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1.
Am J Hum Genet ; 104(6): 1073-1087, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31079899

RESUMO

Cargo transport along the cytoplasmic microtubular network is essential for neuronal function, and cytoplasmic dynein-1 is an established molecular motor that is critical for neurogenesis and homeostasis. We performed whole-exome sequencing, homozygosity mapping, and chromosomal microarray studies in five individuals from three independent pedigrees and identified likely-pathogenic variants in DYNC1I2 (Dynein Cytoplasmic 1 Intermediate Chain 2), encoding a component of the cytoplasmic dynein 1 complex. In a consanguineous Pakistani family with three affected individuals presenting with microcephaly, severe intellectual disability, simplification of cerebral gyration, corpus callosum hypoplasia, and dysmorphic facial features, we identified a homozygous splice donor site variant (GenBank: NM_001378.2:c.607+1G>A). We report two additional individuals who have similar neurodevelopmental deficits and craniofacial features and harbor deleterious variants; one individual bears a c.740A>G (p.Tyr247Cys) change in trans with a 374 kb deletion encompassing DYNC1I2, and an unrelated individual harbors the compound-heterozygous variants c.868C>T (p.Gln290∗) and c.740A>G (p.Tyr247Cys). Zebrafish larvae subjected to CRISPR-Cas9 gene disruption or transient suppression of dync1i2a displayed significantly altered craniofacial patterning with concomitant reduction in head size. We monitored cell death and cell cycle progression in dync1i2a zebrafish models and observed significantly increased apoptosis, likely due to prolonged mitosis caused by abnormal spindle morphology, and this finding offers initial insights into the cellular basis of microcephaly. Additionally, complementation studies in zebrafish demonstrate that p.Tyr247Cys attenuates gene function, consistent with protein structural analysis. Our genetic and functional data indicate that DYNC1I2 dysfunction probably causes an autosomal-recessive microcephaly syndrome and highlight further the critical roles of the dynein-1 complex in neurodevelopment.


Assuntos
Anormalidades Craniofaciais/etiologia , Dineínas/genética , Deficiência Intelectual/etiologia , Malformações Arteriovenosas Intracranianas/etiologia , Microcefalia/etiologia , Mutação , Peixe-Zebra/crescimento & desenvolvimento , Adulto , Alelos , Sequência de Aminoácidos , Animais , Pré-Escolar , Anormalidades Craniofaciais/patologia , Dineínas/química , Dineínas/metabolismo , Exoma , Feminino , Homozigoto , Humanos , Lactente , Deficiência Intelectual/patologia , Malformações Arteriovenosas Intracranianas/patologia , Masculino , Microcefalia/patologia , Linhagem , Fenótipo , Conformação Proteica , Homologia de Sequência , Sequenciamento do Exoma , Adulto Jovem , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
2.
Am J Hum Genet ; 105(5): 907-920, 2019 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-31607425

RESUMO

We report two consanguineous families with probands that exhibit intellectual disability, developmental delay, short stature, aphasia, and hypotonia in which homozygous non-synonymous variants were identified in IQSEC1 (GenBank: NM_001134382.3). In a Pakistani family, the IQSEC1 segregating variant is c.1028C>T (p.Thr343Met), while in a Saudi Arabian family the variant is c.962G>A (p.Arg321Gln). IQSEC1-3 encode guanine nucleotide exchange factors for the small GTPase ARF6 and their loss affects a variety of actin-dependent cellular processes, including AMPA receptor trafficking at synapses. The ortholog of IQSECs in the fly is schizo and its loss affects growth cone guidance at the midline in the CNS, also an actin-dependent process. Overexpression of the reference IQSEC1 cDNA in wild-type flies is lethal, but overexpression of the two variant IQSEC1 cDNAs did not affect viability. Loss of schizo caused embryonic lethality that could be rescued to 2nd instar larvae by moderate expression of the human reference cDNA. However, the p.Arg321Gln and p.Thr343Met variants failed to rescue embryonic lethality. These data indicate that the variants behave as loss-of-function mutations. We also show that schizo in photoreceptors is required for phototransduction. Finally, mice with a conditional Iqsec1 deletion in cortical neurons exhibited an increased density of dendritic spines with an immature morphology. The phenotypic similarity of the affecteds and the functional experiments in flies and mice indicate that IQSEC1 variants are the cause of a recessive disease with intellectual disability, developmental delay, and short stature, and that axonal guidance and dendritic projection defects as well as dendritic spine dysgenesis may underlie disease pathogenesis.


