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1.
Am J Hum Genet ; 102(5): 956-972, 2018 05 03.
Artículo en Inglés | MEDLINE | ID: mdl-29727692

RESUMEN

Primary ciliary dyskinesia (PCD) is a genetically and phenotypically heterogeneous disorder characterized by destructive respiratory disease and laterality abnormalities due to randomized left-right body asymmetry. PCD is mostly caused by mutations affecting the core axoneme structure of motile cilia that is essential for movement. Genes that cause PCD when mutated include a group that encode proteins essential for the assembly of the ciliary dynein motors and the active transport process that delivers them from their cytoplasmic assembly site into the axoneme. We screened a cohort of affected individuals for disease-causing mutations using a targeted next generation sequencing panel and identified two unrelated families (three affected children) with mutations in the uncharacterized C11orf70 gene (official gene name CFAP300). The affected children share a consistent PCD phenotype from early life with laterality defects and immotile respiratory cilia displaying combined loss of inner and outer dynein arms (IDA+ODA). Phylogenetic analysis shows C11orf70 is highly conserved, distributed across species similarly to proteins involved in the intraflagellar transport (IFT)-dependant assembly of axonemal dyneins. Paramecium C11orf70 RNAi knockdown led to combined loss of ciliary IDA+ODA with reduced cilia beating and swim velocity. Tagged C11orf70 in Paramecium and Chlamydomonas localizes mainly in the cytoplasm with a small amount in the ciliary component. IFT139/TTC21B (IFT-A protein) and FLA10 (IFT kinesin) depletion experiments show that its transport within cilia is IFT dependent. During ciliogenesis, C11orf70 accumulates at the ciliary tips in a similar distribution to the IFT-B protein IFT46. In summary, C11orf70 is essential for assembly of dynein arms and C11orf70 mutations cause defective cilia motility and PCD.


Asunto(s)
Dineínas Axonemales/metabolismo , Trastornos de la Motilidad Ciliar/genética , Proteínas del Citoesqueleto/genética , Flagelos/metabolismo , Mutación/genética , Proteínas Nucleares/genética , Alelos , Secuencia de Aminoácidos , Dineínas Axonemales/ultraestructura , Secuencia de Bases , Transporte Biológico , Diferenciación Celular/genética , Chlamydomonas/metabolismo , Secuencia Conservada/genética , Flagelos/ultraestructura , Técnicas de Silenciamiento del Gen , Proteínas Fluorescentes Verdes/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Proteínas Nucleares/química , Paramecium/metabolismo , Paramecium/ultraestructura , Transcripción Genética
2.
J Med Genet ; 57(5): 322-330, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-31879361

RESUMEN

BACKGROUND: Primary ciliary dyskinesia (PCD), a genetically heterogeneous condition enriched in some consanguineous populations, results from recessive mutations affecting cilia biogenesis and motility. Currently, diagnosis requires multiple expert tests. METHODS: The diagnostic utility of multigene panel next-generation sequencing (NGS) was evaluated in 161 unrelated families from multiple population ancestries. RESULTS: Most (82%) families had affected individuals with biallelic or hemizygous (75%) or single (7%) pathogenic causal alleles in known PCD genes. Loss-of-function alleles dominate (73% frameshift, stop-gain, splice site), most (58%) being homozygous, even in non-consanguineous families. Although 57% (88) of the total 155 diagnostic disease variants were novel, recurrent mutations and mutated genes were detected. These differed markedly between white European (52% of families carry DNAH5 or DNAH11 mutations), Arab (42% of families carry CCDC39 or CCDC40 mutations) and South Asian (single LRRC6 or CCDC103 mutations carried in 36% of families) patients, revealing a striking genetic stratification according to population of origin in PCD. Genetics facilitated successful diagnosis of 81% of families with normal or inconclusive ultrastructure and 67% missing prior ultrastructure results. CONCLUSIONS: This study shows the added value of high-throughput targeted NGS in expediting PCD diagnosis. Therefore, there is potential significant patient benefit in wider and/or earlier implementation of genetic screening.


