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1.
Nat Commun ; 11(1): 5520, 2020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-33139725

RESUMO

Axonemal dynein ATPases direct ciliary and flagellar beating via adenosine triphosphate (ATP) hydrolysis. The modulatory effect of adenosine monophosphate (AMP) and adenosine diphosphate (ADP) on flagellar beating is not fully understood. Here, we describe a deficiency of cilia and flagella associated protein 45 (CFAP45) in humans and mice that presents a motile ciliopathy featuring situs inversus totalis and asthenospermia. CFAP45-deficient cilia and flagella show normal morphology and axonemal ultrastructure. Proteomic profiling links CFAP45 to an axonemal module including dynein ATPases and adenylate kinase as well as CFAP52, whose mutations cause a similar ciliopathy. CFAP45 binds AMP in vitro, consistent with structural modelling that identifies an AMP-binding interface between CFAP45 and AK8. Microtubule sliding of dyskinetic sperm from Cfap45-/- mice is rescued with the addition of either AMP or ADP with ATP, compared to ATP alone. We propose that CFAP45 supports mammalian ciliary and flagellar beating via an adenine nucleotide homeostasis module.


Assuntos
Nucleotídeos de Adenina/metabolismo , Astenozoospermia/genética , Proteínas do Citoesqueleto/deficiência , Situs Inversus/genética , Adolescente , Adulto , Animais , Astenozoospermia/patologia , Axonema/ultraestrutura , Sistemas CRISPR-Cas/genética , Cílios/metabolismo , Cílios/ultraestrutura , Proteínas do Citoesqueleto/genética , Análise Mutacional de DNA , Modelos Animais de Doenças , Epididimo/patologia , Feminino , Flagelos/metabolismo , Flagelos/ultraestrutura , Humanos , Mutação com Perda de Função , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Planárias/citologia , Planárias/genética , Planárias/metabolismo , Mucosa Respiratória/citologia , Mucosa Respiratória/patologia , Situs Inversus/diagnóstico por imagem , Situs Inversus/patologia , Motilidade Espermática/genética , Tomografia Computadorizada por Raios X , Sequenciamento Completo do Exoma
2.
Am J Respir Cell Mol Biol ; 62(3): 382-396, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31545650

RESUMO

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous chronic destructive airway disease. PCD is traditionally diagnosed by nasal nitric oxide measurement, analysis of ciliary beating, transmission electron microscopy (TEM), and/or genetic testing. In most genetic PCD variants, laterality defects can occur. However, it is difficult to establish a diagnosis in individuals with PCD and central pair (CP) defects, and alternative strategies are required because of very subtle ciliary beating abnormalities, a normal ciliary ultrastructure, and normal situs composition. Mutations in HYDIN are known to cause CP defects, but the genetic analysis of HYDIN variants is confounded by the pseudogene HYDIN2, which is almost identical in terms of intron/exon structure. We have previously shown that several types of PCD can be diagnosed via immunofluorescence (IF) microscopy analyses. Here, using IF microscopy, we demonstrated that in individuals with PCD and CP defects, the CP-associated protein SPEF2 is absent in HYDIN-mutant cells, revealing its dependence on functional HYDIN. Next, we performed IF analyses of SPEF2 in respiratory cells from 189 individuals with suspected PCD and situs solitus. Forty-one of the 189 individuals had undetectable SPEF2 and were subjected to a genetic analysis, which revealed one novel loss-of-function mutation in SPEF2 and three reported and 13 novel HYDIN mutations in 15 individuals. The remaining 25 individuals are good candidates for new, as-yet uncharacterized PCD variants that affect the CP apparatus. SPEF2 mutations have been associated with male infertility but have not previously been identified to cause PCD. We identified a mutation of SPEF2 that is causative for PCD with a CP defect. We conclude that SPEF2 IF analyses can facilitate the detection of CP defects and evaluation of the pathogenicity of HYDIN variants, thus aiding the molecular diagnosis of CP defects.


Assuntos
Proteínas de Ciclo Celular/deficiência , Cílios/química , Transtornos da Motilidade Ciliar/genética , Proteínas dos Microfilamentos/genética , Axonema/química , Axonema/ultraestrutura , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Transtornos da Motilidade Ciliar/diagnóstico , Transtornos da Motilidade Ciliar/patologia , Códon sem Sentido , Estudos de Coortes , Análise Mutacional de DNA , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Heterogeneidade Genética , Homozigoto , Humanos , Mutação com Perda de Função , Masculino , Proteínas dos Microfilamentos/fisiologia , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Depuração Mucociliar/genética , Mutação , Mutação de Sentido Incorreto , Linhagem , Cultura Primária de Células , Situs Inversus/diagnóstico , Situs Inversus/genética , Situs Inversus/patologia
3.
Artigo em Inglês | MEDLINE | ID: mdl-31638833