Assuntos
Deficiências do Desenvolvimento/genética , Nanismo/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Deficiência Intelectual/genética , Mutação/genética , Adulto , Alelos , Animais , Criança , Espinhas Dendríticas/genética , Drosophila/genética , Feminino , Humanos , Masculino , Camundongos , Arábia Saudita , Sinapses/genética , Adulto Jovem
3.
Hum Mol Genet ; 28(6): 972-979, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30481285

RESUMO

FBXL3 (F-Box and Leucine Rich Repeat Protein 3) encodes a protein that contains an F-box and several tandem leucine-rich repeats (LRR) domains. FBXL3 is part of the SCF (Skp1-Cullin-F box protein) ubiquitin ligase complex that binds and leads to phosphorylation-dependent degradation of the central clock protein cryptochromes (CRY1 and CRY2) by the proteasome and its absence causes circadian phenotypes in mice and behavioral problems. No FBXL3-related phenotypes have been described in humans. By a combination of exome sequencing and homozygosity mapping, we analyzed two consanguineous families with intellectual disability and identified homozygous loss-of-function (LoF) variants in FBXL3. In the first family, from Pakistan, an FBXL3 frameshift variant [NM_012158.2:c.885delT:p.(Leu295Phefs*25)] was the onlysegregating variant in five affected individuals in two family loops (LOD score: 3.12). In the second family, from Lebanon, we identified a nonsense variant [NM_012158.2:c.445C>T:p.(Arg149*)]. In a third patient from Italy, a likely deleterious non-synonymous variant [NM_012158.2:c.1072T>C:p.(Cys358Arg)] was identified in homozygosity. Protein 3D modeling predicted that the Cys358Arg change influences the binding with CRY2 by destabilizing the structure of the FBXL3, suggesting that this variant is also likely to be LoF. The eight affected individuals from the three families presented with a similar phenotype that included intellectual disability, developmental delay, short stature and mild facial dysmorphism, mainly large nose with a bulbous tip. The phenotypic similarity and the segregation analysis suggest that FBXL3 biallelic, LoF variants link this gene with syndromic autosomal recessive developmental delay/intellectual disability.


Assuntos
Alelos , Deficiências do Desenvolvimento/genética , Nanismo/genética , Proteínas F-Box/genética , Variação Genética , Deficiência Intelectual/genética , Adulto , Consanguinidade , Análise Mutacional de DNA , Deficiências do Desenvolvimento/diagnóstico , Nanismo/diagnóstico , Proteínas F-Box/química , Fácies , Feminino , Homozigoto , Humanos , Deficiência Intelectual/diagnóstico , Masculino , Pessoa de Meia-Idade , Modelos Moleculares , Linhagem , Fenótipo , Conformação Proteica , Relação Estrutura-Atividade , Adulto Jovem
4.
Am J Hum Genet ; 103(4): 568-578, 2018 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-30290152

RESUMO

Infantile and childhood-onset cataracts form a heterogeneous group of disorders; among the many genetic causes, numerous pathogenic variants in additional genes associated with autosomal-recessive infantile cataracts remain to be discovered. We identified three consanguineous families affected by bilateral infantile cataracts. Using exome sequencing, we found homozygous loss-of-function variants in DNMBP: nonsense variant c.811C>T (p.Arg271∗) in large family F385 (nine affected individuals; LOD score = 5.18 at θ = 0), frameshift deletion c.2947_2948del (p.Asp983∗) in family F372 (two affected individuals), and frameshift variant c.2852_2855del (p.Thr951Metfs∗41) in family F3 (one affected individual). The phenotypes of all affected individuals include infantile-onset cataracts. RNAi-mediated knockdown of the Drosophila ortholog still life (sif), enriched in lens-secreting cells, affects the development of these cells as well as the localization of E-cadherin, alters the distribution of septate junctions in adjacent cone cells, and leads to a ∼50% reduction in electroretinography amplitudes in young flies. DNMBP regulates the shape of tight junctions, which correspond to the septate junctions in invertebrates, as well as the assembly pattern of E-cadherin in human epithelial cells. E-cadherin has an important role in lens vesicle separation and lens epithelial cell survival in humans. We therefore conclude that DNMBP loss-of-function variants cause infantile-onset cataracts in humans.