Asunto(s)
Cilios/genética , Trastornos de la Motilidad Ciliar/genética , Pruebas Genéticas , Secuenciación de Nucleótidos de Alto Rendimiento , Alelos , Pueblo Asiatico/genética , Cilios/patología , Trastornos de la Motilidad Ciliar/diagnóstico , Trastornos de la Motilidad Ciliar/patología , Estudios de Cohortes , Etnicidad/genética , Femenino , Homocigoto , Humanos , Masculino , Mutación/genética , Fenotipo
3.
Thorax ; 73(2): 157-166, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-28790179

RESUMEN

RATIONALE: Primary ciliary dyskinesia is a genetically heterogeneous inherited condition characterised by progressive lung disease arising from abnormal cilia function. Approximately half of patients have situs inversus. The estimated prevalence of primary ciliary dyskinesia in the UK South Asian population is 1:2265. Early, accurate diagnosis is key to implementing appropriate management but clinical diagnostic tests can be equivocal. OBJECTIVES: To determine the importance of genetic screening for primary ciliary dyskinesia in a UK South Asian population with a typical clinical phenotype, where standard testing is inconclusive. METHODS: Next-generation sequencing was used to screen 86 South Asian patients who had a clinical history consistent with primary ciliary dyskinesia. The effect of a CCDC103 p.His154Pro missense variant compared with other dynein arm-associated gene mutations on diagnostic/phenotypic variability was tested. CCDC103 p.His154Pro variant pathogenicity was assessed by oligomerisation assay. RESULTS: Sixteen of 86 (19%) patients carried a homozygous CCDC103 p.His154Pro mutation which was found to disrupt protein oligomerisation. Variable diagnostic test results were obtained including normal nasal nitric oxide levels, normal ciliary beat pattern and frequency and a spectrum of partial and normal dynein arm retention. Fifteen (94%) patients or their sibling(s) had situs inversus suggesting CCDC103 p.His154Pro patients without situs inversus are missed. CONCLUSIONS: The CCDC103 p.His154Pro mutation is more prevalent than previously thought in the South Asian community and causes primary ciliary dyskinesia that can be difficult to diagnose using pathology-based clinical tests. Genetic testing is critical when there is a strong clinical phenotype with inconclusive standard diagnostic tests.


Asunto(s)
Pueblo Asiatico/genética , Síndrome de Kartagener/etnología , Síndrome de Kartagener/genética , Proteínas Asociadas a Microtúbulos/genética , Mutación/genética , Adolescente , Adulto , Niño , Preescolar , Estudios de Cohortes , Femenino , Humanos , Masculino , Pakistán/etnología , Reino Unido , Adulto Joven
4.
Eur Respir J ; 51(2)2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-29467202

RESUMEN

In primary ciliary dyskinesia (PCD), motile ciliary dysfunction arises from ciliary defects usually confirmed by transmission electron microscopy (TEM). In 30% of patients, such as those with DNAH11 mutations, apparently normal ultrastructure makes diagnosis difficult. Genetic analysis supports diagnosis, but may not identify definitive causal variants. Electron tomography, an extension of TEM, produces three-dimensional ultrastructural ciliary models with superior resolution to TEM. Our hypothesis is that tomography using existing patient samples will enable visualisation of DNAH11-associated ultrastructural defects. Dual axis tomograms from araldite-embedded nasal cilia were collected in 13 PCD patients with normal ultrastructure (DNAH11 n=7, HYDIN n=2, CCDC65 n=3 and DRC1 n=1) and six healthy controls, then analysed using IMOD and Chimera software.DNAH11 protein is localised to the proximal ciliary region. Within this region, electron tomography indicated a deficiency of >25% of proximal outer dynein arm volume in all patients with DNAH11 mutations (n=7) compared to other patients with PCD and normal ultrastructure (n=6) and healthy controls (n=6). DNAH11 mutations cause a shared abnormality in ciliary ultrastructure previously undetectable by TEM. Advantageously, electron tomography can be used on existing diagnostic samples and establishes a structural abnormality where ultrastructural studies were previously normal.


Asunto(s)
Dineínas Axonemales/deficiencia , Dineínas Axonemales/genética , Cilios/ultraestructura , Trastornos de la Motilidad Ciliar/diagnóstico por imagen , Trastornos de la Motilidad Ciliar/genética , Adolescente , Estudios de Casos y Controles , Niño , Tomografía con Microscopio Electrónico , Femenino , Genotipo , Humanos , Imagenología Tridimensional , Masculino , Mutación , Tomografía
5.
Am J Hum Genet ; 95(3): 257-74, 2014 Sep 04.
Artículo en Inglés | MEDLINE | ID: mdl-25192045