RESUMO

Background - Nearly one in 100 live births presents with congenital heart defects (CHD). CHD are frequently associated with laterality defects, such as situs inversus totalis (SIT), a mirrored positioning of internal organs. Body laterality is established by a complex process: monocilia at the embryonic left-right organizer (LRO) facilitate both the generation and sensing of a leftward fluid flow. This induces the conserved left-sided Nodal signaling cascade to initiate asymmetric organogenesis. Primary ciliary dyskinesia (PCD) originates from dysfunction of motile cilia, causing symptoms such as chronic sinusitis, bronchiectasis and frequently SIT. The most frequently mutated gene in PCD, DNAH5 is associated with randomization of body asymmetry resulting in SIT in half of the patients; however, its relation to CHD occurrence in humans has not been investigated in detail so far. Methods - We performed genotype / phenotype correlations in 132 PCD patients carrying disease-causing DNAH5 mutations, focusing on situs defects and CHD. Using high speed video microscopy-, immunofluorescence-, and in situ hybridization analyses, we investigated the initial steps of left-right axis establishment in embryos of a Dnah5 mutant mouse model. Results - 65.9% (87 / 132) of the PCD patients carrying disease-causing DNAH5 mutations had laterality defects: 88.5% (77 / 87) presented with SIT, 11.5% (10 / 87) presented with situs ambiguus; and 6.1% (8 / 132) presented with CHD. In Dnah5mut/mut mice, embryonic LRO monocilia lack outer dynein arms resulting in immotile cilia, impaired flow at the LRO, and randomization of Nodal signaling with normal, reversed or bilateral expression of key molecules. Conclusions - For the first time, we directly demonstrate the disease-mechanism of laterality defects linked to DNAH5 deficiency at the molecular level during embryogenesis. We highlight that mutations in DNAH5 are not only associated with classical randomization of left-right body asymmetry but also with severe laterality defects including CHD.

4.
BMC Cancer ; 19(1): 787, 2019 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-31395037

RESUMO

BACKGROUND: Inherited pathogenic variants in BRCA1 and BRCA2 are the most common causes of hereditary breast and ovarian cancer (HBOC). The risk of developing breast cancer by age 80 in women carrying a BRCA1 pathogenic variant is 72%. The lifetime risk varies between families and even within affected individuals of the same family. The cause of this variability is largely unknown, but it is hypothesized that additional genetic factors contribute to differences in age at onset (AAO). Here we investigated whether truncating and rare missense variants in genes of different DNA-repair pathways contribute to this phenomenon. METHODS: We used extreme phenotype sampling to recruit 133 BRCA1-positive patients with either early breast cancer onset, below 35 (early AAO cohort) or cancer-free by age 60 (controls). Next Generation Sequencing (NGS) was used to screen for variants in 311 genes involved in different DNA-repair pathways. RESULTS: Patients with an early AAO (73 women) had developed breast cancer at a median age of 27 years (interquartile range (IQR); 25.00-27.00 years). A total of 3703 variants were detected in all patients and 43 of those (1.2%) were truncating variants. The truncating variants were found in 26 women of the early AAO group (35.6%; 95%-CI 24.7 - 47.7%) compared to 16 women of controls (26.7%; 95%-CI 16.1 to 39.7%). When adjusted for environmental factors and family history, the odds ratio indicated an increased breast cancer risk for those carrying an additional truncating DNA-repair variant to BRCA1 mutation (OR: 3.1; 95%-CI 0.92 to 11.5; p-value = 0.07), although it did not reach the conventionally acceptable significance level of 0.05. CONCLUSIONS: To our knowledge this is the first time that the combined effect of truncating variants in DNA-repair genes on AAO in patients with hereditary breast cancer is investigated. Our results indicate that co-occurring truncating variants might be associated with an earlier onset of breast cancer in BRCA1-positive patients. Larger cohorts are needed to confirm these results.


Assuntos
Proteína BRCA1/genética , Biomarcadores Tumorais , Neoplasias da Mama/genética , Reparo do DNA , Predisposição Genética para Doença , Deleção de Sequência , Adulto , Idade de Início , Idoso , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/epidemiologia , Bases de Dados Genéticas , Feminino , Estudos de Associação Genética , Loci Gênicos , Alemanha/epidemiologia , Humanos , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Vigilância da População , Medição de Risco , Fatores de Risco
5.
Am J Hum Genet ; 103(6): 995-1008, 2018 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-30471718

RESUMO

Dysfunction of motile monocilia, altering the leftward flow at the embryonic node essential for determination of left-right body asymmetry, is a major cause of laterality defects. Laterality defects are also often associated with reduced mucociliary clearance caused by defective multiple motile cilia of the airway and are responsible for destructive airway disease. Outer dynein arms (ODAs) are essential for ciliary beat generation, and human respiratory cilia contain different ODA heavy chains (HCs): the panaxonemally distributed γ-HC DNAH5, proximally located ß-HC DNAH11 (defining ODA type 1), and the distally localized ß-HC DNAH9 (defining ODA type 2). Here we report loss-of-function mutations in DNAH9 in five independent families causing situs abnormalities associated with subtle respiratory ciliary dysfunction. Consistent with the observed subtle respiratory phenotype, high-speed video microscopy demonstrates distally impaired ciliary bending in DNAH9 mutant respiratory cilia. DNAH9-deficient cilia also lack other ODA components such as DNAH5, DNAI1, and DNAI2 from the distal axonemal compartment, demonstrating an essential role of DNAH9 for distal axonemal assembly of ODAs type 2. Yeast two-hybrid and co-immunoprecipitation analyses indicate interaction of DNAH9 with the ODA components DNAH5 and DNAI2 as well as the ODA-docking complex component CCDC114. We further show that during ciliogenesis of respiratory cilia, first proximally located DNAH11 and then distally located DNAH9 is assembled in the axoneme. We propose that the ß-HC paralogs DNAH9 and DNAH11 achieved specific functional roles for the distinct axonemal compartments during evolution with human DNAH9 function matching that of ancient ß-HCs such as that of the unicellular Chlamydomonas reinhardtii.