Assuntos
Catarata/genética , Proteínas do Citoesqueleto/genética , Predisposição Genética para Doença/genética , Variação Genética/genética , Perda de Heterozigosidade/genética , Adulto , Alelos , Animais , Caderinas/genética , Criança , Drosophila/genética , Células Epiteliais/patologia , Exoma/genética , Feminino , Homozigoto , Humanos , Escore Lod , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Junções Íntimas/patologia
5.
Proc Natl Acad Sci U S A ; 115(51): 13015-13020, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30510006

RESUMO

X-chromosome inactivation (XCI) provides a dosage compensation mechanism where, in each female cell, one of the two X chromosomes is randomly silenced. However, some genes on the inactive X chromosome and outside the pseudoautosomal regions escape from XCI and are expressed from both alleles (escapees). We investigated XCI at single-cell resolution combining deep single-cell RNA sequencing with whole-genome sequencing to examine allelic-specific expression in 935 primary fibroblast and 48 lymphoblastoid single cells from five female individuals. In this framework we integrated an original method to identify and exclude doublets of cells. In fibroblast cells, we have identified 55 genes as escapees including five undescribed escapee genes. Moreover, we observed that all genes exhibit a variable propensity to escape XCI in each cell and cell type and that each cell displays a distinct expression profile of the escapee genes. A metric, the Inactivation Score-defined as the mean of the allelic expression profiles of the escapees per cell-enables us to discover a heterogeneous and continuous degree of cellular XCI with extremes represented by "inactive" cells, i.e., cells exclusively expressing the escaping genes from the active X chromosome and "escaping" cells expressing the escapees from both alleles. We found that this effect is associated with cell-cycle phases and, independently, with the XIST expression level, which is higher in the quiescent phase (G0). Single-cell allele-specific expression is a powerful tool to identify novel escapees in different tissues and provide evidence of an unexpected cellular heterogeneity of XCI.


Assuntos
Cromossomos Humanos X/genética , Fibroblastos/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Célula Única/métodos , Inativação do Cromossomo X , Alelos , Células Cultivadas , Feminino , Fibroblastos/citologia , Humanos , Transcriptoma
6.
Hum Mol Genet ; 27(15): 2703-2711, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29771303

RESUMO

Developmental eye defects often severely reduce vision. Despite extensive efforts, for a substantial fraction of these cases the molecular causes are unknown. Recessive eye disorders are frequent in consanguineous populations and such large families with multiple affected individuals provide an opportunity to identify recessive causative genes. We studied a Pakistani consanguineous family with three affected individuals with congenital vision loss and progressive eye degeneration. The family was analyzed by exome sequencing of one affected individual and genotyping of all family members. We have identified a non-synonymous homozygous variant (NM_001128918.2: c.1708C > G: p.Arg570Gly) in the MARK3 gene as the likely cause of the phenotype. Given that MARK3 is highly conserved in flies (I: 55%; S: 67%) we knocked down the MARK3 homologue, par-1, in the eye during development. This leads to a significant reduction in eye size, a severe loss of photoreceptors and loss of vision based on electroretinogram (ERG) recordings. Expression of the par-1 p.Arg792Gly mutation (equivalent to the MARK3 variant found in patients) in developing fly eyes also induces loss of eye tissue and reduces the ERG signals. The data in flies and human indicate that the MARK3 variant corresponds to a loss of function. We conclude that the identified mutation in MARK3 establishes a new gene-disease link, since it likely causes structural abnormalities during eye development and visual impairment in humans, and that the function of MARK3/par-1 is evolutionarily conserved in eye development.


