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1.
J Biomed Inform ; 100: 103308, 2019 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-31622800

RESUMO

Rare diseases are often hard and long to be diagnosed precisely, and most of them lack approved treatment. For some complex rare diseases, precision medicine approach is further required to stratify patients into homogeneous subgroups based on the clinical, biological or molecular features. In such situation, deep phenotyping of these patients and comparing their profiles based on subjacent similarities are thus essential to help fast and precise diagnoses and better understanding of pathophysiological processes in order to develop therapeutic solutions. In this article, we developed a new pipeline of using deep phenotyping to define patient similarity and applied it to ciliopathies, a group of rare and severe diseases caused by ciliary dysfunction. As a French national reference center for rare and undiagnosed diseases, the Necker-Enfants Malades Hospital (Necker Children's Hospital) hosts the Imagine Institute, a research institute focusing on genetic diseases. The clinical data warehouse contains on one hand EHR data, and on the other hand, clinical research data. The similarity metrics were computed on both data sources, and were evaluated with two tasks: diagnoses with EHRs and subtyping with ciliopathy specific research data. We obtained a precision of 0.767 in the top 30 most similar patients with diagnosed ciliopathies. Subtyping ciliopathy patients with phenotypic similarity showed concordances with expert knowledge. Similarity metrics applied to rare disease offer new perspectives in a translational context that may help to recruit patients for research, reduce the length of the diagnostic journey, and better understand the mechanisms of the disease.

2.
Hum Mol Genet ; 28(16): 2720-2737, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31042281

RESUMO

Mutations in genes encoding components of the intraflagellar transport (IFT) complexes have previously been associated with a spectrum of diseases collectively termed ciliopathies. Ciliopathies relate to defects in the formation or function of the cilium, a sensory or motile organelle present on the surface of most cell types. IFT52 is a key component of the IFT-B complex and ensures the interaction of the two subcomplexes, IFT-B1 and IFT-B2. Here, we report novel IFT52 biallelic mutations in cases with a short-rib thoracic dysplasia (SRTD) or a congenital anomaly of kidney and urinary tract (CAKUT). Combining in vitro and in vivo studies in zebrafish, we showed that SRTD-associated missense mutation impairs IFT-B complex assembly and IFT-B2 ciliary localization, resulting in decreased cilia length. In comparison, CAKUT-associated missense mutation has a mild pathogenicity, thus explaining the lack of skeletal defects in CAKUT case. In parallel, we demonstrated that the previously reported homozygous nonsense IFT52 mutation associated with Sensenbrenner syndrome [Girisha et al. (2016) A homozygous nonsense variant in IFT52 is associated with a human skeletal ciliopathy. Clin. Genet., 90, 536-539] leads to exon skipping and results in a partially functional protein. Finally, our work uncovered a novel role for IFT52 in microtubule network regulation. We showed that IFT52 interacts and partially co-localized with centrin at the distal end of centrioles where it is involved in its recruitment and/or maintenance. Alteration of this function likely contributes to centriole splitting observed in Ift52-/- cells. Altogether, our findings allow a better comprehensive genotype-phenotype correlation among IFT52-related cases and revealed a novel, extra-ciliary role for IFT52, i.e. disruption may contribute to pathophysiological mechanisms.

3.
Proc Natl Acad Sci U S A ; 115(49): 12489-12494, 2018 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-30446612

RESUMO

Genetic treatments of renal ciliopathies leading to cystic kidney disease would provide a real advance in current therapies. Mutations in CEP290 underlie a ciliopathy called Joubert syndrome (JBTS). Human disease phenotypes include cerebral, retinal, and renal disease, which typically progresses to end stage renal failure (ESRF) within the first two decades of life. While currently incurable, there is often a period of years between diagnosis and ESRF that provides a potential window for therapeutic intervention. By studying patient biopsies, patient-derived kidney cells, and a mouse model, we identify abnormal elongation of primary cilia as a key pathophysiological feature of CEP290-associated JBTS and show that antisense oligonucleotide (ASO)-induced splicing of the mutated exon (41, G1890*) restores protein expression in patient cells. We demonstrate that ASO-induced splicing leading to exon skipping is tolerated, resulting in correct localization of CEP290 protein to the ciliary transition zone, and restoration of normal cilia length in patient kidney cells. Using a gene trap Cep290 mouse model of JBTS, we show that systemic ASO treatment can reduce the cystic burden of diseased kidneys in vivo. These findings indicate that ASO treatment may represent a promising therapeutic approach for kidney disease in CEP290-associated ciliopathy syndromes.