Assuntos
Dineínas do Axonema/genética , Cílios/genética , Dineínas/genética , Mutação/genética , Axonema/genética , Transtornos da Motilidade Ciliar/genética , Humanos , Síndrome de Kartagener/genética , Fenótipo
6.
Am J Hum Genet ; 102(5): 973-984, 2018 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-29727693

RESUMO

Primary ciliary dyskinesia (PCD) is characterized by chronic airway disease, male infertility, and randomization of the left/right body axis as a result of defects of motile cilia and sperm flagella. We identified loss-of-function mutations in the open-reading frame C11orf70 in PCD individuals from five distinct families. Transmission electron microscopy analyses and high-resolution immunofluorescence microscopy demonstrate that loss-of-function mutations in C11orf70 cause immotility of respiratory cilia and sperm flagella, respectively, as a result of the loss of axonemal outer (ODAs) and inner dynein arms (IDAs), indicating that C11orf70 is involved in cytoplasmic assembly of dynein arms. Expression analyses of C11orf70 showed that C11orf70 is expressed in ciliated respiratory cells and that the expression of C11orf70 is upregulated during ciliogenesis, similar to other previously described cytoplasmic dynein-arm assembly factors. Furthermore, C11orf70 shows an interaction with cytoplasmic ODA/IDA assembly factor DNAAF2, supporting our hypothesis that C11orf70 is a preassembly factor involved in the pathogenesis of PCD. The identification of additional genetic defects that cause PCD and male infertility is of great importance for the clinic as well as for genetic counselling.


Assuntos
Padronização Corporal , Dineínas/genética , Síndrome de Kartagener/genética , Mutação/genética , Proteínas Nucleares/genética , Cílios/metabolismo , Cílios/ultraestrutura , Dineínas/ultraestrutura , Feminino , Genes Recessivos , Humanos , Mutação com Perda de Função/genética , Masculino , Cauda do Espermatozoide/metabolismo
7.
Cancer Med ; 7(4): 1349-1358, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29522266

RESUMO

The prevalence of germ line mutations in non-BRCA1/2 genes associated with hereditary breast cancer (BC) is low, and the role of some of these genes in BC predisposition and pathogenesis is conflicting. In this study, 5589 consecutive BC index patients negative for pathogenic BRCA1/2 mutations and 2189 female controls were screened for germ line mutations in eight cancer predisposition genes (ATM, CDH1, CHEK2, NBN, PALB2, RAD51C, RAD51D, and TP53). All patients met the inclusion criteria of the German Consortium for Hereditary Breast and Ovarian Cancer for germ line testing. The highest mutation prevalence was observed in the CHEK2 gene (2.5%), followed by ATM (1.5%) and PALB2 (1.2%). The mutation prevalence in each of the remaining genes was 0.3% or lower. Using Exome Aggregation Consortium control data, we confirm significant associations of heterozygous germ line mutations with BC for ATM (OR: 3.63, 95%CI: 2.67-4.94), CDH1 (OR: 17.04, 95%CI: 3.54-82), CHEK2 (OR: 2.93, 95%CI: 2.29-3.75), PALB2 (OR: 9.53, 95%CI: 6.25-14.51), and TP53 (OR: 7.30, 95%CI: 1.22-43.68). NBN germ line mutations were not significantly associated with BC risk (OR:1.39, 95%CI: 0.73-2.64). Due to their low mutation prevalence, the RAD51C and RAD51D genes require further investigation. Compared with control datasets, predicted damaging rare missense variants were significantly more prevalent in CHEK2 and TP53 in BC index patients. Compared with the overall sample, only TP53 mutation carriers show a significantly younger age at first BC diagnosis. We demonstrate a significant association of deleterious variants in the CHEK2, PALB2, and TP53 genes with bilateral BC. Both, ATM and CHEK2, were negatively associated with triple-negative breast cancer (TNBC) and estrogen receptor (ER)-negative tumor phenotypes. A particularly high CHEK2 mutation prevalence (5.2%) was observed in patients with human epidermal growth factor receptor 2 (HER2)-positive tumors.


Assuntos
Biomarcadores Tumorais , Genes BRCA1 , Genes BRCA2 , Síndrome Hereditária de Câncer de Mama e Ovário/diagnóstico , Síndrome Hereditária de Câncer de Mama e Ovário/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Testes Genéticos/métodos , Variação Genética , Síndrome Hereditária de Câncer de Mama e Ovário/epidemiologia , Humanos , Pessoa de Meia-Idade , Razão de Chances , Prevalência , Adulto Jovem
8.
Breast Cancer Res ; 20(1): 7, 2018 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-29368626