Assuntos
Oftalmopatias/genética , Proteínas Serina-Treonina Quinases/genética , Transtornos da Visão/genética , Animais , Animais Geneticamente Modificados , Consanguinidade , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Anormalidades do Olho/genética , Feminino , Genes Recessivos , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Masculino , Mutação de Sentido Incorreto , Linhagem , Transtornos da Visão/diagnóstico por imagem , Sequenciamento do Exoma
7.
Am J Hum Genet ; 100(3): 444-453, 2017 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-28190458

RESUMO

Genomic imprinting results in parental-specific gene expression. Imprinted genes are involved in the etiology of rare syndromes and have been associated with common diseases such as diabetes and cancer. Standard RNA bulk cell sequencing applied to whole-tissue samples has been used to detect imprinted genes in human and mouse models. However, lowly expressed genes cannot be detected by using RNA bulk approaches. Here, we report an original and robust method that combines single-cell RNA-seq and whole-genome sequencing into an optimized statistical framework to analyze genomic imprinting in specific cell types and in different individuals. Using samples from the probands of 2 family trios and 3 unrelated individuals, 1,084 individual primary fibroblasts were RNA sequenced and more than 700,000 informative heterozygous single-nucleotide variations (SNVs) were genotyped. The allele-specific coverage per gene of each SNV in each single cell was used to fit a beta-binomial distribution to model the likelihood of a gene being expressed from one and the same allele. Genes presenting a significant aggregate allelic ratio (between 0.9 and 1) were retained to identify of the allelic parent of origin. Our approach allowed us to validate the imprinting status of all of the known imprinted genes expressed in fibroblasts and the discovery of nine putative imprinted genes, thereby demonstrating the advantages of single-cell over bulk RNA-seq to identify imprinted genes. The proposed single-cell methodology is a powerful tool for establishing a cell type-specific map of genomic imprinting.


Assuntos
Alelos , Expressão Gênica , Impressão Genômica , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Linhagem Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Estudos de Associação Genética , Técnicas de Genotipagem , Heterozigoto , Humanos , Modelos Teóricos , Polimorfismo de Nucleotídeo Único , Transcriptoma
8.
Nature ; 508(7496): 345-50, 2014 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-24740065

RESUMO

Trisomy 21 is the most frequent genetic cause of cognitive impairment. To assess the perturbations of gene expression in trisomy 21, and to eliminate the noise of genomic variability, we studied the transcriptome of fetal fibroblasts from a pair of monozygotic twins discordant for trisomy 21. Here we show that the differential expression between the twins is organized in domains along all chromosomes that are either upregulated or downregulated. These gene expression dysregulation domains (GEDDs) can be defined by the expression level of their gene content, and are well conserved in induced pluripotent stem cells derived from the twins' fibroblasts. Comparison of the transcriptome of the Ts65Dn mouse model of Down's syndrome and normal littermate mouse fibroblasts also showed GEDDs along the mouse chromosomes that were syntenic in human. The GEDDs correlate with the lamina-associated (LADs) and replication domains of mammalian cells. The overall position of LADs was not altered in trisomic cells; however, the H3K4me3 profile of the trisomic fibroblasts was modified and accurately followed the GEDD pattern. These results indicate that the nuclear compartments of trisomic cells undergo modifications of the chromatin environment influencing the overall transcriptome, and that GEDDs may therefore contribute to some trisomy 21 phenotypes.


Assuntos
Síndrome de Down/genética , Regulação da Expressão Gênica/genética , Genoma/genética , Transcriptoma/genética , Animais , Células Cultivadas , Cromatina/química , Cromatina/metabolismo , Cromossomos Humanos Par 21/genética , Cromossomos de Mamíferos/genética , Período de Replicação do DNA , Síndrome de Down/patologia , Feminino , Feto/citologia , Fibroblastos , Histonas/química , Histonas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Lisina/metabolismo , Masculino , Metilação , Camundongos , Gêmeos Monozigóticos/genética
9.
Am J Hum Genet ; 96(1): 70-80, 2015 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-25557783

RESUMO

The study of gene expression in mammalian single cells via genomic technologies now provides the possibility to investigate the patterns of allelic gene expression. We used single-cell RNA sequencing to detect the allele-specific mRNA level in 203 single human primary fibroblasts over 133,633 unique heterozygous single-nucleotide variants (hetSNVs). We observed that at the snapshot of analyses, each cell contained mostly transcripts from one allele from the majority of genes; indeed, 76.4% of the hetSNVs displayed stochastic monoallelic expression in single cells. Remarkably, adjacent hetSNVs exhibited a haplotype-consistent allelic ratio; in contrast, distant sites located in two different genes were independent of the haplotype structure. Moreover, the allele-specific expression in single cells correlated with the abundance of the cellular transcript. We observed that genes expressing both alleles in the majority of the single cells at a given time point were rare and enriched with highly expressed genes. The relative abundance of each allele in a cell was controlled by some regulatory mechanisms given that we observed related single-cell allelic profiles according to genes. Overall, these results have direct implications in cellular phenotypic variability.


