RESUMEN
Ciliopathies are a large group of clinically and genetically heterogeneous disorders caused by defects in primary cilia. Here we identified mutations in TRAF3IP1 (TNF Receptor-Associated Factor Interacting Protein 1) in eight patients from five families with nephronophthisis (NPH) and retinal degeneration, two of the most common manifestations of ciliopathies. TRAF3IP1 encodes IFT54, a subunit of the IFT-B complex required for ciliogenesis. The identified mutations result in mild ciliary defects in patients but also reveal an unexpected role of IFT54 as a negative regulator of microtubule stability via MAP4 (microtubule-associated protein 4). Microtubule defects are associated with altered epithelialization/polarity in renal cells and with pronephric cysts and microphthalmia in zebrafish embryos. Our findings highlight the regulation of cytoplasmic microtubule dynamics as a role of the IFT54 protein beyond the cilium, contributing to the development of NPH-related ciliopathies.
Asunto(s)
Proteínas Portadoras/genética , Enfermedades Renales Quísticas/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mutación , Degeneración Retiniana/genética , Proteínas de Pez Cebra/genética , Animales , Western Blotting , Proteínas Portadoras/metabolismo , Polaridad Celular/genética , Dicroismo Circular , Embrión no Mamífero , Femenino , Técnica del Anticuerpo Fluorescente , Técnicas de Inactivación de Genes , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunoprecipitación , Enfermedades Renales Quísticas/metabolismo , Masculino , Microftalmía/genética , Linaje , Degeneración Retiniana/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Pez Cebra , Proteínas de Pez Cebra/metabolismoRESUMEN
Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder caused by cilia and sperm dysmotility. About 12% of cases show perturbed 9+2 microtubule cilia structure and inner dynein arm (IDA) loss, historically termed "radial spoke defect." We sequenced CCDC39 and CCDC40 in 54 "radial spoke defect" families, as these are the two genes identified so far to cause this defect. We discovered biallelic mutations in a remarkable 69% (37/54) of families, including identification of 25 (19 novel) mutant alleles (12 in CCDC39 and 13 in CCDC40). All the mutations were nonsense, splice, and frameshift predicting early protein truncation, which suggests this defect is caused by "null" alleles conferring complete protein loss. Most families (73%; 27/37) had homozygous mutations, including families from outbred populations. A major putative hotspot mutation was identified, CCDC40 c.248delC, as well as several other possible hotspot mutations. Together, these findings highlight the key role of CCDC39 and CCDC40 in PCD with axonemal disorganization and IDA loss, and these genes represent major candidates for genetic testing in families affected by this ciliary phenotype. We show that radial spoke structures are largely intact in these patients and propose this ciliary ultrastructural abnormality be referred to as "IDA and microtubular disorganisation defect," rather than "radial spoke defect."
Asunto(s)
Axonema/genética , Dineínas/genética , Síndrome de Kartagener/genética , Mutación , Proteínas/genética , Alelos , Axonema/patología , Cilios/genética , Cilios/patología , Proteínas del Citoesqueleto/genética , Exoma , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Masculino , Microscopía Electrónica , Linaje , FenotipoRESUMEN
Cilia are essential for fertilization, respiratory clearance, cerebrospinal fluid circulation and establishing laterality. Cilia motility defects cause primary ciliary dyskinesia (PCD, MIM244400), a disorder affecting 1:15,000-30,000 births. Cilia motility requires the assembly of multisubunit dynein arms that drive ciliary bending. Despite progress in understanding the genetic basis of PCD, mutations remain to be identified for several PCD-linked loci. Here we show that the zebrafish cilia paralysis mutant schmalhans (smh(tn222)) encodes the coiled-coil domain containing 103 protein (Ccdc103), a foxj1a-regulated gene product. Screening 146 unrelated PCD families identified individuals in six families with reduced outer dynein arms who carried mutations in CCDC103. Dynein arm assembly in smh mutant zebrafish was rescued by wild-type but not mutant human CCDC103. Chlamydomonas Ccdc103/Pr46b functions as a tightly bound, axoneme-associated protein. These results identify Ccdc103 as a dynein arm attachment factor that causes primary ciliary dyskinesia when mutated.