RESUMO

BACKGROUND: Germline mutations in the BRIP1 gene have been described as conferring a moderate risk for ovarian cancer (OC), while the role of BRIP1 in breast cancer (BC) pathogenesis remains controversial. METHODS: To assess the role of deleterious BRIP1 germline mutations in BC/OC predisposition, 6341 well-characterized index patients with BC, 706 index patients with OC, and 2189 geographically matched female controls were screened for loss-of-function (LoF) mutations and potentially damaging missense variants. All index patients met the inclusion criteria of the German Consortium for Hereditary Breast and Ovarian Cancer for germline testing and tested negative for pathogenic BRCA1/2 variants. RESULTS: BRIP1 LoF mutations confer a high OC risk in familial index patients (odds ratio (OR) = 20.97, 95% confidence interval (CI) = 12.02-36.57, P < 0.0001) and in the subgroup of index patients with late-onset OC (OR = 29.91, 95% CI = 14.99-59.66, P < 0.0001). No significant association of BRIP1 LoF mutations with familial BC was observed (OR = 1.81 95% CI = 1.00-3.30, P = 0.0623). In the subgroup of familial BC index patients without a family history of OC there was also no apparent association (OR = 1.42, 95% CI = 0.70-2.90, P = 0.3030). In 1027 familial BC index patients with a family history of OC, the BRIP1 mutation prevalence was significantly higher than that observed in controls (OR = 3.59, 95% CI = 1.43-9.01; P = 0.0168). Based on the negative association between BRIP1 LoF mutations and familial BC in the absence of an OC family history, we conclude that the elevated mutation prevalence in the latter cohort was driven by the occurrence of OC in these families. Compared with controls, predicted damaging rare missense variants were significantly more prevalent in OC (P = 0.0014) but not in BC (P = 0.0693) patients. CONCLUSIONS: To avoid ambiguous results, studies aimed at assessing the impact of candidate predisposition gene mutations on BC risk might differentiate between BC index patients with an OC family history and those without. In familial cases, we suggest that BRIP1 is a high-risk gene for late-onset OC but not a BC predisposition gene, though minor effects cannot be excluded.


Assuntos
Neoplasias da Mama/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Predisposição Genética para Doença , Neoplasias Ovarianas/genética , RNA Helicases/genética , Adulto , Idoso , Neoplasias da Mama/patologia , Feminino , Estudos de Associação Genética , Mutação em Linhagem Germinativa , Humanos , Mutação com Perda de Função/genética , Pessoa de Meia-Idade , Neoplasias Ovarianas/patologia , Linhagem , Fatores de Risco
9.
PLoS One ; 11(7): e0158801, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27463617

RESUMO

Population-based genome wide association studies have identified a locus at 9p22.2 associated with ovarian cancer risk, which also modifies ovarian cancer risk in BRCA1 and BRCA2 mutation carriers. We conducted fine-scale mapping at 9p22.2 to identify potential causal variants in BRCA1 and BRCA2 mutation carriers. Genotype data were available for 15,252 (2,462 ovarian cancer cases) BRCA1 and 8,211 (631 ovarian cancer cases) BRCA2 mutation carriers. Following genotype imputation, ovarian cancer associations were assessed for 4,873 and 5,020 SNPs in BRCA1 and BRCA 2 mutation carriers respectively, within a retrospective cohort analytical framework. In BRCA1 mutation carriers one set of eight correlated candidate causal variants for ovarian cancer risk modification was identified (top SNP rs10124837, HR: 0.73, 95%CI: 0.68 to 0.79, p-value 2× 10-16). These variants were located up to 20 kb upstream of BNC2. In BRCA2 mutation carriers one region, up to 45 kb upstream of BNC2, and containing 100 correlated SNPs was identified as candidate causal (top SNP rs62543585, HR: 0.69, 95%CI: 0.59 to 0.80, p-value 1.0 × 10-6). The candidate causal in BRCA1 mutation carriers did not include the strongest associated variant at this locus in the general population. In sum, we identified a set of candidate causal variants in a region that encompasses the BNC2 transcription start site. The ovarian cancer association at 9p22.2 may be mediated by different variants in BRCA1 mutation carriers and in the general population. Thus, potentially different mechanisms may underlie ovarian cancer risk for mutation carriers and the general population.


Assuntos
Cromossomos Humanos Par 9 , Genes BRCA1 , Genes BRCA2 , Triagem de Portadores Genéticos , Predisposição Genética para Doença , Neoplasias Ovarianas/genética , Mapeamento Cromossômico , Feminino , Humanos , Polimorfismo de Nucleotídeo Único
10.
Hum Mol Genet ; 25(11): 2256-2268, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27008870

RESUMO

A recent analysis using family history weighting and co-observation classification modeling indicated that BRCA1 c.594-2A > C (IVS9-2A > C), previously described to cause exon 10 skipping (a truncating alteration), displays characteristics inconsistent with those of a high risk pathogenic BRCA1 variant. We used large-scale genetic and clinical resources from the ENIGMA, CIMBA and BCAC consortia to assess pathogenicity of c.594-2A > C. The combined odds for causality considering case-control, segregation and breast tumor pathology information was 3.23 × 10-8 Our data indicate that c.594-2A > C is always in cis with c.641A > G. The spliceogenic effect of c.[594-2A > C;641A > G] was characterized using RNA analysis of human samples and splicing minigenes. As expected, c.[594-2A > C; 641A > G] caused exon 10 skipping, albeit not due to c.594-2A > C impairing the acceptor site but rather by c.641A > G modifying exon 10 splicing regulatory element(s). Multiple blood-based RNA assays indicated that the variant allele did not produce detectable levels of full-length transcripts, with a per allele BRCA1 expression profile composed of ≈70-80% truncating transcripts, and ≈20-30% of in-frame Δ9,10 transcripts predicted to encode a BRCA1 protein with tumor suppression function.We confirm that BRCA1c.[594-2A > C;641A > G] should not be considered a high-risk pathogenic variant. Importantly, results from our detailed mRNA analysis suggest that BRCA-associated cancer risk is likely not markedly increased for individuals who carry a truncating variant in BRCA1 exons 9 or 10, or any other BRCA1 allele that permits 20-30% of tumor suppressor function. More generally, our findings highlight the importance of assessing naturally occurring alternative splicing for clinical evaluation of variants in disease-causing genes.


