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1.
PLoS Genet ; 15(8): e1008315, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31425546

RESUMO

Cilia are evolutionarily conserved hair-like structures with a wide spectrum of key biological roles, and their dysfunction has been linked to a growing class of genetic disorders, known collectively as ciliopathies. Many strides have been made towards deciphering the molecular causes for these diseases, which have in turn expanded the understanding of cilia and their functional roles. One recently-identified ciliary gene is ARL2BP, encoding the ADP-Ribosylation Factor Like 2 Binding Protein. In this study, we have identified multiple ciliopathy phenotypes associated with mutations in ARL2BP in human patients and in a mouse knockout model. Our research demonstrates that spermiogenesis is impaired, resulting in abnormally shaped heads, shortened and mis-assembled sperm tails, as well as in loss of axonemal doublets. Additional phenotypes in the mouse included enlarged ventricles of the brain and situs inversus. Mouse embryonic fibroblasts derived from knockout animals revealed delayed depolymerization of primary cilia. Our results suggest that ARL2BP is required for the structural maintenance of cilia as well as of the sperm flagellum, and that its deficiency leads to syndromic ciliopathy.

2.
Genet Med ; 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31263216

RESUMO

PURPOSE: We observed four individuals in two unrelated but consanguineous families from Portugal and Brazil affected by early-onset retinal degeneration, sensorineural hearing loss, microcephaly, intellectual disability, and skeletal dysplasia with scoliosis and short stature. The phenotype precisely matched that of an individual of Azorean descent published in 1986 by Liberfarb and coworkers. METHODS: Patients underwent specialized clinical examinations (including ophthalmological, audiological, orthopedic, radiological, and developmental assessment). Exome and targeted sequencing was performed on selected individuals. Minigene constructs were assessed by quantitative polymerase chain reaction (qPCR) and Sanger sequencing. RESULTS: Affected individuals shared a 3.36-Mb region of autozygosity on chromosome 22q12.2, including a 10-bp deletion (NM_014338.3:c.904-12_904-3delCTATCACCAC), immediately upstream of the last exon of the PISD (phosphatidylserine decarboxylase) gene. Sequencing of PISD from paraffin-embedded tissue from the 1986 case revealed the identical homozygous variant. In HEK293T cells, this variant led to aberrant splicing of PISD transcripts. CONCLUSION: We have identified the genetic etiology of the Liberfarb syndrome, affecting brain, eye, ear, bone, and connective tissue. Our work documents the migration of a rare Portuguese founder variant to two continents and highlights the link between phospholipid metabolism and bone formation, sensory defects, and cerebral development, while raising the possibility of therapeutic phospholipid replacement.

3.
Nat Commun ; 10(1): 2884, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253780

RESUMO

Hereditary retinal degenerations (HRDs) are Mendelian diseases characterized by progressive blindness and caused by ultra-rare mutations. In a genomic screen of 331 unrelated Japanese patients, we identify a disruptive Alu insertion and a nonsense variant (p.Arg1933*) in the ciliary gene RP1, neither of which are rare alleles in Japan. p.Arg1933* is almost polymorphic (frequency = 0.6%, amongst 12,000 individuals), does not cause disease in homozygosis or heterozygosis, and yet is significantly enriched in HRD patients (frequency = 2.1%, i.e., a 3.5-fold enrichment; p-value = 9.2 × 10-5). Familial co-segregation and association analyses show that p.Arg1933* can act as a Mendelian mutation in trans with the Alu insertion, but might also associate with disease in combination with two alleles in the EYS gene in a non-Mendelian pattern of heredity. Our results suggest that rare conditions such as HRDs can be paradoxically determined by relatively common variants, following a quasi-Mendelian model linking monogenic and complex inheritance.