RESUMEN

A diverse family of cytoskeletal dynein motors powers various cellular transport systems, including axonemal dyneins generating the force for ciliary and flagellar beating essential to movement of extracellular fluids and of cells through fluid. Multisubunit outer dynein arm (ODA) motor complexes, produced and preassembled in the cytosol, are transported to the ciliary or flagellar compartment and anchored into the axonemal microtubular scaffold via the ODA docking complex (ODA-DC) system. In humans, defects in ODA assembly are the major cause of primary ciliary dyskinesia (PCD), an inherited disorder of ciliary and flagellar dysmotility characterized by chronic upper and lower respiratory infections and defects in laterality. Here, by combined high-throughput mapping and sequencing, we identified CCDC151 loss-of-function mutations in five affected individuals from three independent families whose cilia showed a complete loss of ODAs and severely impaired ciliary beating. Consistent with the laterality defects observed in these individuals, we found Ccdc151 expressed in vertebrate left-right organizers. Homozygous zebrafish ccdc151(ts272a) and mouse Ccdc151(Snbl) mutants display a spectrum of situs defects associated with complex heart defects. We demonstrate that CCDC151 encodes an axonemal coiled coil protein, mutations in which abolish assembly of CCDC151 into respiratory cilia and cause a failure in axonemal assembly of the ODA component DNAH5 and the ODA-DC-associated components CCDC114 and ARMC4. CCDC151-deficient zebrafish, planaria, and mice also display ciliary dysmotility accompanied by ODA loss. Furthermore, CCDC151 coimmunoprecipitates CCDC114 and thus appears to be a highly evolutionarily conserved ODA-DC-related protein involved in mediating assembly of both ODAs and their axonemal docking machinery onto ciliary microtubules.


Asunto(s)
Dineínas Axonemales/metabolismo , Cilios/patología , Síndrome de Kartagener/genética , Proteínas Asociadas a Microtúbulos/fisiología , Mutación/genética , Animales , Dineínas Axonemales/genética , Axonema/genética , Células Cultivadas , Cilios/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Exoma/genética , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Inmunoprecipitación , Hibridación in Situ , Síndrome de Kartagener/metabolismo , Síndrome de Kartagener/patología , Masculino , Ratones , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Linaje , Fenotipo , Técnicas del Sistema de Dos Híbridos , Pez Cebra/genética , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismo
6.
Pediatr Res ; 81(4): 632-638, 2017 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-27855150

RESUMEN

BACKGROUND: The aim was to identify susceptibility alleles for infantile hypertrophic pyloric stenosis (IHPS) in a pedigree previously linked to IHPS5 on chromosome 16q24. METHODS: We screened the positional and functional candidate gene FOXF1 by Sanger sequencing in a single affected individual. All family members for whom DNA was available were genotyped to determine cosegregation status of the putative causal variant. Immunofluorescence studies were performed to compare the cellular localization of wildtype and mutant form of the protein. Transcriptional activity was compared using a luciferase assay. RESULTS: A single novel substitution in FOXF1 (c.416G>A) predicted to result in a missense mutation (R139Q) was shown to cosegregate with disease trait. It was not seen in 560 control chromosomes nor has it been reported in ExAC or ESP. The R139Q substitution affects a conserved arginine residue within the DNA-binding domain of FOXF1. The transcriptional activity of the mutant FOXF1 protein is significantly reduced in comparison to wild-type. CONCLUSION: These results provide strong evidence that the R139Q substitution in FOXF1 causes IHPS in this family and imply a novel pathological pathway for the condition. They further support a role for FOXF1 in the regulation of embryonic and neonatal development of the gastro-intestinal tract.


Asunto(s)
Cromosomas Humanos Par 16 , Factores de Transcripción Forkhead/genética , Mutación Missense , Estenosis Hipertrófica del Piloro/genética , Alelos , Arginina/química , Mapeo Cromosómico , Femenino , Variación Genética , Genotipo , Células HEK293 , Haplotipos , Células Hep G2 , Humanos , Masculino , Microscopía Fluorescente , Linaje , Estenosis Hipertrófica del Piloro/metabolismo , Análisis de Secuencia de ADN , Activación Transcripcional
7.
Hum Mol Genet ; 23(13): 3362-74, 2014 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-24518672