Assuntos
Proteína BRCA1/genética , Neoplasias da Mama/genética , Mutação/genética , Neoplasias Ovarianas/genética , Proteínas Supressoras de Tumor/genética , Adulto , Idoso , Processamento Alternativo/genética , Neoplasias da Mama/patologia , Análise Mutacional de DNA , Éxons/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Neoplasias Ovarianas/patologia , Sítios de Splice de RNA/genética , Processamento de RNA/genética
11.
Am J Respir Cell Mol Biol ; 55(2): 213-24, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-26909801

RESUMO

Primary ciliary dyskinesia (PCD) is a recessively inherited disease that leads to chronic respiratory disorders owing to impaired mucociliary clearance. Conventional transmission electron microscopy (TEM) is a diagnostic standard to identify ultrastructural defects in respiratory cilia but is not useful in approximately 30% of PCD cases, which have normal ciliary ultrastructure. DNAH11 mutations are a common cause of PCD with normal ciliary ultrastructure and hyperkinetic ciliary beating, but its pathophysiology remains poorly understood. We therefore characterized DNAH11 in human respiratory cilia by immunofluorescence microscopy (IFM) in the context of PCD. We used whole-exome and targeted next-generation sequence analysis as well as Sanger sequencing to identify and confirm eight novel loss-of-function DNAH11 mutations. We designed and validated a monoclonal antibody specific to DNAH11 and performed high-resolution IFM of both control and PCD-affected human respiratory cells, as well as samples from green fluorescent protein (GFP)-left-right dynein mice, to determine the ciliary localization of DNAH11. IFM analysis demonstrated native DNAH11 localization in only the proximal region of wild-type human respiratory cilia and loss of DNAH11 in individuals with PCD with certain loss-of-function DNAH11 mutations. GFP-left-right dynein mice confirmed proximal DNAH11 localization in tracheal cilia. DNAH11 retained proximal localization in respiratory cilia of individuals with PCD with distinct ultrastructural defects, such as the absence of outer dynein arms (ODAs). TEM tomography detected a partial reduction of ODAs in DNAH11-deficient cilia. DNAH11 mutations result in a subtle ODA defect in only the proximal region of respiratory cilia, which is detectable by IFM and TEM tomography.


Assuntos
Dineínas do Axonema/metabolismo , Cílios/metabolismo , Dineínas/metabolismo , Pulmão/metabolismo , Sequência de Bases , Cílios/ultraestrutura , Dineínas/ultraestrutura , Homozigoto , Humanos , Síndrome de Kartagener/genética , Mutação/genética , Transporte Proteico
12.
Hum Mol Genet ; 25(22): 4898-4910, 2016 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-28171660

RESUMO

The X-linked reproductive homeobox (RHOX) gene cluster encodes transcription factors preferentially expressed in reproductive tissues. This gene cluster has important roles in male fertility based on phenotypic defects of Rhox-mutant mice and the finding that aberrant RHOX promoter methylation is strongly associated with abnormal human sperm parameters. However, little is known about the molecular mechanism of RHOX function in humans. Using gene expression profiling, we identified genes regulated by members of the human RHOX gene cluster. Some genes were uniquely regulated by RHOXF1 or RHOXF2/2B, while others were regulated by both of these transcription factors. Several of these regulated genes encode proteins involved in processes relevant to spermatogenesis; e.g. stress protection and cell survival. One of the target genes of RHOXF2/2B is RHOXF1, suggesting cross-regulation to enhance transcriptional responses. The potential role of RHOX in human infertility was addressed by sequencing all RHOX exons in a group of 250 patients with severe oligozoospermia. This revealed two mutations in RHOXF1 (c.515G > A and c.522C > T) and four in RHOXF2/2B (-73C > G, c.202G > A, c.411C > T and c.679G > A), of which only one (c.202G > A) was found in a control group of men with normal sperm concentration. Functional analysis demonstrated that c.202G > A and c.679G > A significantly impaired the ability of RHOXF2/2B to regulate downstream genes. Molecular modelling suggested that these mutations alter RHOXF2/F2B protein conformation. By combining clinical data with in vitro functional analysis, we demonstrate how the X-linked RHOX gene cluster may function in normal human spermatogenesis and we provide evidence that it is impaired in human male fertility.