4.
Hum Mol Genet ; 2018 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-30388224

RESUMO

Mutations in KIF14havepreviously been associated with either severe, isolated or syndromic microcephaly with renal hypodysplasia (RHD). Syndromic microcephaly-RHD was strongly reminiscent of clinical ciliopathies, relating to defects of the primary cilium, a signalling organelle present on the surface of many quiescent cells.KIF14 encodes a mitotic kinesin which plays a key role at the midbody during cytokinesisand has not previously been shown to be involved in cilia-related functions. Here, we analysed four families with foetuses presenting with the syndromic form and harbouring biallelic variantsin KIF14. Our functional analyses showthat the identified variantsseverely impact the activity of KIF14 and likely correspond to loss-of-function mutations. Analysis in human foetal tissues further revealed the accumulation of KIF14-positive midbody remnants in the lumen of ureteric bud tips indicating a shared function of KIF14 during brain and kidney development. Subsequently, analysis of a kif14mutant zebrafish line showed a conserved role for this mitotic kinesin. Interestingly, ciliopathy-associated phenotypes were also present in mutant embryos,supportinga potential direct or indirect role for KIF14 at cilia. However, our in vitro and in vivoanalyses did not provide evidence of a direct role for KIF14 in ciliogenesis and suggestedthat loss ofkif14causesciliopathy-like phenotypesthrough an accumulation ofmitotic cells in ciliated tissues. Altogether, our results demonstrate that KIF14 mutations result in a severe syndrome associating microcephaly and RHD through its conserved functionin cytokinesis during kidney and brain development.

5.
Hum Mutat ; 39(7): 983-992, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29688594

RESUMO

Ciliopathies represent a wide spectrum of rare diseases with overlapping phenotypes and a high genetic heterogeneity. Among those, IFT140 is implicated in a variety of phenotypes ranging from isolated retinis pigmentosa to more syndromic cases. Using whole-genome sequencing in patients with uncharacterized ciliopathies, we identified a novel recurrent tandem duplication of exon 27-30 (6.7 kb) in IFT140, c.3454-488_4182+2588dup p.(Tyr1152_Thr1394dup), missed by whole-exome sequencing. Pathogenicity of the mutation was assessed on the patients' skin fibroblasts. Several hundreds of patients with a ciliopathy phenotype were screened and biallelic mutations were identified in 11 families representing 12 pathogenic variants of which seven are novel. Among those unrelated families especially with a Mainzer-Saldino syndrome, eight carried the same tandem duplication (two at the homozygous state and six at the heterozygous state). In conclusion, we demonstrated the implication of structural variations in IFT140-related diseases expanding its mutation spectrum. We also provide evidences for a unique genomic event mediated by an Alu-Alu recombination occurring on a shared haplotype. We confirm that whole-genome sequencing can be instrumental in the ability to detect structural variants for genomic disorders.

6.
EMBO J ; 37(10)2018 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-29650680

RESUMO

Ciliopathies are life-threatening human diseases caused by defective cilia. They can often be traced back to mutations of genes encoding transition zone (TZ) proteins demonstrating that the understanding of TZ organisation is of paramount importance. The TZ consists of multimeric protein modules that are subject to a stringent assembly hierarchy. Previous reports place Rpgrip1l at the top of the TZ assembly hierarchy in Caenorhabditis elegans By performing quantitative immunofluorescence studies in RPGRIP1L-/- mouse embryos and human embryonic cells, we recognise a different situation in vertebrates in which Rpgrip1l deficiency affects TZ assembly in a cell type-specific manner. In cell types in which the loss of Rpgrip1l alone does not affect all modules, additional truncation or removal of vertebrate-specific Rpgrip1 results in an impairment of all modules. Consequently, Rpgrip1l and Rpgrip1 synergistically ensure the TZ composition in several vertebrate cell types, revealing a higher complexity of TZ assembly in vertebrates than in invertebrates.