Assuntos
Alelos , Fibroblastos/citologia , Genoma Humano , Análise de Sequência de RNA , DNA Complementar/genética , DNA Complementar/metabolismo , Haplótipos , Heterozigoto , Humanos , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Célula Única
10.
Nature ; 489(7414): 101-8, 2012 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-22955620

RESUMO

Eukaryotic cells make many types of primary and processed RNAs that are found either in specific subcellular compartments or throughout the cells. A complete catalogue of these RNAs is not yet available and their characteristic subcellular localizations are also poorly understood. Because RNA represents the direct output of the genetic information encoded by genomes and a significant proportion of a cell's regulatory capabilities are focused on its synthesis, processing, transport, modification and translation, the generation of such a catalogue is crucial for understanding genome function. Here we report evidence that three-quarters of the human genome is capable of being transcribed, as well as observations about the range and levels of expression, localization, processing fates, regulatory regions and modifications of almost all currently annotated and thousands of previously unannotated RNAs. These observations, taken together, prompt a redefinition of the concept of a gene.


Assuntos
DNA/genética , Enciclopédias como Assunto , Genoma Humano/genética , Anotação de Sequência Molecular , Sequências Reguladoras de Ácido Nucleico/genética , Transcrição Gênica/genética , Transcriptoma/genética , Alelos , Linhagem Celular , DNA Intergênico/genética , Elementos Facilitadores Genéticos , Éxons/genética , Perfilação da Expressão Gênica , Genes/genética , Genômica , Humanos , Poliadenilação/genética , Isoformas de Proteínas/genética , RNA/biossíntese , RNA/genética , Edição de RNA/genética , Splicing de RNA/genética , Sequências Repetitivas de Ácido Nucleico/genética , Análise de Sequência de RNA
11.
PLoS Genet ; 11(1): e1004958, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25634236

RESUMO

Understanding how genetic variation affects distinct cellular phenotypes, such as gene expression levels, alternative splicing and DNA methylation levels, is essential for better understanding of complex diseases and traits. Furthermore, how inter-individual variation of DNA methylation is associated to gene expression is just starting to be studied. In this study, we use the GenCord cohort of 204 newborn Europeans' lymphoblastoid cell lines, T-cells and fibroblasts derived from umbilical cords. The samples were previously genotyped for 2.5 million SNPs, mRNA-sequenced, and assayed for methylation levels in 482,421 CpG sites. We observe that methylation sites associated to expression levels are enriched in enhancers, gene bodies and CpG island shores. We show that while the correlation between DNA methylation and gene expression can be positive or negative, it is very consistent across cell-types. However, this epigenetic association to gene expression appears more tissue-specific than the genetic effects on gene expression or DNA methylation (observed in both sharing estimations based on P-values and effect size correlations between cell-types). This predominance of genetic effects can also be reflected by the observation that allele specific expression differences between individuals dominate over tissue-specific effects. Additionally, we discover genetic effects on alternative splicing and interestingly, a large amount of DNA methylation correlating to alternative splicing, both in a tissue-specific manner. The locations of the SNPs and methylation sites involved in these associations highlight the participation of promoter proximal and distant regulatory regions on alternative splicing. Overall, our results provide high-resolution analyses showing how genome sequence variation has a broad effect on cellular phenotypes across cell-types, whereas epigenetic factors provide a secondary layer of variation that is more tissue-specific. Furthermore, the details of how this tissue-specificity may vary across inter-relations of molecular traits, and where these are occurring, can yield further insights into gene regulation and cellular biology as a whole.