Assuntos
Proteínas de Homeodomínio/genética , Infertilidade Masculina/genética , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Genes Homeobox , Genes Ligados ao Cromossomo X , Células HEK293 , Proteínas de Homeodomínio/metabolismo , Humanos , Infertilidade Masculina/metabolismo , Infertilidade Masculina/patologia , Masculino , Família Multigênica , Regiões Promotoras Genéticas , Espermatogênese/genética , Espermatozoides/patologia , Fatores de Transcrição/genética
13.
Cilia ; 4(1): 1, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25589952

RESUMO

Heterotaxy (also known as situs ambiguous) and situs inversus totalis describe disorders of laterality in which internal organs do not display their typical pattern of asymmetry. First described around 1600 by Girolamo Fabrizio, numerous case reports about laterality disorders in humans were published without any idea about the underlying cause. Then, in 1976, immotile cilia were described as the cause of a human syndrome that was previously clinically described, both in 1904 by AK Siewert and in 1933 by Manes Kartagener, as an association of situs inversus with chronic sinusitis and bronchiectasis, now commonly known as Kartagener's syndrome. Despite intense research, the underlying defect of laterality disorders remained unclear. Nearly 20 years later in 1995, Björn Afzelius discussed five hypotheses to explain the connection between ciliary defects and loss of laterality control in a paper published in the International Journal of Developmental Biology asking: 'Situs inversus and ciliary abnormalities: What is the connection?'. Here, nearly 20 research years later, we revisit some of the key findings that led to the current knowledge about the connection between situs inversus and ciliary abnormalities.

14.
BMC Nephrol ; 15: 3, 2014 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-24397250

RESUMO

BACKGROUND: Nephrotic syndrome (NS) is pathological condition characterized by heavy proteinuria. Our study investigates hypothesis that change in cell proliferation of proximal tubules influences primary cilia structure and function and promotes cystogenesis in congenital nephrotic syndrome of the Finnish type (CNF) and focal segmental glomerulosclerosis (FSGS). METHODS: CNF kidneys were analyzed genetically. Proliferation (Ki-67), apoptosis (caspase-3), and primary cilia (α-tubulin) length and structure were analyzed immunohistochemically and ultrastructurally in healthy, CNF and FSGS kidneys. Cyst diameters were measured and correlated with proliferation index. RESULTS: Proximal tubules cells of healthy kidneys did not proliferate. In nephrotic kidneys, tubules with apparently normal diameter covered by cuboidal/columnar epithelium (PTNC) contained 81.54% of proliferating cells in CNF and 36.18% in FSGS, while cysts covered with columnar epithelium (CC) contained 37.52% of proliferating cells in CNF and 45.23% in FSGS. The largest cysts, covered with squamous epithelium (CS) had 11.54% of proliferating cells in CNF and 13.76% in FSGS. Increase in cysts diameter correlated with changes in proliferation index, tubular cells shape, primary cilia formation and appearance of apoptotic cells. CONCLUSIONS: We present a novel histopathological data on the structure and possible changes in function of tubular cell in NS kidneys during cystogenesis. We suggest existence of common principles of cystogenesis in CNF and FSGS kidneys, including serious disturbances of tubular cells proliferation and apoptosis, and faulty primary cilia signaling leading to deterioration of proteinuria in NS kidneys.


Assuntos
Glomerulosclerose Segmentar e Focal/patologia , Túbulos Renais Proximais/patologia , Síndrome Nefrótica/patologia , Proteinúria/patologia , Criança , Feminino , Humanos , Lactente , Masculino
15.
Eur J Hum Genet ; 22(4): 497-504, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23942199

RESUMO

Despite recent progress in our understanding of renal magnesium (Mg(2+)) handling, the molecular mechanisms accounting for transepithelial Mg(2+) transport are still poorly understood. Mutations in the TRPM6 gene, encoding the epithelial Mg(2+) channel TRPM6 (transient receptor potential melastatin 6), have been proven to be the molecular cause of hypomagnesemia with secondary hypocalcemia (HSH; OMIM 602014). HSH manifests in the newborn period being characterized by very low serum Mg(2+) levels (<0.4 mmol/l) accompanied by low serum calcium (Ca(2+)) concentrations. A proportion of previously described TRPM6 mutations lead to a truncated TRPM6 protein resulting in a complete loss-of-function of the ion channel. In addition, five-point mutations have been previously described. The aim of this study was to complement the current clinical picture by adding the molecular data from five new missense mutations found in five patients with HSH. To this end, patch-clamp analysis and cell surface measurements were performed to assess the effect of the various mutations on TRPM6 channel function. All mutant channels, expressed in HEK293 cells, showed loss-of-function, whereas no severe trafficking impairment to the plasma membrane surface was observed. We conclude that the new TRPM6 missense mutations lead to dysregulated intestinal/renal Mg(2+) (re)absorption as a consequence of loss of TRPM6 channel function.