RESUMEN

Primary ciliary dyskinesia (PCD) is an inherited chronic respiratory obstructive disease with randomized body laterality and infertility, resulting from cilia and sperm dysmotility. PCD is characterized by clinical variability and extensive genetic heterogeneity, associated with different cilia ultrastructural defects and mutations identified in >20 genes. Next generation sequencing (NGS) technologies therefore present a promising approach for genetic diagnosis which is not yet in routine use. We developed a targeted panel-based NGS pipeline to identify mutations by sequencing of selected candidate genes in 70 genetically undefined PCD patients. This detected loss-of-function RSPH1 mutations in four individuals with isolated central pair (CP) agenesis and normal body laterality, from two unrelated families. Ultrastructural analysis in RSPH1-mutated cilia revealed transposition of peripheral outer microtubules into the 'empty' CP space, accompanied by a distinctive intermittent loss of the central pair microtubules. We find that mutations in RSPH1, RSPH4A and RSPH9, which all encode homologs of components of the 'head' structure of ciliary radial spoke complexes identified in Chlamydomonas, cause clinical phenotypes that appear to be indistinguishable except at the gene level. By high-resolution immunofluorescence we identified a loss of RSPH4A and RSPH9 along with RSPH1 from RSPH1-mutated cilia, suggesting RSPH1 mutations may result in loss of the entire spoke head structure. CP loss is seen in up to 28% of PCD cases, in whom laterality determination specified by CP-less embryonic node cilia remains undisturbed. We propose this defect could arise from instability or agenesis of the ciliary central microtubules due to loss of their normal radial spoke head tethering.


Asunto(s)
Proteínas de Unión al ADN/genética , Síndrome de Kartagener/genética , Axonema/metabolismo , Axonema/fisiología , Proteínas del Citoesqueleto/genética , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Síndrome de Kartagener/fisiopatología , Microscopía Electrónica , Microscopía Fluorescente , Mutación , Proteínas/genética
8.
Am J Hum Genet ; 92(1): 88-98, 2013 Jan 10.
Artículo en Inglés | MEDLINE | ID: mdl-23261303

RESUMEN

Defects in motile cilia and sperm flagella cause primary ciliary dyskinesia (PCD), characterized by chronic airway disease, infertility, and left-right laterality disturbances, usually as a result of loss of the outer dynein arms (ODAs) that power cilia/flagella beating. Here, we identify loss-of-function mutations in CCDC114 causing PCD with laterality malformations involving complex heart defects. CCDC114 is homologous to DCC2, an ODA microtubule-docking complex component of the biflagellate alga Chlamydomonas. We show that CCDC114 localizes along the entire length of human cilia and that its deficiency causes a complete absence of ciliary ODAs, resulting in immotile cilia. Thus, CCDC114 is an essential ciliary protein required for microtubular attachment of ODAs in the axoneme. Fertility is apparently not greatly affected by CCDC114 deficiency, and qPCR shows that this may explained by low transcript expression in testis compared to ciliated respiratory epithelium. One CCDC114 mutation, c.742G>A, dating back to at least the 1400s, presents an important diagnostic and therapeutic target in the isolated Dutch Volendam population.


Asunto(s)
Axonema/genética , Síndrome de Kartagener/genética , Proteínas Asociadas a Microtúbulos/genética , Mutación , Sitios de Empalme de ARN , Secuencia de Bases , Dineínas , Femenino , Humanos , Masculino , Datos de Secuencia Molecular , Linaje
9.
Am J Hum Genet ; 93(2): 346-56, 2013 Aug 08.
Artículo en Inglés | MEDLINE | ID: mdl-23891471

RESUMEN

Primary ciliary dyskinesia (PCD) is a ciliopathy characterized by airway disease, infertility, and laterality defects, often caused by dual loss of the inner dynein arms (IDAs) and outer dynein arms (ODAs), which power cilia and flagella beating. Using whole-exome and candidate-gene Sanger resequencing in PCD-affected families afflicted with combined IDA and ODA defects, we found that 6/38 (16%) carried biallelic mutations in the conserved zinc-finger gene BLU (ZMYND10). ZMYND10 mutations conferred dynein-arm loss seen at the ultrastructural and immunofluorescence level and complete cilia immotility, except in hypomorphic p.Val16Gly (c.47T>G) homozygote individuals, whose cilia retained a stiff and slowed beat. In mice, Zmynd10 mRNA is restricted to regions containing motile cilia. In a Drosophila model of PCD, Zmynd10 is exclusively expressed in cells with motile cilia: chordotonal sensory neurons and sperm. In these cells, P-element-mediated gene silencing caused IDA and ODA defects, proprioception deficits, and sterility due to immotile sperm. Drosophila Zmynd10 with an equivalent c.47T>G (p.Val16Gly) missense change rescued mutant male sterility less than the wild-type did. Tagged Drosophila ZMYND10 is localized primarily to the cytoplasm, and human ZMYND10 interacts with LRRC6, another cytoplasmically localized protein altered in PCD. Using a fly model of PCD, we conclude that ZMYND10 is a cytoplasmic protein required for IDA and ODA assembly and that its variants cause ciliary dysmotility and PCD with laterality defects.