7.
Am J Physiol Renal Physiol ; 315(1): F57-F73, 2018 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-29537311

RESUMO

Following the discovery of (R)-roscovitine's beneficial effects in three polycystic kidney disease (PKD) mouse models, cyclin-dependent kinases (CDKs) inhibitors have been investigated as potential treatments. We have used various affinity chromatography approaches to identify the molecular targets of roscovitine and its more potent analog (S)-CR8 in human and murine polycystic kidneys. These methods revealed casein kinases 1 (CK1) as additional targets of the two drugs. CK1ε expression at the mRNA and protein levels is enhanced in polycystic kidneys of 11 different PKD mouse models as well as in human polycystic kidneys. A shift in the pattern of CK1α isoforms is observed in all PKD mouse models. Furthermore, the catalytic activities of both CK1ε and CK1α are increased in mouse polycystic kidneys. Inhibition of CK1ε and CK1α may thus contribute to the long-lasting attenuating effects of roscovitine and (S)-CR8 on cyst development. CDKs and CK1s may constitute a dual therapeutic target to develop kinase inhibitory PKD drug candidates.

8.
Hum Mol Genet ; 27(2): 266-282, 2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29121203

RESUMO

A child presenting with Mainzer-Saldino syndrome (MZSDS), characterized by renal, retinal and skeletal involvements, was also diagnosed with lung infections and airway ciliary dyskinesia. These manifestations suggested dysfunction of both primary and motile cilia, respectively. Targeted exome sequencing identified biallelic mutations in WDR19, encoding an IFT-A subunit previously associated with MZSDS-related chondrodysplasia, Jeune asphyxiating thoracic dysplasia and cranioectodermal dysplasia, linked to primary cilia dysfunction, and in TEKT1 which encodes tektin-1 an uncharacterized member of the tektin family, mutations of which may cause ciliary dyskinesia. Tektin-1 localizes at the centrosome in cycling cells, at basal bodies of both primary and motile cilia and to the axoneme of motile cilia in airway cells. The identified mutations impaired these localizations. In addition, airway cells from the affected individual showed severe motility defects without major ultrastructural changes. Knockdown of tekt1 in zebrafish resulted in phenotypes consistent with a function for tektin-1 in ciliary motility, which was confirmed by live imaging. Finally, experiments in the zebrafish also revealed a synergistic effect of tekt1 and wdr19. Altogether, our data show genetic interactions between WDR19 and TEKT1 likely contributing to the overall clinical phenotype observed in the affected individual and provide strong evidence for TEKT1 as a new candidate gene for primary ciliary dyskinesia.

9.
Am J Hum Genet ; 101(5): 803-814, 2017 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-29100091

RESUMO

Congenital anomalies of the kidney and urinary tract (CAKUT) constitute a major cause of chronic kidney disease in children and 20% of prenatally detected anomalies. CAKUT encompass a spectrum of developmental kidney defects, including renal agenesis, hypoplasia, and cystic and non-cystic dysplasia. More than 50 genes have been reported as mutated in CAKUT-affected case subjects. However, the pathophysiological mechanisms leading to bilateral kidney agenesis (BKA) remain largely elusive. Whole-exome or targeted exome sequencing of 183 unrelated familial and/or severe CAKUT-affected case subjects, including 54 fetuses with BKA, led to the identification of 16 heterozygous variants in GREB1L (growth regulation by estrogen in breast cancer 1-like), a gene reported as a target of retinoic acid signaling. Four loss-of-function and 12 damaging missense variants, 14 being absent from GnomAD, were identified. Twelve of them were present in familial or simplex BKA-affected case subjects. Female BKA-affected fetuses also displayed uterus agenesis. We demonstrated a significant association between GREB1L variants and BKA. By in situ hybridization, we showed expression of Greb1l in the nephrogenic zone in developing mouse kidney. We generated a Greb1l knock-out mouse model by CRISPR-Cas9. Analysis at E13.5 revealed lack of kidneys and genital tract anomalies in male and female Greb1l-/- embryos and a slight decrease in ureteric bud branching in Greb1l+/- embryos. We showed that Greb1l invalidation in mIMCD3 cells affected tubulomorphogenesis in 3D-collagen culture, a phenotype rescued by expression of the wild-type human protein. This demonstrates that GREB1L plays a major role in early metanephros and genital development in mice and humans.