Assuntos
Hipocalcemia/genética , Mutação de Sentido Incorreto , Erros Inatos do Transporte Tubular Renal/genética , Canais de Cátion TRPM/genética , Transporte Biológico , Cálcio/sangue , Membrana Celular/metabolismo , Fenômenos Eletrofisiológicos , Células Epiteliais/metabolismo , Feminino , Seguimentos , Células HEK293 , Humanos , Hipocalcemia/diagnóstico , Lactente , Recém-Nascido , Absorção Intestinal , Mucosa Intestinal/metabolismo , Rim/metabolismo , Magnésio/sangue , Magnésio/farmacocinética , Deficiência de Magnésio/congênito , Masculino , Erros Inatos do Transporte Tubular Renal/diagnóstico , Estudos Retrospectivos , Análise de Sequência de DNA
16.
Science ; 338(6104): 226-31, 2012 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-22983710

RESUMO

Unidirectional fluid flow plays an essential role in the breaking of left-right (L-R) symmetry in mouse embryos, but it has remained unclear how the flow is sensed by the embryo. We report that the Ca(2+) channel Polycystin-2 (Pkd2) is required specifically in the perinodal crown cells for sensing the nodal flow. Examination of mutant forms of Pkd2 shows that the ciliary localization of Pkd2 is essential for correct L-R patterning. Whereas Kif3a mutant embryos, which lack all cilia, failed to respond to an artificial flow, restoration of primary cilia in crown cells rescued the response to the flow. Our results thus suggest that nodal flow is sensed in a manner dependent on Pkd2 by the cilia of crown cells located at the edge of the node.


Assuntos
Padronização Corporal , Embrião de Mamíferos/fisiologia , Fatores de Determinação Direita-Esquerda/metabolismo , Organizadores Embrionários/fisiologia , Canais de Cátion TRPP/metabolismo , Animais , Líquidos Corporais/fisiologia , Cálcio/metabolismo , Cílios/metabolismo , Cílios/fisiologia , Embrião de Mamíferos/anatomia & histologia , Embrião de Mamíferos/citologia , Regulação da Expressão Gênica no Desenvolvimento , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Cinesina/genética , Fatores de Determinação Direita-Esquerda/genética , Camundongos , Camundongos Mutantes , Mutação , Organizadores Embrionários/citologia , Transdução de Sinais , Canais de Cátion TRPP/genética
17.
PLoS One ; 6(4): e19426, 2011 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-21559371

RESUMO

A genetic origin is estimated in 30% of infertile men with the common phenotypes of oligo- or azoospermia, but the pathogenesis of spermatogenic failure remains frequently obscure. To determine the involvement of Copy Number Variants (CNVs) in the origin of male infertility, patients with idiopathic severe oligozoospermia (N = 89), Sertoli-cell-only syndrome (SCOS, N = 37)) and controls with normozoospermia (N = 100) were analysed by array-CGH using the 244A/400K array sets (Agilent Technologies). The mean number of CNVs and the amount of DNA gain/loss were comparable between all groups. Ten recurring CNVs were only found in patients with severe oligozoospermia, three only in SCOS and one CNV in both groups with spermatogenic failure but not in normozoospermic men. Sex-chromosomal, mostly private CNVs were significantly overrepresented in patients with SCOS. CNVs found several times in all groups were analysed in a case-control design and four additional candidate genes and two regions without known genes were associated with SCOS (P<1×10(-3)). In conclusion, by applying array-CGH to study male infertility for the first time, we provide a number of candidate genes possibly causing or being risk factors for the men's spermatogenic failure. The recurring, patient-specific and private, sex-chromosomal CNVs as well as those associated with SCOS are candidates for further, larger case-control and re-sequencing studies.


Assuntos
Azoospermia/genética , Dosagem de Genes , Infertilidade Masculina/genética , Oligospermia/genética , Síndrome de Células de Sertoli/genética , Adulto , Estudos de Casos e Controles , Deleção Cromossômica , Cromossomos Humanos X/genética , Hibridização Genômica Comparativa , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Análise de Sequência de DNA , Espermatogênese/genética
18.
Clin J Am Soc Nephrol ; 5(11): 2075-84, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20798252

RESUMO

BACKGROUND AND OBJECTIVES: Mutations in podocyte genes are associated with steroid-resistant nephrotic syndrome (SRNS), mostly affecting younger age groups. To date, it is unclear whether these patients benefit from intensified immunosuppression with cyclosporine A (CsA). The aim of this study was to evaluate the influence of podocyte gene defects in congenital nephrotic syndrome (CNS) and pediatric SRNS on the efficacy of CsA therapy and preservation of renal function. DESIGN, SETTINGS, PARTICIPANTS, & MEASUREMENTS: Genotyping was performed in 91 CNS/SRNS patients, irrespective of age at manifestation or response to CsA. RESULTS: Mutations were identified in 52% of families (11 NPHS1, 17 NPHS2, 11 WT1, 1 LAMB2, 3 TRPC6). Sixty-eight percent of patients with nongenetic SRNS responded to CsA, most of them achieved complete remission. In contrast, none of the patients with genetic CNS/SRNS experienced a complete remission and only two (17%) achieved a partial response, both affected by a WT1 mutation. Preservation of renal function was significantly better in children with nongenetic disease after a mean follow-up time of 8.6 years (ESRD in 29% versus 71%). CONCLUSIONS: The mutation detection rate in our population was high (52%). Most patients with genetic CNS/SRNS did not benefit from CsA with significantly lower response rates compared with nongenetic patients and showed rapid progression to end-stage renal failure. These data strongly support the idea not to expose CNS/SRNS patients with inherited defects related to podocyte function to intensified immunosuppression with CsA.