Asunto(s)
Cilios/genética , Dineínas/genética , Infertilidad Masculina/genética , Síndrome de Kartagener/genética , Proteínas/genética , Sistema Respiratorio/metabolismo , Proteínas Supresoras de Tumor/genética , Animales , Axonema/genética , Axonema/metabolismo , Axonema/patología , Cilios/metabolismo , Cilios/patología , Proteínas del Citoesqueleto , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Dineínas/metabolismo , Exoma , Femenino , Regulación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Infertilidad Masculina/metabolismo , Infertilidad Masculina/patología , Síndrome de Kartagener/metabolismo , Síndrome de Kartagener/patología , Masculino , Ratones , Mutación , Linaje , Estructura Terciaria de Proteína , Proteínas/metabolismo , Sistema Respiratorio/patología , Proteínas Supresoras de Tumor/metabolismo
10.
Am J Hum Genet ; 91(4): 672-84, 2012 Oct 05.
Artículo en Inglés | MEDLINE | ID: mdl-23022101

RESUMEN

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous recessive disorder characterized by defective cilia and flagella motility. Chronic destructive-airway disease is caused by abnormal respiratory-tract mucociliary clearance. Abnormal propulsion of sperm flagella contributes to male infertility. Genetic defects in most individuals affected by PCD cause randomization of left-right body asymmetry; approximately half show situs inversus or situs ambiguous. Almost 70 years after the hy3 mouse possessing Hydin mutations was described as a recessive hydrocephalus model, we report HYDIN mutations in PCD-affected persons without hydrocephalus. By homozygosity mapping, we identified a PCD-associated locus, chromosomal region 16q21-q23, which contains HYDIN. However, a nearly identical 360 kb paralogous segment (HYDIN2) in chromosomal region 1q21.1 complicated mutational analysis. In three affected German siblings linked to HYDIN, we identified homozygous c.3985G>T mutations that affect an evolutionary conserved splice acceptor site and that subsequently cause aberrantly spliced transcripts predicting premature protein termination in respiratory cells. Parallel whole-exome sequencing identified a homozygous nonsense HYDIN mutation, c.922A>T (p.Lys307(∗)), in six individuals from three Faroe Island PCD-affected families that all carried an 8.8 Mb shared haplotype across HYDIN, indicating an ancestral founder mutation in this isolated population. We demonstrate by electron microscopy tomography that, consistent with the effects of loss-of-function mutations, HYDIN mutant respiratory cilia lack the C2b projection of the central pair (CP) apparatus; similar findings were reported in Hydin-deficient Chlamydomonas and mice. High-speed videomicroscopy demonstrated markedly reduced beating amplitudes of respiratory cilia and stiff sperm flagella. Like the hy3 mouse model, all nine PCD-affected persons had normal body composition because nodal cilia function is apparently not dependent on the function of the CP apparatus.


Asunto(s)
Genes Recesivos , Síndrome de Kartagener/genética , Proteínas de Microfilamentos/genética , Mutación , Adulto , Secuencia de Bases , Cromosomas Humanos Par 1 , Cromosomas Humanos Par 16 , Cilios/genética , Análisis Mutacional de ADN/métodos , Femenino , Sitios Genéticos/genética , Predisposición Genética a la Enfermedad , Haplotipos , Homocigoto , Humanos , Hidrocefalia/genética , Masculino , Datos de Secuencia Molecular , Linaje , Empalme del ARN/genética , Hermanos , Situs Inversus/genética
11.
J Med Genet ; 51(1): 61-7, 2014 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24203976