Assuntos
Anormalidades Congênitas/genética , Nefropatias/congênito , Rim/anormalidades , Mutação/genética , Proteínas de Neoplasias/genética , Proteínas/genética , Animais , Criança , Exoma/genética , Feminino , Feto/anormalidades , Heterozigoto , Humanos , Nefropatias/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Sistema Urinário/anormalidades , Anormalidades Urogenitais/genética
10.
Hum Mol Genet ; 26(23): 4657-4667, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-28973549

RESUMO

Joubert syndrome (JBTS) is the archetypal ciliopathy caused by mutation of genes encoding ciliary proteins leading to multi-system phenotypes, including a cerebello-retinal-renal syndrome. JBTS is genetically heterogeneous, however mutations in CEP290 are a common underlying cause. The renal manifestation of JBTS is a juvenile-onset cystic kidney disease, known as nephronophthisis, typically progressing to end-stage renal failure within the first two decades of life, thus providing a potential window for therapeutic intervention. In order to increase understanding of JBTS and its associated kidney disease and to explore potential treatments, we conducted a comprehensive analysis of primary renal epithelial cells directly isolated from patient urine (human urine-derived renal epithelial cells, hURECs). We demonstrate that hURECs from a JBTS patient with renal disease have elongated and disorganized primary cilia and that this ciliary phenotype is specifically associated with an absence of CEP290 protein. Treatment with the Sonic hedgehog (Shh) pathway agonist purmorphamine or cyclin-dependent kinase inhibition (using roscovitine and siRNA directed towards cyclin-dependent kinase 5) ameliorated the cilia phenotype. In addition, purmorphamine treatment was shown to reduce cyclin-dependent kinase 5 in patient cells, suggesting a convergence of these signalling pathways. To our knowledge, this is the most extensive analysis of primary renal epithelial cells from JBTS patients to date. It demonstrates the feasibility and power of this approach to directly assess the consequences of patient-specific mutations in a physiologically relevant context and a previously unrecognized convergence of Shh agonism and cyclin-dependent kinase inhibition as potential therapeutic targets.


Assuntos
Anormalidades Múltiplas/tratamento farmacológico , Anormalidades Múltiplas/patologia , Cerebelo/anormalidades , Cílios/patologia , Anormalidades do Olho/tratamento farmacológico , Anormalidades do Olho/patologia , Doenças Renais Císticas/tratamento farmacológico , Doenças Renais Císticas/patologia , Morfolinas/uso terapêutico , Purinas/uso terapêutico , Retina/anormalidades , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/metabolismo , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Cerebelo/metabolismo , Cerebelo/patologia , Criança , Pré-Escolar , Cílios/efeitos dos fármacos , Cílios/genética , Cílios/metabolismo , Ciliopatias/tratamento farmacológico , Ciliopatias/genética , Ciliopatias/metabolismo , Quinase 5 Dependente de Ciclina/genética , Quinase 5 Dependente de Ciclina/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Anormalidades do Olho/genética , Anormalidades do Olho/metabolismo , Humanos , Doenças Renais Císticas/genética , Doenças Renais Císticas/metabolismo , Falência Renal Crônica/genética , Falência Renal Crônica/metabolismo , Falência Renal Crônica/patologia , Masculino , Mutação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Linhagem , Doenças Renais Policísticas/genética , Cultura Primária de Células , Retina/metabolismo , Retina/patologia , Roscovitina , Transdução de Sinais
11.
J Am Soc Nephrol ; 28(10): 2901-2914, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28566479

