulmonares/terapiaRESUMEN
Primary ciliary dyskinesia (PCD) is an autosomal-recessive disease due to functional or ultra-structural defects of motile cilia. Affected individuals display recurrent respiratory-tract infections; most males are infertile as a result of sperm flagellar dysfunction. The great majority of the PCD-associated genes identified so far encode either components of dynein arms (DAs), which are multiprotein-ATPase complexes essential for ciliary motility, or proteins involved in DA assembly. To identify the molecular basis of a PCD phenotype characterized by central complex (CC) defects but normal DA structure, a phenotype found in â¼15% of cases, we performed whole-exome sequencing in a male individual with PCD and unexplained CC defects. This analysis, combined with whole-genome SNP genotyping, identified a homozygous mutation in DNAJB13 (c.833T>G), a gene encoding a HSP40 co-chaperone whose ortholog in the flagellated alga Chlamydomonas localizes to the radial spokes. In vitro studies showed that this missense substitution (p.Met278Arg), which involves a highly conserved residue of several HSP40 family members, leads to protein instability and triggers proteasomal degradation, a result confirmed by the absence of endogenous DNAJB13 in cilia and sperm from this individual. Subsequent DNAJB13 analyses identified another homozygous mutation in a second family; the study of DNAJB13 transcripts obtained from airway cells showed that this mutation (c.68+1G>C) results in a splicing defect consistent with a loss-of-function mutation. Overall, this study, which establishes mutations in DNAJB13 as a cause of PCD, unveils the key role played by DNAJB13 in the proper formation and function of ciliary and flagellar axonemes in humans.
Asunto(s)
Trastornos de la Motilidad Ciliar/genética , Proteínas de Choque Térmico/genética , Infertilidad Masculina/genética , Mutación , Adolescente , Proteínas Reguladoras de la Apoptosis , Axonema/genética , Cilios/genética , Trastornos de la Motilidad Ciliar/patología , Exoma/genética , Femenino , Flagelos/genética , Flagelos/patología , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico/metabolismo , Homocigoto , Humanos , Infertilidad Masculina/patología , Síndrome de Kartagener/genética , Masculino , Persona de Mediana Edad , Chaperonas Moleculares , Mutación Missense/genética , Fenotipo , Polimorfismo de Nucleótido Simple/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica , Empalme del ARN/genética , Semen , Espermatozoides/metabolismo , Espermatozoides/patologíaRESUMEN
Primary ciliary dyskinesia (PCD) is an autosomal recessive disease characterized by chronic respiratory infections of the upper and lower airways, hypofertility, and, in approximately half of the cases, situs inversus. This complex phenotype results from defects in motile cilia and sperm flagella. Among the numerous genes involved in PCD, very few-including CCDC39 and CCDC40-carry mutations that lead to a disorganization of ciliary axonemes with microtubule misalignment. Focusing on this particular phenotype, we identified bi-allelic loss-of-function mutations in GAS8, a gene that encodes a subunit of the nexin-dynein regulatory complex (N-DRC) orthologous to DRC4 of the flagellated alga Chlamydomonas reinhardtii. Unlike the majority of PCD patients, individuals with GAS8 mutations have motile cilia, which, as documented by high-speed videomicroscopy, display a subtle beating pattern defect characterized by slightly reduced bending amplitude. Immunofluorescence studies performed on patients' respiratory cilia revealed that GAS8 is not required for the proper expression of CCDC39 and CCDC40. Rather, mutations in GAS8 affect the subcellular localization of another N-DRC subunit called DRC3. Overall, this study, which identifies GAS8 as a PCD gene, unveils the key importance of the corresponding protein in N-DRC integrity and in the proper alignment of axonemal microtubules in humans.
Asunto(s)
Axonema/patología , Proteínas del Citoesqueleto/genética , Síndrome de Kartagener/genética , Mutación , Proteínas de Neoplasias/genética , Adulto , Niño , Proteínas del Citoesqueleto/metabolismo , Femenino , Predisposición Genética a la Enfermedad , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Síndrome de Kartagener/metabolismo , Síndrome de Kartagener/patología , Masculino , Proteínas de Neoplasias/metabolismo , Análisis de Secuencia de ADNRESUMEN
Hematopoiesis generated from human embryonic stem cells (ES) and induced pluripotent stem cells (iPS) are unprecedented resources for cell therapy. We compared hematopoietic differentiation potentials from ES and iPS cell lines originated from various donors and derived them using integrative and non-integrative vectors. Significant differences in differentiation toward hematopoietic lineage were observed among ES and iPS. The ability of engraftment of iPS or ES-derived cells in NOG mice varied among the lines with low levels of chimerism. iPS generated from ES cell-derived mesenchymal stem cells (MSC) reproduce a similar hematopoietic outcome compared to their parental ES cell line. We were not able to identify any specific hematopoietic transcription factors that allow to distinguish between good versus poor hematopoiesis in undifferentiated ES or iPS cell lines. There is a relatively unpredictable variation in hematopoietic differentiation between ES and iPS cell lines that could not be predicted based on phenotype or gene expression of the undifferentiated cells. These results demonstrate the influence of genetic background in variation of hematopoietic potential rather than the reprogramming process.