RESUMEN

BACKGROUND: Primary ciliary dyskinesia (PCD) is a rare, genetically heterogeneous ciliopathy disorder affecting cilia and sperm motility. A range of ultrastructural defects of the axoneme underlie the disease, which is characterised by chronic respiratory symptoms and obstructive lung disease, infertility and body axis laterality defects. We applied a next-generation sequencing approach to identify the gene responsible for this phenotype in two consanguineous families. METHODS AND RESULTS: Data from whole-exome sequencing in a consanguineous Turkish family, and whole-genome sequencing in the obligate carrier parents of a consanguineous Pakistani family was combined to identify homozygous loss-of-function mutations in ARMC4, segregating in all five affected individuals from both families. Both families carried nonsense mutations within the highly conserved armadillo repeat region of ARMC4: c.2675C>A; pSer892* and c.1972G>T; p.Glu658*. A deficiency of ARMC4 protein was seen in patient's respiratory cilia accompanied by loss of the distal outer dynein arm motors responsible for generating ciliary beating, giving rise to cilia immotility. ARMC4 gene expression is upregulated during ciliogenesis, and we found a predicted interaction with the outer dynein arm protein DNAI2, mutations in which also cause PCD. CONCLUSIONS: We report the first use of whole-genome sequencing to identify gene mutations causing PCD. Loss-of-function mutations in ARMC4 cause PCD with situs inversus and cilia immotility, associated with a loss of the distal outer (but not inner) dynein arms. This addition of ARMC4 to the list of genes associated with ciliary outer dynein arm defects expands our understanding of the complexities of PCD genetics.


Asunto(s)
Proteínas del Dominio Armadillo/genética , Dineínas/genética , Estudio de Asociación del Genoma Completo , Síndrome de Kartagener/genética , Síndrome de Kartagener/metabolismo , Mutación , Proteínas del Dominio Armadillo/química , Proteínas del Dominio Armadillo/metabolismo , Cilios/genética , Cilios/metabolismo , Cilios/ultraestructura , Dineínas/química , Dineínas/metabolismo , Exoma , Femenino , Genoma Humano , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Modelos Moleculares , Linaje , Fenotipo , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas
12.
Hum Mutat ; 34(3): 462-72, 2013 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-23255504

RESUMEN

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 , Fenotipo
13.
J Hum Genet ; 58(4): 236-7, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23426030

RESUMEN

Infantile hypertrophic pyloric stenosis (IHPS) is a multifactorial heritable condition affecting infants in the first 3 months of life. It is characterized by hypertrophy of the pylorus resulting in blockage of the pyloric canal. Patients present with projectile vomiting, weight loss and dehydration. Five susceptibility loci have been identified through genome-wide linkage analysis and candidate gene approaches. The first genome-wide association study was recently performed and three statistically significant associations identified. Here, we report our confirmation of two of these significant results thus providing further support for new loci for IHPS on chromosome 3p25.1 and chromosome 5q35.2.


Asunto(s)
Cromosomas Humanos Par 3/genética , Cromosomas Humanos Par 5/genética , Sitios Genéticos , Estenosis Hipertrófica del Piloro/genética , Femenino , Ligamiento Genético , Estudio de Asociación del Genoma Completo , Genotipo , Humanos , Lactante , Masculino
14.
Nat Genet ; 30(2): 143-4, 2002 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-11788826

RESUMEN

Primary ciliary dyskinesia (PCD, MIM 242650) is characterized by recurrent infections of the respiratory tract due to reduced mucociliary clearance and by sperm immobility. Half of the affected offspring have situs inversus (reversed organs), which results from randomization of left-right (LR) asymmetry. We previously localized to chromosome 5p a PCD locus containing DNAH5, which encodes a protein highly similar to the Chlamydomonas gamma-dynein heavy chain. Here we characterize the full-length 14-kb transcript of DNAH5. Sequence analysis in individuals with PCD with randomization of LR asymmetry identified mutations resulting in non-functional DNAH5 proteins.