RESUMO

Congenital anomalies of the kidney and urinary tract (CAKUT) occur in three to six of 1000 live births, represent about 20% of the prenatally detected anomalies, and constitute the main cause of CKD in children. These disorders are phenotypically and genetically heterogeneous. Monogenic causes of CAKUT in humans and mice have been identified. However, despite high-throughput sequencing studies, the cause of the disease remains unknown in most patients, and several studies support more complex inheritance and the role of environmental factors and/or epigenetics in the pathophysiology of CAKUT. Here, we report the targeted exome sequencing of 330 genes, including genes known to be involved in CAKUT and candidate genes, in a cohort of 204 unrelated patients with CAKUT; 45% of the patients were severe fetal cases. We identified pathogenic mutations in 36 of 204 (17.6%) patients. These mutations included five de novo heterozygous loss of function mutations/deletions in the PBX homeobox 1 gene (PBX1), a gene known to have a crucial role in kidney development. In contrast, the frequency of SOX17 and DSTYK variants recently reported as pathogenic in CAKUT did not indicate causality. These findings suggest that PBX1 is involved in monogenic CAKUT in humans and call into question the role of some gene variants recently reported as pathogenic in CAKUT. Targeted exome sequencing also proved to be an efficient and cost-effective strategy to identify pathogenic mutations and deletions in known CAKUT genes.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas Proto-Oncogênicas/genética , Anormalidades Urogenitais/genética , Estudos de Coortes , Análise Mutacional de DNA , Exoma , Feminino , Humanos , Masculino , Fator de Transcrição 1 de Leucemia de Células Pré-B
12.
J Med Genet ; 54(6): 371-380, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28289185

RESUMO

Oral-facial-digital syndromes (OFDS) gather rare genetic disorders characterised by facial, oral and digital abnormalities associated with a wide range of additional features (polycystic kidney disease, cerebral malformations and several others) to delineate a growing list of OFDS subtypes. The most frequent, OFD type I, is caused by a heterozygous mutation in the OFD1 gene encoding a centrosomal protein. The wide clinical heterogeneity of OFDS suggests the involvement of other ciliary genes. For 15 years, we have aimed to identify the molecular bases of OFDS. This effort has been greatly helped by the recent development of whole-exome sequencing (WES). Here, we present all our published and unpublished results for WES in 24 cases with OFDS. We identified causal variants in five new genes (C2CD3, TMEM107, INTU, KIAA0753 and IFT57) and related the clinical spectrum of four genes in other ciliopathies (C5orf42, TMEM138, TMEM231 and WDPCP) to OFDS. Mutations were also detected in two genes previously implicated in OFDS. Functional studies revealed the involvement of centriole elongation, transition zone and intraflagellar transport defects in OFDS, thus characterising three ciliary protein modules: the complex KIAA0753-FOPNL-OFD1, a regulator of centriole elongation; the Meckel-Gruber syndrome module, a major component of the transition zone; and the CPLANE complex necessary for IFT-A assembly. OFDS now appear to be a distinct subgroup of ciliopathies with wide heterogeneity, which makes the initial classification obsolete. A clinical classification restricted to the three frequent/well-delineated subtypes could be proposed, and for patients who do not fit one of these three main subtypes, a further classification could be based on the genotype.


Assuntos
Face/anormalidades , Síndromes Orofaciodigitais/genética , Anormalidades Múltiplas/genética , Transtornos da Motilidade Ciliar/genética , Encefalocele/genética , Feminino , Heterozigoto , Humanos , Masculino , Mutação/genética , Doenças Renais Policísticas/genética , Proteínas/genética , Retinite Pigmentosa
14.
Nat Commun ; 8: 14177, 2017 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-28134340

RESUMO

Ciliary membrane composition is controlled by transition zone (TZ) proteins such as RPGRIP1, RPGRIPL and NPHP4, which are vital for balanced coordination of diverse signalling systems like the Sonic hedgehog (Shh) pathway. Activation of this pathway involves Shh-induced ciliary accumulation of Smoothened (SMO), which is disrupted by disease-causing mutations in TZ components. Here we identify kinesin-3 motor protein KIF13B as a novel member of the RPGRIP1N-C2 domain-containing protein family and show that KIF13B regulates TZ membrane composition and ciliary SMO accumulation. KIF13B is upregulated during ciliogenesis and is recruited to the ciliary base by NPHP4, which binds to two distinct sites in the KIF13B tail region, including an RPGRIP1N-C2 domain. KIF13B and NPHP4 are both essential for establishment of a CAV1 membrane microdomain at the TZ, which in turn is required for Shh-induced ciliary SMO accumulation. Thus KIF13B is a novel regulator of ciliary TZ configuration, membrane composition and Shh signalling.