Assuntos
Ciliopatias/genética , Proteínas do Olho/genética , Predisposição Genética para Doença , Doenças Retinianas/genética , Elementos Alu/genética , Grupo com Ancestrais do Continente Asiático/genética , Genômica , Humanos , Japão , Mutação , Linhagem
4.
Ophthalmic Genet ; 40(2): 177-181, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31012789

RESUMO

BACKGROUND: Inherited retinal degenerations (IRDs) encompass a wide spectrum of genetic ocular diseases characterized by considerable genetic and clinical heterogeneity. METHODS: Complete ophthalmic examination and next-generation sequencing. RESULTS: We describe a patient with no family history of vision loss, who at the age of 28 years developed visual impairment consistent with a severe form of retinitis pigmentosa. Genetic testing by means of whole exome sequencing identified a homozygous variant in the gene IDH3A. To date, only three papers have reported mutations in IDH3A, in families with early-onset retinal degeneration with or without the presence of macular pseudocoloboma. CONCLUSION: This study highlights the importance of including this rarely-mutated gene in the molecular diagnostic set-ups for IRDs, and further delineates the phenotypic spectrum elicited by mutations in IDH3A.

5.
EBioMedicine ; 29: 128-145, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29503225

RESUMO

Metastasis is a multi-step process in which direct crosstalk between cancer cells and their microenvironment plays a key role. Here, we assessed the effect of paired tumor-associated and normal lung tissue mesenchymal stem cells (MSCs) on the growth and dissemination of primary human lung carcinoma cells isolated from the same patients. We show that the tumor microenvironment modulates MSC gene expression and identify a four-gene MSC signature that is functionally implicated in promoting metastasis. We also demonstrate that tumor-associated MSCs induce the expression of genes associated with an aggressive phenotype in primary lung cancer cells and selectively promote their dissemination rather than local growth. Our observations provide insight into mechanisms by which the stroma promotes lung cancer metastasis.


Assuntos
Comunicação Celular , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Células-Tronco Mesenquimais/metabolismo , Microambiente Tumoral , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Gradação de Tumores , Metástase Neoplásica , Estadiamento de Neoplasias , Transcriptoma , Microambiente Tumoral/genética , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Genes (Basel) ; 9(1)2018 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-29320387

RESUMO

Inherited retinal diseases (IRDs) display an enormous genetic heterogeneity. Whole exome sequencing (WES) recently identified genes that were mutated in a small proportion of IRD cases. Consequently, finding a second case or family carrying pathogenic variants in the same candidate gene often is challenging. In this study, we searched for novel candidate IRD gene-associated variants in isolated IRD families, assessed their causality, and searched for novel genotype-phenotype correlations. Whole exome sequencing was performed in 11 probands affected with IRDs. Homozygosity mapping data was available for five cases. Variants with minor allele frequencies ≤ 0.5% in public databases were selected as candidate disease-causing variants. These variants were ranked based on their: (a) presence in a gene that was previously implicated in IRD; (b) minor allele frequency in the Exome Aggregation Consortium database (ExAC); (c) in silico pathogenicity assessment using the combined annotation dependent depletion (CADD) score; and (d) interaction of the corresponding protein with known IRD-associated proteins. Twelve unique variants were found in 11 different genes in 11 IRD probands. Novel autosomal recessive and dominant inheritance patterns were found for variants in Small Nuclear Ribonucleoprotein U5 Subunit 200 (SNRNP200) and Zinc Finger Protein 513 (ZNF513), respectively. Using our pathogenicity assessment, a variant in DEAH-Box Helicase 32 (DHX32) was the top ranked novel candidate gene to be associated with IRDs, followed by eight medium and lower ranked candidate genes. The identification of candidate disease-associated sequence variants in 11 single families underscores the notion that the previously identified IRD-associated genes collectively carry > 90% of the defects implicated in IRDs. To identify multiple patients or families with variants in the same gene and thereby provide extra proof for pathogenicity, worldwide data sharing is needed.