Asunto(s)
Tipificación del Cuerpo/genética , Dineínas/genética , Síndrome de Kartagener/genética , Mutación , Animales , Cilios/ultraestructura , Femenino , Humanos , Masculino , Ratones , Proteínas Motoras Moleculares/genética , Situs Inversus/genética
15.
Am J Hum Genet ; 84(2): 197-209, 2009 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19200523

RESUMEN

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous inherited disorder arising from dysmotility of motile cilia and sperm. This is associated with a variety of ultrastructural defects of the cilia and sperm axoneme that affect movement, leading to clinical consequences on respiratory-tract mucociliary clearance and lung function, fertility, and left-right body-axis determination. We performed whole-genome SNP-based linkage analysis in seven consanguineous families with PCD and central-microtubular-pair abnormalities. This identified two loci, in two families with intermittent absence of the central-pair structure (chromosome 6p21.1, Zmax 6.7) and in five families with complete absence of the central pair (chromosome 6q22.1, Zmax 7.0). Mutations were subsequently identified in two positional candidate genes, RSPH9 on chromosome 6p21.1 and RSPH4A on chromosome 6q22.1. Haplotype analysis identified a common ancestral founder effect RSPH4A mutation present in UK-Pakistani pedigrees. Both RSPH9 and RSPH4A encode protein components of the axonemal radial spoke head. In situ hybridization of murine Rsph9 shows gene expression restricted to regions containing motile cilia. Investigation of the effect of knockdown or mutations of RSPH9 orthologs in zebrafish and Chlamydomonas indicate that radial spoke head proteins are important in maintaining normal movement in motile, "9+2"-structure cilia and flagella. This effect is rescued by reintroduction of gene expression for restoration of a normal beat pattern in zebrafish. Disturbance in function of these genes was not associated with defects in left-right axis determination in humans or zebrafish.


Asunto(s)
Cilios/patología , Anomalías Congénitas/genética , Proteínas del Citoesqueleto/genética , Proteínas de Unión al ADN/genética , Síndrome de Kartagener/genética , Mutación , Animales , Chlamydomonas/genética , Aberraciones Cromosómicas , Mapeo Cromosómico , Cromosomas Humanos/genética , Cromosomas Humanos Par 1 , Cilios/genética , Femenino , Humanos , Hibridación in Situ , Masculino , Linaje , Polimorfismo de Nucleótido Simple , Pez Cebra/genética
16.
Am J Hum Genet ; 82(3): 756-62, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18308288

RESUMEN

Infantile hypertrophic pyloric stenosis (IHPS) has an incidence of 1-8 per 1000 live births and is inherited as a complex sex-modified multifactorial trait with a striking male preponderance. Syndromic and monogenic forms exist, and two loci have been identified. Infants present with vomiting due to gastric-outlet obstruction caused by hypertrophy of the smooth muscle of the pylorus. A genome-wide SNP-based high-density linkage scan was carried out on 81 IHPS pedigrees. Nonparametric and parametric linkage analysis identified loci on chromosomes 11q14-q22 (Z(max) = 3.9, p < 0.0001; HLOD(max) = 3.4, alpha = 0.34) and Xq23 (Z(max) = 4.3, p < 0.00001; HLOD(max) = 4.8, alpha = 0.56). The two linked chromosomal regions each harbor functional candidate genes that are members of the canonical transient receptor potential (TRPC) family of ion channels and have a potential role in smooth-muscle control and hypertrophy.


Asunto(s)
Cromosomas Humanos Par 11/genética , Ligamiento Genético , Predisposición Genética a la Enfermedad , Genoma Humano/genética , Estenosis Hipertrófica del Piloro/genética , Mapeo Cromosómico , Femenino , Humanos , Lactante , Masculino , Linaje , Polimorfismo de Nucleótido Simple , Razón de Masculinidad
17.
Am J Hum Genet ; 83(5): 547-58, 2008 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-18950741

RESUMEN

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder characterized by chronic destructive airway disease and randomization of left/right body asymmetry. Males often have reduced fertility due to impaired sperm tail function. The complex PCD phenotype results from dysfunction of cilia of the airways and the embryonic node and the structurally related motile sperm flagella. This is associated with underlying ultrastructural defects that frequently involve the outer dynein arm (ODA) complexes that generate cilia and flagella movement. Applying a positional and functional candidate-gene approach, we identified homozygous loss-of-function DNAI2 mutations (IVS11+1G > A) in four individuals from a family with PCD and ODA defects. Further mutational screening of 105 unrelated PCD families detected two distinct homozygous mutations, including a nonsense (c.787C > T) and a splicing mutation (IVS3-3T > G) resulting in out-of-frame transcripts. Analysis of protein expression of the ODA intermediate chain DNAI2 showed sublocalization throughout respiratory cilia. Electron microscopy showed that mutant respiratory cells from these patients lacked DNAI2 protein expression and exhibited ODA defects. High-resolution immunofluorescence imaging demonstrated absence of the ODA heavy chains DNAH5 and DNAH9 from all DNAI2 mutant ciliary axonemes. In addition, we demonstrated complete or distal absence of DNAI2 from ciliary axonemes in respiratory cells of patients with mutations in genes encoding the ODA chains DNAH5 and DNAI1, respectively. Thus, DNAI2 and DNAH5 mutations affect assembly of proximal and distal ODA complexes, whereas DNAI1 mutations mainly disrupt assembly of proximal ODA complexes.