Asunto(s)
Reprogramación Celular/genética , Células Madre Embrionarias/citología , Heterogeneidad Genética , Supervivencia de Injerto , Hematopoyesis/genética , Células Madre Pluripotentes Inducidas/citología , Animales , Biomarcadores/metabolismo , Diferenciación Celular , Línea Celular , Linaje de la Célula/genética , Quimerismo , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/trasplante , Expresión Génica , Vectores Genéticos , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/trasplante , Lentivirus/genética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Retroviridae/genética , Donantes de Tejidos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Trasplante HeterólogoRESUMEN
Idiopathic interstitial pneumonias (IIPs) comprise a heterogeneous group of rare lung parenchyma disorders with high morbidity and mortality, which can occur at all ages. In adults, the most common form of IIPs, idiopathic pulmonary fibrosis (IPF), has been associated with an increased frequency of lung cancer. The molecular basis of IIPs remains unknown in most cases. This study investigates IIP pathophysiology in 12 families affected by IPF and lung cancer. We identified, in a multigenerational family, nine members carrying a heterozygous missense mutation with evidence of pathogenicity in SFTPA1 that encodes the surfactant protein (SP)-A1. The mutation (p.Trp211Arg), which segregates with a disease phenotype characterized by either isolated IIP/IPF, or IPF associated with lung adenocarcinoma, is located in the carbohydrate recognition domain (CRD) of SP-A1 and involves a residue invariant throughout evolution, not only in SP-A1, but also in its close paralog SP-A2 and other CRD-containing proteins. As shown through functional studies, the p.Trp211Arg mutation impairs SP-A1 secretion. Immunohistochemistry studies on patient alveolar epithelium showed an altered SP-A expression pattern. Overall, this first report of a germline molecular defect in SFTPA1 unveils the key role of SP-A1 in the occurrence of several chronic respiratory diseases, ranging from severe respiratory insufficiency occurring early in life to the association of lung fibrosis and cancer in adult patients. These data also clearly show that, in spite of their structural and functional similarities, SP-A1 and SP-A2 are not redundant.
Asunto(s)
Mutación de Línea Germinal , Neumonías Intersticiales Idiopáticas/genética , Neoplasias Pulmonares/genética , Mutación Missense , Proteína A Asociada a Surfactante Pulmonar/genética , Adulto , Anciano , Femenino , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Neumonías Intersticiales Idiopáticas/patología , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Linaje , Proteína A Asociada a Surfactante Pulmonar/metabolismo , Análisis de Secuencia de ADNRESUMEN
POU1F1, a pituitary-specific POU-homeo domain transcription factor, plays an essential role in the specification of the somatotroph, lactotroph and thyrotroph lineages and in the activation of GH1, PRL and TSHß transcription. Individuals with mutations in POU1F1 present with combined deficiency of GH, PRL and TSH. Here, we identified a heterozygous missense mutation with evidence of pathogenicity, at the POU1F1 locus, in a large family in which an isolated growth hormone deficiency segregates as an autosomal dominant trait. The corresponding p.Pro76Leu mutation maps to a conserved site within the POU1F1 transactivation domain. Bandshift assays revealed that the mutation alters wild-type POU1F1 binding to cognate sites within the hGH-LCR and hGH1 promoter, but not to sites within the PRL promoter, and it selectively increases binding affinity to sites within the hGH-LCR. Co-immunoprecipitation studies reveal that this substitution enhances interactions of POU1F1 with three of its cofactors, PITX1, LHX3a and ELK1, and that residue 76 plays a critical role in these interactions. The insertion of the mutation at the mouse Pou1f1 locus results in a dramatic loss of protein expression despite normal mRNA concentrations. Mice heterozygous for the p.Pro76Leu mutation were phenotypically normal while homozygotes demonstrated a dwarf phenotype. Overall, this study unveils the involvement of POU1F1 in dominantly inherited isolated GH deficiency and demonstrates a significant impact of the Pro76Leu mutation on DNA-binding activities, alterations in transactivating functions and interactions with cofactors. Our data further highlight difficulties in modeling human genetic disorders in the mouse despite apparent conservation of gene expression pathways and physiologic functions.