7.
Am J Hum Genet ; 101(4): 623-629, 2017 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-28985496

RESUMO

In contrast to recessive conditions with biallelic inheritance, identification of dominant (monoallelic) mutations for Mendelian disorders is more difficult, because of the abundance of benign heterozygous variants that act as massive background noise (typically, in a 400:1 excess ratio). To reduce this overflow of false positives in next-generation sequencing (NGS) screens, we developed DOMINO, a tool assessing the likelihood for a gene to harbor dominant changes. Unlike commonly-used predictors of pathogenicity, DOMINO takes into consideration features that are the properties of genes, rather than of variants. It uses a machine-learning approach to extract discriminant information from a broad array of features (N = 432), including: genomic data, intra-, and interspecies conservation, gene expression, protein-protein interactions, protein structure, etc. DOMINO's iterative architecture includes a training process on 985 genes with well-established inheritance patterns for Mendelian conditions, and repeated cross-validation that optimizes its discriminant power. When validated on 99 newly-discovered genes with pathogenic mutations, the algorithm displays an excellent final performance, with an area under the curve (AUC) of 0.92. Furthermore, unsupervised analysis by DOMINO of real sets of NGS data from individuals with intellectual disability or epilepsy correctly recognizes known genes and predicts 9 new candidates, with very high confidence. In summary, DOMINO is a robust and reliable tool that can infer dominance of candidate genes with high sensitivity and specificity, making it a useful complement to any NGS pipeline dealing with the analysis of the morbid human genome.


Assuntos
Genes Dominantes , Doenças Genéticas Inatas/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Aprendizado de Máquina , Mutação , Software , Bases de Dados Genéticas , Genoma Humano , Genômica , Humanos
8.
Genetics ; 203(2): 893-904, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27052569

RESUMO

Methods that bypass analytical evaluations of the likelihood function have become an indispensable tool for statistical inference in many fields of science. These so-called likelihood-free methods rely on accepting and rejecting simulations based on summary statistics, which limits them to low-dimensional models for which the value of the likelihood is large enough to result in manageable acceptance rates. To get around these issues, we introduce a novel, likelihood-free Markov chain Monte Carlo (MCMC) method combining two key innovations: updating only one parameter per iteration and accepting or rejecting this update based on subsets of statistics approximately sufficient for this parameter. This increases acceptance rates dramatically, rendering this approach suitable even for models of very high dimensionality. We further derive that for linear models, a one-dimensional combination of statistics per parameter is sufficient and can be found empirically with simulations. Finally, we demonstrate that our method readily scales to models of very high dimensionality, using toy models as well as by jointly inferring the effective population size, the distribution of fitness effects (DFE) of segregating mutations, and selection coefficients for each locus from data of a recent experiment on the evolution of drug resistance in influenza.


Assuntos
Farmacorresistência Viral/genética , Modelos Genéticos , Aptidão Genética , Loci Gênicos , Mutação , Orthomyxoviridae/efeitos dos fármacos , Orthomyxoviridae/genética , Probabilidade , Seleção Genética
9.
Genome Biol ; 15(6): R85, 2014 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-24972996

RESUMO

BACKGROUND: In mammals, ChIP-seq studies of RNA polymerase II (PolII) occupancy have been performed to reveal how recruitment, initiation and pausing of PolII may control transcription rates, but the focus is rarely on obtaining finely resolved profiles that can portray the progression of PolII through sequential promoter states. RESULTS: Here, we analyze PolII binding profiles from high-coverage ChIP-seq on promoters of actively transcribed genes in mouse and humans. We show that the enrichment of PolII near transcription start sites exhibits a stereotypical bimodal structure, with one peak near active transcription start sites and a second peak 110 base pairs downstream from the first. Using an empirical model that reliably quantifies the spatial PolII signal, gene by gene, we show that the first PolII peak allows for refined positioning of transcription start sites, which is corroborated by mRNA sequencing. This bimodal signature is found both in mouse and humans. Analysis of the pausing-related factors NELF and DSIF suggests that the downstream peak reflects widespread pausing at the +1 nucleosome barrier. Several features of the bimodal pattern are correlated with sequence features such as CpG content and TATA boxes, as well as the histone mark H3K4me3. CONCLUSIONS: We thus show how high coverage DNA sequencing experiments can reveal as-yet unnoticed bimodal spatial features of PolII accumulation that are frequent at individual mammalian genes and reminiscent of transcription initiation and pausing. The initiation-pausing hypothesis is corroborated by evidence from run-on sequencing and immunoprecipitation in other cell types and species.


Assuntos
Regiões Promotoras Genéticas , RNA Polimerase II/fisiologia , Animais , Imunoprecipitação da Cromatina , Células HeLa , Histonas/metabolismo , Humanos , Camundongos , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sítio de Iniciação de Transcrição , Transcrição Genética
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