Asunto(s)
Cilios/genética , Dineínas/genética , Dineínas/ultraestructura , Síndrome de Kartagener/genética , Mutación , Adolescente , Adulto , Anciano , Alelos , Niño , Preescolar , Cilios/ultraestructura , Consanguinidad , Análisis Mutacional de ADN , Dineínas/química , Exones , Femenino , Flagelos/genética , Frecuencia de los Genes , Ligamiento Genético , Humanos , Masculino , Persona de Mediana Edad , Linaje , Polimorfismo de Nucleótido Simple , Empalme del ARN , Análisis de Secuencia de ADN , Adulto Joven
18.
Hum Genet ; 126(6): 819-31, 2009 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19701773

RESUMEN

Infantile hypertrophic pyloric stenosis (IHPS) is the most common inherited form of gastrointestinal obstruction in infancy with a striking male preponderance. Infants present with vomiting due to gastric outlet obstruction caused by hypertrophy of the smooth muscle of the pylorus. Two loci specific to extended pedigrees displaying autosomal dominant inheritance have been identified. A genome scan identified loci on chromosomes 11q14-q22 and Xq23-q24 which are predicted to be responsible for a subset of smaller families with IHPS demonstrating non-Mendelian inheritance. The two linked chromosomal regions both harbour functional candidate genes which are members of the canonical transient receptor potential (TRPC) family of ion channels. Both TRPC5 (Xq23-q24) and TRPC6 (11q14-q22) have a potential role in smooth muscle control and hypertrophy. Here, we report suggestive evidence for a third locus on chromosome 3q12-q25 (Zmax = 2.7, p < 0.004), a region which harbours a third TRPC gene, TRPC1. Fine mapping of all three genes using a tagSNP approach and re-sequencing identified a SNP in the promoter region of TRPC6 and a missense variant in exon 4 of TRPC6 which may be putative causal variants.


Asunto(s)
Estenosis Hipertrófica del Piloro/genética , Canales Catiónicos TRPC/genética , Femenino , Ligamiento Genético , Predisposición Genética a la Enfermedad , Humanos , Masculino , Linaje , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN/métodos , Canal Catiónico TRPC6
19.
Med Educ ; 42(6): 600-6, 2008 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18482091

RESUMEN

CONTEXT: Following a 15-week attachment in paediatrics and child health, general practice and dermatology medical students in their second clinical year at this medical school undertake a high-stakes assessment including an objective structured clinical examination (OSCE). There were 2 hypotheses. Firstly, groups of similar stations map to competency domains identifiable by factor analysis. Secondly, poor performance in individual domains is compensated for by achieving the required standard of performance across the whole assessment. METHODS: A total of 647 medical students were assessed by an OSCE during 5 individual examination sittings (diets) over 2 years. Ten scoring stations in the OSCE were analysed and confirmatory factor analysis performed comparing a 1-factor model (where all the stations are discrete entities related to one underlying domain) with a 3-factor model (where the stations load onto 3 domains from a previously reported exploratory factor analysis). RESULTS: The 3-factor model yielded a significantly better fit to the data (chi(2 )=( )15.3, P < 0.01). Assessing the compensation data of 1 diet, 29 of 127 students failed in 1 or more domains described, whereas only 5 failed if compensation was allowed across all domains. DISCUSSION: Confirmatory factor analysis showed a significant fit of the data to previously described competency domains for a high-stakes undergraduate OSCE. Compensation within but not between competency domains would provide a more robust standard, improve validity, and substantially reduce the pass rate.


Asunto(s)
Competencia Clínica/normas , Medicina Clínica/educación , Educación de Pregrado en Medicina , Medicina Familiar y Comunitaria/educación , Pediatría/educación , Inglaterra , Anamnesis , Examen Físico
20.
Nat Commun ; 8: 14279, 2017 02 08.
Artículo en Inglés | MEDLINE | ID: mdl-28176794

RESUMEN

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.


Asunto(s)
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 Cebra
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