Assuntos
Ciclosporina/uso terapêutico , Resistência a Medicamentos , Imunossupressores/uso terapêutico , Falência Renal Crônica/prevenção & controle , Rim/efeitos dos fármacos , Síndrome Nefrótica/tratamento farmacológico , Esteroides/uso terapêutico , Adolescente , Criança , Pré-Escolar , Análise Mutacional de DNA , Progressão da Doença , Feminino , Genes do Tumor de Wilms , Predisposição Genética para Doença , Alemanha , Hereditariedade , Hospitais Pediátricos , Hospitais Universitários , Humanos , Lactente , Peptídeos e Proteínas de Sinalização Intracelular/genética , Estimativa de Kaplan-Meier , Rim/fisiopatologia , Falência Renal Crônica/etiologia , Falência Renal Crônica/genética , Falência Renal Crônica/fisiopatologia , Laminina/genética , Masculino , Proteínas de Membrana/genética , Mutação , Síndrome Nefrótica/congênito , Síndrome Nefrótica/etiologia , Síndrome Nefrótica/fisiopatologia , Seleção de Pacientes , Fenótipo , Estudos Retrospectivos , Canais de Cátion TRPC/genética , Canal de Cátion TRPC6 , Fatores de Tempo , Resultado do Tratamento
19.
Int J Cancer ; 126(12): 2858-62, 2010 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-19856316

RESUMO

To validate common low-risk variants predisposing for breast cancer (BC) in a large set of BRCA1/2 negative familial or genetically enriched cases from Germany, we genotyped 1,415 cases and 1,830 healthy women by MALDI-TOF in 105 candidate SNPs. Significantly higher ORs than previously reported for heterozygous unselected cases were found for the minor allele in FGFR2 (OR = 1.43, 95% CI 1.30-1.59, p-value = 1.24 x 10(-12)) and for TNRC9 (OR = 1.33, 95% CI 1.19-1.46, p-value = 1.54 x 10(-7)). Most intriguing, however, were the ORs for homozygous carriers from high-risk families for FGFR2 (OR = 2.05, 95% CI 1.68-2.51, LSP1 (OR = 0.49, 95% CI 0.28-0.86) and TNRC9 (OR = 1.62, 95% CI 1.27-2.07). Moreover, the additional validation of 99 CGEMS-SNPs identified putative novel susceptibility alleles within the LSP1 gene (OR = 0.73, 95% CI 0.61-0.87, p-value = 5.23 x 10(-4)). Finally, we provide evidence for the first time that a low-risk variant located at 6q22.33 (rs6569479) is associated with estrogen receptor negative BC in familial cases (OR = 1.33, 95% CI 1.06-1.66; p-value = 0.012). Our data confirm the impact of the previously identified susceptibility loci and provide preliminary evidence for novel susceptibility loci in familial BC cases and correlate them to specific histopathological subtypes defined by estrogen receptor status.


Assuntos
Neoplasias da Mama/genética , Predisposição Genética para Doença , Proteínas dos Microfilamentos/genética , Polimorfismo de Nucleotídeo Único/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptores de Progesterona/genética , Adulto , Idoso , Proteínas Reguladoras de Apoptose , Neoplasias da Mama/patologia , Estudos de Casos e Controles , Cromossomos Humanos Par 6/genética , Feminino , Genótipo , Alemanha , Heterozigoto , Proteínas de Grupo de Alta Mobilidade , Humanos , Pessoa de Meia-Idade , Fenótipo , Prognóstico , Receptores Estrogênicos/metabolismo , Transativadores
20.
J Am Soc Nephrol ; 20(1): 48-56, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18945944

RESUMO

Autosomal dominant polycystic kidney disease (ADPKD) is a common hereditary disease associated with progressive renal failure. Although cyst growth and compression of surrounding tissue may account for some loss of renal tissue, the other factors contributing to the progressive renal failure in patients with ADPKD are incompletely understood. Here, we report that secreted frizzled-related protein 4 (sFRP4) is upregulated in human ADPKD and in four different animal models of PKD, suggesting that sFRP4 expression is triggered by a common mechanism that underlies cyst formation. Cyst fluid from ADPKD kidneys activated the sFRP4 promoter and induced production of sFRP4 protein in renal tubular epithelial cell lines. Antagonism of the vasopressin 2 receptor blocked both promoter activity and tubular sFRP4 expression. In addition, sFRP4 selectively influenced members of the canonical Wnt signaling cascade and promoted cystogenesis of the zebrafish pronephros. sFRP4 was detected in the urine of both patients and animals with PKD, suggesting that sFRP4 may be a potential biomarker for monitoring the progression of ADPKD. Taken together, these observations suggest a potential role for SFRP4 in the pathogenesis of ADPKD.


Assuntos
Rim/metabolismo , Rim Policístico Autossômico Dominante/etiologia , Proteínas Proto-Oncogênicas/fisiologia , Animais , Células Cultivadas , Líquido Cístico/fisiologia , Modelos Animais de Doenças , Humanos , Camundongos , Morfolinas/farmacologia , Néfrons/embriologia , Doenças Renais Policísticas/metabolismo , Rim Policístico Autossômico Dominante/metabolismo , Proteínas Proto-Oncogênicas/análise , Transdução de Sinais , Compostos de Espiro/farmacologia , Canais de Cátion TRPP/fisiologia , Fatores de Transcrição/fisiologia , Proteínas Wnt/fisiologia , Xenopus , Peixe-Zebra
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