Assuntos
Processamento Alternativo/genética , Metilação de DNA/genética , Epigênese Genética , Regulação da Expressão Gênica/genética , Variação Genética , Alelos , Ilhas de CpG , Humanos , Recém-Nascido , Especificidade de Órgãos , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico/genética
13.
Genome Res ; 23(9): 1410-21, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23783273

RESUMO

Congenital heart defect (CHD) occurs in 40% of Down syndrome (DS) cases. While carrying three copies of chromosome 21 increases the risk for CHD, trisomy 21 itself is not sufficient to cause CHD. Thus, additional genetic variation and/or environmental factors could contribute to the CHD risk. Here we report genomic variations that in concert with trisomy 21, determine the risk for CHD in DS. This case-control GWAS includes 187 DS with CHD (AVSD = 69, ASD = 53, VSD = 65) as cases, and 151 DS without CHD as controls. Chromosome 21-specific association studies revealed rs2832616 and rs1943950 as CHD risk alleles (adjusted genotypic P-values <0.05). These signals were confirmed in a replication cohort of 92 DS-CHD cases and 80 DS-without CHD (nominal P-value 0.0022). Furthermore, CNV analyses using a customized chromosome 21 aCGH of 135K probes in 55 DS-AVSD and 53 DS-without CHD revealed three CNV regions associated with AVSD risk (FDR ≤ 0.05). Two of these regions that are located within the previously identified CHD region on chromosome 21 were further confirmed in a replication study of 49 DS-AVSD and 45 DS- without CHD (FDR ≤ 0.05). One of these CNVs maps near the RIPK4 gene, and the second includes the ZBTB21 (previously ZNF295) gene, highlighting the potential role of these genes in the pathogenesis of CHD in DS. We propose that the genetic architecture of the CHD risk of DS is complex and includes trisomy 21, and SNP and CNV variations in chromosome 21. In addition, a yet-unidentified genetic variation in the rest of the genome may contribute to this complex genetic architecture.


Assuntos
Variações do Número de Cópias de DNA , Síndrome de Down/diagnóstico , Cardiopatias Congênitas/genética , Polimorfismo de Nucleotídeo Único , Estudos de Casos e Controles , Cromossomos Humanos Par 21/genética , Proteínas de Ligação a DNA/genética , Síndrome de Down/complicações , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Cardiopatias Congênitas/etiologia , Humanos , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Fatores de Transcrição/genética
14.
Blood ; 122(4): 554-61, 2013 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-23733339

RESUMO

Some neonates with Down syndrome (DS) are diagnosed with self-regressing transient myeloproliferative disorder (TMD), and 20% to 30% of those progress to acute megakaryoblastic leukemia (AMKL). We performed exome sequencing in 7 TMD/AMKL cases and copy-number analysis in these and 10 additional cases. All TMD/AMKL samples contained GATA1 mutations. No exome-sequenced TMD/AMKL sample had other recurrently mutated genes. However, 2 of 5 TMD cases, and all AMKL cases, showed mutations/deletions other than GATA1, in genes proven as transformation drivers in non-DS leukemia (EZH2, APC, FLT3, JAK1, PARK2-PACRG, EXT1, DLEC1, and SMC3). One patient at the TMD stage revealed 2 clonal expansions with different GATA1 mutations, of which 1 clone had an additional driver mutation. Interestingly, it was the other clone that gave rise to AMKL after accumulating mutations in 7 other genes. Data suggest that GATA1 mutations alone are sufficient for clonal expansions, and additional driver mutations at the TMD stage do not necessarily predict AMKL progression. Later in infancy, leukemic progression requires "third-hit driver" mutations/somatic copy-number alterations found in non-DS leukemias. Putative driver mutations affecting WNT (wingless-related integration site), JAK-STAT (Janus kinase/signal transducer and activator of transcription), or MAPK/PI3K (mitogen-activated kinase/phosphatidylinositol-3 kinase) pathways were found in all cases, aberrant activation of which converges on overexpression of MYC.


Assuntos
Transformação Celular Neoplásica/genética , Síndrome de Down/genética , Leucemia Megacarioblástica Aguda/genética , Transtornos Mieloproliferativos/genética , Progressão da Doença , Síndrome de Down/complicações , Exoma/genética , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla , Instabilidade Genômica/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Lactente , Recém-Nascido , Leucemia Megacarioblástica Aguda/complicações , Leucemia Megacarioblástica Aguda/patologia , Análise em Microsséries , Transtornos Mieloproliferativos/complicações , Transtornos Mieloproliferativos/patologia , Polimorfismo de Nucleotídeo Único , Transcriptoma
15.
Genome Res ; 21(1): 68-73, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21147911

RESUMO

MicroRNAs (miRNAs) are regulatory noncoding RNAs that affect the production of a significant fraction of human mRNAs via post-transcriptional regulation. Interindividual variation of the miRNA expression levels is likely to influence the expression of miRNA target genes and may therefore contribute to phenotypic differences in humans, including susceptibility to common disorders. The extent to which miRNA levels are genetically controlled is largely unknown. In this report, we assayed the expression levels of miRNAs in primary fibroblasts from 180 European newborns of the GenCord project and performed association analysis to identify eQTLs (expression quantitative traits loci). We detected robust expression for 121 miRNAs out of 365 interrogated. We have identified significant cis- (10%) and trans- (11%) eQTLs. Furthermore, we detected one genomic locus (rs1522653) that influences the expression levels of five miRNAs, thus unraveling a novel mechanism for coregulation of miRNA expression.