Assuntos
Caveolina 1/metabolismo , Cílios/fisiologia , Cinesina/metabolismo , Proteínas/metabolismo , Transdução de Sinais/fisiologia , Receptor Smoothened/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Biologia Computacional , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/fisiologia , Técnicas de Inativação de Genes , Células HEK293 , Proteínas Hedgehog/metabolismo , Humanos , Cinesina/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Células NIH 3T3 , Domínios Proteicos/fisiologia , Regulação para Cima , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismo
15.
Am J Hum Genet ; 100(2): 323-333, 2017 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-28089251

RESUMO

Nephronophthisis (NPH), an autosomal-recessive tubulointerstitial nephritis, is the most common cause of hereditary end-stage renal disease in the first three decades of life. Since most NPH gene products (NPHP) function at the primary cilium, NPH is classified as a ciliopathy. We identified mutations in a candidate gene in eight individuals from five families presenting late-onset NPH with massive renal fibrosis. This gene encodes MAPKBP1, a poorly characterized scaffolding protein for JNK signaling. Immunofluorescence analyses showed that MAPKBP1 is not present at the primary cilium and that fibroblasts from affected individuals did not display ciliogenesis defects, indicating that MAPKBP1 may represent a new family of NPHP not involved in cilia-associated functions. Instead, MAPKBP1 is recruited to mitotic spindle poles (MSPs) during the early phases of mitosis where it colocalizes with its paralog WDR62, which plays a key role at MSP. Detected mutations compromise recruitment of MAPKBP1 to the MSP and/or its interaction with JNK2 or WDR62. Additionally, we show increased DNA damage response signaling in fibroblasts from affected individuals and upon knockdown of Mapkbp1 in murine cell lines, a phenotype previously associated with NPH. In conclusion, we identified mutations in MAPKBP1 as a genetic cause of juvenile or late-onset and cilia-independent NPH.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Doenças Renais Císticas/congênito , Adolescente , Alelos , Animais , Criança , Cílios/genética , Dano ao DNA/genética , Modelos Animais de Doenças , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibrose , Regulação da Expressão Gênica , Humanos , Rim/citologia , Rim/metabolismo , Doenças Renais Císticas/diagnóstico , Doenças Renais Císticas/genética , Falência Renal Crônica/diagnóstico , Falência Renal Crônica/genética , Camundongos , Camundongos Knockout , Mitose , Mutação , Células NIH 3T3 , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Linhagem , Fenótipo , Transdução de Sinais , Polos do Fuso/metabolismo , Adulto Jovem , Peixe-Zebra
16.
Clin Chem Lab Med ; 55(6): 809-816, 2017 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-28002029

RESUMO

BACKGROUND: Nephronophthisis, an autosomal recessive nephropathy, is responsible for 10% of childhood chronic renal failure. The deletion of its major gene, NPHP1, with a minor allele frequency of 0.24% in the general population, is the most common mutation leading to a monogenic form of childhood chronic renal failure. It is challenging to detect it in the heterozygous state. We aimed to evaluate the sensitivity and the specificity of the quantitative multiplex PCR of short fluorescent fragments (QMPSF) in its detection. METHODS: After setting up the protocol of QMPSF, we validated it on 39 individuals diagnosed by multiplex ligation-dependent probe amplification (MLPA) with normal NPHP1 copy number (n=17), with heterozygous deletion (n=13, seven parents and six patients), or with homozygous deletion (n=9). To assess the rate of the deletions that arise from independent events, deleted alleles were haplotyped. RESULTS: The results of QMPSF and MLPA correlated perfectly in the identification of 76 heterozygously deleted and 56 homozygously deleted exons. The inter-experimental variability of the dosage quotient obtained by QMPSF was low: control, 1.05 (median; range, 0.86-1.33, n = 102 exons); heterozygous deletion, 0.51 (0.42-0.67, n = 76 exons); homozygous deletion, 0 (0-0, n = 56 exons). All patients harboring a heterozygous deletion were found to carry a hemizygous mutation. At least 15 out of 18 deletions appeared on different haplotypes and one deletion appeared de novo. CONCLUSIONS: The cost- and time-effective QMPSF has a 100% sensitivity and specificity in the detection of NPHP1 deletion. The potential de novo appearance of NPHP1 deletions makes its segregation analysis highly recommended in clinical practice.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Deleção de Genes , Heterozigoto , Proteínas de Membrana/genética , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Éxons/genética , Humanos , Doenças Renais Císticas/congênito , Doenças Renais Císticas/genética , Limite de Detecção
17.
PLoS Genet ; 12(7): e1006220, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27472056