Asunto(s)
Dineínas/metabolismo , Síndrome de Kartagener/genética , Animales , Cilios/metabolismo , Femenino , Humanos , Masculino , Mutación , Linaje , Pez CebraRESUMEN
RATIONALE: Primary ciliary dyskinesia (PCD) is an autosomal recessive, genetically heterogeneous disorder characterised by oto-sino-pulmonary disease and situs abnormalities (Kartagener syndrome) due to abnormal structure and/or function of cilia. Most patients currently recognised to have PCD have ultrastructural defects of cilia; however, some patients have clinical manifestations of PCD and low levels of nasal nitric oxide, but normal ultrastructure, including a few patients with biallelic mutations in dynein axonemal heavy chain 11 (DNAH11). OBJECTIVES: To test further for mutant DNAH11 as a cause of PCD, DNAH11 was sequenced in patients with a PCD clinical phenotype, but no known genetic aetiology. METHODS: 82 exons and intron/exon junctions in DNAH11 were sequenced in 163 unrelated patients with a clinical phenotype of PCD, including those with normal ciliary ultrastructure (n=58), defects in outer and/or inner dynein arms (n=76), radial spoke/central pair defects (n=6), and 23 without definitive ultrastructural results, but who had situs inversus (n=17), or bronchiectasis and/or low nasal nitric oxide (n=6). Additionally, DNAH11 was sequenced in 13 subjects with isolated situs abnormalities to see if mutant DNAH11 could cause situs defects without respiratory disease. RESULTS: Of the 58 unrelated patients with PCD with normal ultrastructure, 13 (22%) had two (biallelic) mutations in DNAH11; and two patients without ultrastructural analysis had biallelic mutations. All mutations were novel and private. None of the patients with dynein arm or radial spoke/central pair defects, or isolated situs abnormalities, had mutations in DNAH11. Of the 35 identified mutant alleles, 24 (69%) were nonsense, insertion/deletion or loss-of-function splice-site mutations. CONCLUSIONS: Mutations in DNAH11 are a common cause of PCD in patients without ciliary ultrastructural defects; thus, genetic analysis can be used to ascertain the diagnosis of PCD in this challenging group of patients.
Asunto(s)
Dineínas Axonemales/genética , Cilios/ultraestructura , Trastornos de la Motilidad Ciliar/genética , Mutación , Adolescente , Adulto , Niño , Preescolar , Trastornos de la Motilidad Ciliar/diagnóstico , Trastornos de la Motilidad Ciliar/patología , Femenino , Genotipo , Humanos , Lactante , Masculino , Linaje , Fenotipo , Polimorfismo Genético , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Adulto JovenRESUMEN
Primary ciliary dyskinesia (PCD) is an inherited disorder characterized by recurrent infections of the upper and lower respiratory tract, reduced fertility in males and situs inversus in about 50% of affected individuals (Kartagener syndrome). It is caused by motility defects in the respiratory cilia that are responsible for airway clearance, the flagella that propel sperm cells and the nodal monocilia that determine left-right asymmetry. Recessive mutations that cause PCD have been identified in genes encoding components of the outer dynein arms, radial spokes and cytoplasmic pre-assembly factors of axonemal dyneins, but these mutations account for only about 50% of cases of PCD. We exploited the unique properties of dog populations to positionally clone a new PCD gene, CCDC39. We found that loss-of-function mutations in the human ortholog underlie a substantial fraction of PCD cases with axonemal disorganization and abnormal ciliary beating. Functional analyses indicated that CCDC39 localizes to ciliary axonemes and is essential for assembly of inner dynein arms and the dynein regulatory complex.
Asunto(s)
Cilios/fisiología , Trastornos de la Motilidad Ciliar/genética , Dineínas/genética , Proteínas/genética , Animales , Secuencia de Bases , Células Cultivadas , Proteínas del Citoesqueleto , Perros , Humanos , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Mutación , Proteínas/análisis , Proteínas/fisiologíaRESUMEN
Primary ciliary dyskinesia (PCD) is a genetically heterogeneous autosomal recessive disorder characterized by recurrent infections of the respiratory tract associated with the abnormal function of motile cilia. Approximately half of individuals with PCD also have alterations in the left-right organization of their internal organ positioning, including situs inversus and situs ambiguous (Kartagener's syndrome). Here, we identify an uncharacterized coiled-coil domain containing a protein, CCDC40, essential for correct left-right patterning in mouse, zebrafish and human. In mouse and zebrafish, Ccdc40 is expressed in tissues that contain motile cilia, and mutations in Ccdc40 result in cilia with reduced ranges of motility. We further show that CCDC40 mutations in humans result in a variant of PCD characterized by misplacement of the central pair of microtubules and defective assembly of inner dynein arms and dynein regulatory complexes. CCDC40 localizes to motile cilia and the apical cytoplasm and is required for axonemal recruitment of CCDC39, disruption of which underlies a similar variant of PCD.
Asunto(s)
Trastornos de la Motilidad Ciliar/genética , Proteínas/genética , Animales , Cilios/genética , Dineínas/genética , Humanos , Síndrome de Kartagener/genética , Ratones , Ratones Endogámicos , Mutación , Proteínas/fisiología , Situs Inversus/genética , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Genetic defects affecting motility of cilia and flagella cause chronic destructive airway disease, randomization of left-right body asymmetry, and, frequently, male infertility in primary ciliary dyskinesia (PCD). The most frequent defects involve outer and inner dynein arms (ODAs and IDAs) that are large multiprotein complexes responsible for cilia-beat generation and regulation, respectively. Here, we demonstrate that large genomic deletions, as well as point mutations involving LRRC50, are responsible for a distinct PCD variant that is characterized by a combined defect involving assembly of the ODAs and IDAs. Functional analyses showed that LRRC50 deficiency disrupts assembly of distally and proximally DNAH5- and DNAI2-containing ODA complexes, as well as DNALI1-containing IDA complexes, resulting in immotile cilia. On the basis of these findings, we assume that LRRC50 plays a role in assembly of distinct dynein-arm complexes.