Assuntos
Elementos Facilitadores Genéticos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , MicroRNAs/metabolismo , Locos de Características Quantitativas/genética , Processamento Pós-Transcricional do RNA , Linhagem Celular , Europa (Continente) , Perfilação da Expressão Gênica , Variação Genética , Estudo de Associação Genômica Ampla , Humanos , Recém-Nascido , MicroRNAs/genética
16.
Hum Mutat ; 33(3): 495-503, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22102620

RESUMO

Primary ciliary dyskinesia (PCD) is an inherited disorder causing significant upper and lower respiratory tract morbidity and impaired fertility. Half of PCD patients show abnormal situs. Human disease loci have been identified but a mouse model without additional deleterious defects is elusive. The inversus viscerum mouse, mutated at the outer arm dynein heavy chain 11 locus (Dnahc11) is a known model of heterotaxy. We demonstrated immotile tracheal cilia with normal ultrastructure and reduced sperm motility in the Dnahc11(iv) mouse. This is accompanied by gross rhinitis, sinusitis, and otitis media, all indicators of human PCD. Strikingly, age-related progression of the disease is evident. The Dnahc11(iv) mouse is robust, lacks secondary defects, and requires no intervention to precipitate the phenotype. Together these findings show the Dnahc11(iv) mouse to be an excellent model of many aspects of human PCD. Mutation of the homologous human locus has previously been associated with hyperkinetic tracheal cilia in PCD. Two PCD patients with normal ciliary ultrastructure, one with immotile and one with hyperkinetic cilia were found to carry DNAH11 mutations. Three novel DNAH11 mutations were detected indicating that this gene should be investigated in patients with normal ciliary ultrastructure and static, as well as hyperkinetic cilia.


Assuntos
Dineínas do Axonema/genética , Síndrome de Kartagener/genética , Animais , Criança , Pré-Escolar , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Mutação
17.
Nat Commun ; 10(1): 4495, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31582743

RESUMO

Aneuploidy is a major source of gene dosage imbalance due to copy number alterations (CNA), and viable human trisomies are model disorders of altered gene expression. We study gene and allele-specific expression (ASE) of 9668 single-cell fibroblasts from trisomy 21 (T21) discordant twins and from mosaic T21, T18, T13 and T8. We examine 928 single cells with deep scRNAseq. Expected and observed overexpression of trisomic genes in trisomic vs. diploid bulk RNAseq is not detectable in trisomic vs. diploid single cells. Instead, for trisomic genes with low-to-average expression, their altered gene dosage is mainly due to the higher fraction of trisomic cells simultaneously expressing these genes, in agreement with a stochastic 2-state burst-like model of transcription. These results, confirmed in a further analysis of 8740 single fibroblasts with shallow scRNAseq, suggest that the specific transcriptional profile of each gene contributes to the phenotypic variability of trisomies. We propose an improved model to understand the effects of CNA and, generally, of gene regulation on gene dosage imbalance.


Assuntos
Variações do Número de Cópias de DNA , Dosagem de Genes , Modelos Genéticos , Transcriptoma/genética , Trissomia/genética , Alelos , Cromossomos Humanos Par 13/genética , Cromossomos Humanos Par 18/genética , Cromossomos Humanos Par 8/genética , Síndrome de Down/genética , Feminino , Fibroblastos , Perfilação da Expressão Gênica , Humanos , Masculino , Mosaicismo , Fenótipo , RNA-Seq , Análise de Célula Única
18.
Respiration ; 76(2): 198-204, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18434704