RESUMO

Ciliopathies are genetic disorders arising from dysfunction of microtubule-based cellular appendages called cilia. Different cilia types possess distinct stereotypic microtubule doublet arrangements with non-motile or 'primary' cilia having a 9+0 and motile cilia have a 9+2 array of microtubule doublets. Primary cilia are critical sensory and signaling centers needed for normal mammalian development. Defects in their structure/function result in a spectrum of clinical and developmental pathologies including abnormal neural tube and limb patterning. Altered patterning phenotypes in the limb and neural tube are due to perturbations in the hedgehog (Hh) signaling pathway. Motile cilia are important in fluid movement and defects in motility result in chronic respiratory infections, altered left-right asymmetry, and infertility. These features are the hallmarks of Primary Ciliary Dyskinesia (PCD, OMIM 244400). While mutations in several genes are associated with PCD in patients and animal models, the genetic lesion in many cases is unknown. We assessed the in vivo functions of Growth Arrest Specific 8 (GAS8). GAS8 shares strong sequence similarity with the Chlamydomonas Nexin-Dynein Regulatory Complex (NDRC) protein 4 (DRC4) where it is needed for proper flagella motility. In mammalian cells, the GAS8 protein localizes not only to the microtubule axoneme of motile cilia, but also to the base of non-motile cilia. Gas8 was recently implicated in the Hh signaling pathway as a regulator of Smoothened trafficking into the cilium. Here, we generate the first mouse with a Gas8 mutation and show that it causes severe PCD phenotypes; however, there were no overt Hh pathway phenotypes. In addition, we identified two human patients with missense variants in Gas8. Rescue experiments in Chlamydomonas revealed a subtle defect in swim velocity compared to controls. Further experiments using CRISPR/Cas9 homology driven repair (HDR) to generate one of these human missense variants in mice demonstrated that this allele is likely pathogenic.


Assuntos
Padronização Corporal/genética , Cílios/genética , Síndrome de Kartagener/genética , Proteínas/genética , Animais , Movimento Celular/genética , Chlamydomonas/genética , Cílios/patologia , Citoesqueleto/genética , Modelos Animais de Doenças , Extremidades/crescimento & desenvolvimento , Extremidades/patologia , Predisposição Genética para Doença , Humanos , Síndrome de Kartagener/patologia , Camundongos , Microtúbulos/genética , Mutação , Tubo Neural/crescimento & desenvolvimento , Tubo Neural/patologia , Transdução de Sinais/genética
18.
Hum Mutat ; 37(10): 1025-9, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27319779

RESUMO

Neonatal sclerosing cholangitis (NSC) is a rare biliary disease leading to liver transplantation in childhood. Patients with NSC and ichtyosis have already been identified with a CLDN1 mutation, encoding a tight-junction protein. However, for the majority of patients, the molecular basis of NSC remains unknown. We identified biallelic missense mutations or in-frame deletion in DCDC2 in four affected children. Mutations involve highly conserved amino acids in the doublecortin domains of the protein. In cholangiocytes, DCDC2 protein is normally located in the cytoplasm and cilia, whereas in patients the mutated protein is accumulated in the cytoplasm, absent from cilia, and associated with ciliogenesis defect. This is the first report of DCDC2 mutations in NSC. This data expands the molecular spectrum of NSC, that can be considered as a ciliopathy and also expands the clinical spectrum of the DCDC2 mutations, previously reported in dyslexia, deafness, and nephronophtisis.


Assuntos
Colangite Esclerosante/genética , Cílios/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Colangite Esclerosante/metabolismo , Citoplasma/metabolismo , Feminino , Humanos , Masculino , Proteínas Associadas aos Microtúbulos/química , Mutação de Sentido Incorreto , Linhagem , Domínios Proteicos , Deleção de Sequência
19.
PLoS Genet ; 12(3): e1005894, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26967905

RESUMO

Ciliopathies are a group of genetic multi-systemic disorders related to dysfunction of the primary cilium, a sensory organelle present at the cell surface that regulates key signaling pathways during development and tissue homeostasis. In order to identify novel genes whose mutations would cause severe developmental ciliopathies, >500 patients/fetuses were analyzed by a targeted high throughput sequencing approach allowing exome sequencing of >1200 ciliary genes. NEK8/NPHP9 mutations were identified in five cases with severe overlapping phenotypes including renal cystic dysplasia/hypodysplasia, situs inversus, cardiopathy with hypertrophic septum and bile duct paucity. These cases highlight a genotype-phenotype correlation, with missense and nonsense mutations associated with hypodysplasia and enlarged cystic organs, respectively. Functional analyses of NEK8 mutations in patient fibroblasts and mIMCD3 cells showed that these mutations differentially affect ciliogenesis, proliferation/apoptosis/DNA damage response, as well as epithelial morphogenesis. Notably, missense mutations exacerbated some of the defects due to NEK8 loss of function, highlighting their likely gain-of-function effect. We also showed that NEK8 missense and loss-of-function mutations differentially affect the regulation of the main Hippo signaling effector, YAP, as well as the expression of its target genes in patient fibroblasts and renal cells. YAP imbalance was also observed in enlarged spheroids of Nek8-invalidated renal epithelial cells grown in 3D culture, as well as in cystic kidneys of Jck mice. Moreover, co-injection of nek8 MO with WT or mutated NEK8-GFP RNA in zebrafish embryos led to shortened dorsally curved body axis, similar to embryos injected with human YAP RNA. Finally, treatment with Verteporfin, an inhibitor of YAP transcriptional activity, partially rescued the 3D spheroid defects of Nek8-invalidated cells and the abnormalities of NEK8-overexpressing zebrafish embryos. Altogether, our study demonstrates that NEK8 human mutations cause major organ developmental defects due to altered ciliogenesis and cell differentiation/proliferation through deregulation of the Hippo pathway.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Cílios/genética , Fosfoproteínas/genética , Doenças Renais Policísticas/genética , Proteínas Quinases/genética , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Animais , Diferenciação Celular/genética , Cílios/patologia , Feminino , Estudos de Associação Genética , Humanos , Rim/metabolismo , Rim/patologia , Camundongos , Morfogênese/genética , Mutação , Quinases Relacionadas a NIMA , Fosfoproteínas/antagonistas & inibidores , Fosfoproteínas/biossíntese , Doenças Renais Policísticas/patologia , Porfirinas/administração & dosagem , Transdução de Sinais , Verteporfina , Peixe-Zebra
20.
Nat Cell Biol ; 18(1): 122-31, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26595381

RESUMO

The transition zone (TZ) ciliary subcompartment is thought to control cilium composition and signalling by facilitating a protein diffusion barrier at the ciliary base. TZ defects cause ciliopathies such as Meckel-Gruber syndrome (MKS), nephronophthisis (NPHP) and Joubert syndrome (JBTS). However, the molecular composition and mechanisms underpinning TZ organization and barrier regulation are poorly understood. To uncover candidate TZ genes, we employed bioinformatics (coexpression and co-evolution) and identified TMEM107 as a TZ protein mutated in oral-facial-digital syndrome and JBTS patients. Mechanistic studies in Caenorhabditis elegans showed that TMEM-107 controls ciliary composition and functions redundantly with NPHP-4 to regulate cilium integrity, TZ docking and assembly of membrane to microtubule Y-link connectors. Furthermore, nematode TMEM-107 occupies an intermediate layer of the TZ-localized MKS module by organizing recruitment of the ciliopathy proteins MKS-1, TMEM-231 (JBTS20) and JBTS-14 (TMEM237). Finally, MKS module membrane proteins are immobile and super-resolution microscopy in worms and mammalian cells reveals periodic localizations within the TZ. This work expands the MKS module of ciliopathy-causing TZ proteins associated with diffusion barrier formation and provides insight into TZ subdomain architecture.


Assuntos
Cerebelo/anormalidades , Cílios/metabolismo , Proteínas de Membrana/metabolismo , Retina/anormalidades , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Cerebelo/metabolismo , Anormalidades do Olho/genética , Anormalidades do Olho/metabolismo , Humanos , Doenças Renais Císticas/genética , Doenças Renais Císticas/metabolismo , Proteínas de Membrana/genética , Retina/metabolismo
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