RESUMO

BACKGROUND: Primary ciliary dyskinesia (PCD) is a rare recessive hereditary disorder characterized by dysmotility to immotility of ciliated and flagellated structures. Its main symptoms are respiratory, caused by defective ciliary beating in the epithelium of the upper airways (nose, bronchi and paranasal sinuses). Impairing the drainage of inhaled microorganisms and particles leads to recurrent infections and pulmonary complications. To date, 5 genes encoding 3 dynein protein arm subunits (DNAI1, DNAH5 and DNAH11), the kinase TXNDC3 and the X-linked RPGR have been found to be mutated in PCD. OBJECTIVES: We proposed to determine the impact of the DNAI1 gene on a cohort of unrelated PCD patients (n = 104) recruited without any phenotypic preselection. METHODS: We used denaturing high-performance liquid chromatography and sequencing to screen for mutations in the coding and splicing site sequences of the gene DNAI1. RESULTS: Three mutations were identified: a novel missense variant (p.Glu174Lys) was found in 1 patient and 2 previously reported variants were identified (p.Trp568Ser in 1 patient and IVS1+2_3insT in 3 patients). Overall, mutations on both alleles of gene DNAI1 were identified in only 2% of our clinically heterogeneous cohort of patients. CONCLUSION: We conclude that DNAI1 gene mutation is not a common cause of PCD, and that major or several additional disease gene(s) still remain to be identified before a sensitive molecular diagnostic test can be developed for PCD.


Assuntos
Dineínas/genética , Síndrome de Kartagener/genética , Sequência de Aminoácidos , Dineínas do Axonema , Análise Mutacional de DNA , Humanos , Dados de Sequência Molecular
19.
Elife ; 62017 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-28869038

RESUMO

The importance of natural gene expression variation for human behavior is undisputed, but its impact on circadian physiology remains mostly unexplored. Using umbilical cord fibroblasts, we have determined by genome-wide association how common genetic variation impacts upon cellular circadian function. Gene set enrichment points to differences in protein catabolism as one major source of clock variation in humans. The two most significant alleles regulated expression of COPS7B, a subunit of the COP9 signalosome. We further show that the signalosome complex is imported into the nucleus in timed fashion to stabilize the essential circadian protein BMAL1, a novel mechanism to oppose its proteasome-mediated degradation. Thus, circadian clock properties depend in part upon a genetically-encoded competition between stabilizing and destabilizing forces, and genetic alterations in these mechanisms provide one explanation for human chronotype.


Assuntos
Variação Biológica da População , Ritmo Circadiano , Regulação da Expressão Gênica , Variação Genética , Fatores de Transcrição ARNTL/metabolismo , Complexo do Signalossomo COP9/metabolismo , Estudo de Associação Genômica Ampla , Humanos , Estabilidade Proteica , Proteínas/metabolismo
20.
PLoS One ; 10(5): e0126475, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25955728

RESUMO

The HSA21 encoded Single-minded 2 (SIM2) transcription factor has key neurological functions and is a good candidate to be involved in the cognitive impairment of Down syndrome. We aimed to explore the functional capacity of SIM2 by mapping its DNA binding sites in mouse embryonic stem cells. ChIP-sequencing revealed 1229 high-confidence SIM2-binding sites. Analysis of the SIM2 target genes confirmed the importance of SIM2 in developmental and neuronal processes and indicated that SIM2 may be a master transcription regulator. Indeed, SIM2 DNA binding sites share sequence specificity and overlapping domains of occupancy with master transcription factors such as SOX2, OCT4 (Pou5f1), NANOG or KLF4. The association between SIM2 and these pioneer factors is supported by co-immunoprecipitation of SIM2 with SOX2, OCT4, NANOG or KLF4. Furthermore, the binding of SIM2 marks a particular sub-category of enhancers known as super-enhancers. These regions are characterized by typical DNA modifications and Mediator co-occupancy (MED1 and MED12). Altogether, we provide evidence that SIM2 binds a specific set of enhancer elements thus explaining how SIM2 can regulate its gene network in neuronal features.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , DNA/metabolismo , Elementos Facilitadores Genéticos , Células-Tronco Embrionárias Murinas/metabolismo , Fatores de Transcrição/genética , Animais , Sítios de Ligação , Células Cultivadas , Imunoprecipitação da Cromatina/métodos , DNA/genética , Fator 4 Semelhante a Kruppel , Camundongos , Análise de Sequência de RNA , Fatores de Transcrição/metabolismo
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