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1.
Nat Commun ; 11(1): 4593, 2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32929070

RESUMO

Gene-targeted animal models that are generated by injecting Cas9 and sgRNAs into zygotes are often accompanied by undesired double-strand break (DSB)-induced byproducts and random biallelic targeting due to uncontrollable Cas9 targeting activity. Here, we establish a parental allele-specific gene-targeting (Past-CRISPR) method, based on the detailed observation that pronuclear transfer-mediated cytoplasmic dilution can effectively terminate Cas9 activity. We apply this method in embryos to efficiently target the given parental alleles of a gene of interest and observed little genomic mosaicism because of the spatiotemporal control of Cas9 activity. This method allows us to rapidly explore the function of individual parent-of-origin effects and to construct animal models with a single genomic change. More importantly, Past-CRISPR could also be used for therapeutic applications or disease model construction.


Assuntos
Alelos , Sistemas CRISPR-Cas/genética , Núcleo Celular/genética , Edição de Genes , Terapia de Substituição Mitocondrial , Animais , Sequência de Bases , Modelos Animais de Doenças , Nanismo/genética , Perda do Embrião/genética , Feminino , Marcação de Genes , Genes Dominantes , Impressão Genômica , Heterozigoto , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Reprodutibilidade dos Testes , Fatores de Tempo
2.
Gene ; 757: 144940, 2020 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-32640303

RESUMO

OBJECTIVE: We sought to analyze the association between miR-146a rs2910164 G > C polymorphism and susceptibility to lung cancer using a meta-analysis of case-control studies. METHODS: We systematically searched for studies reporting on the relationship between miR-146a rs2910164 polymorphism and the risk of lung cancer in PubMed, Embase, Web of Science and Chinese National Knowledge Infrastructure databases. We then calculated pooled odds ratios (ORs), at 95% confidence interval (CI) to assess the aforementioned relationship. All the data were analyzed using statistical packages implemented in R version 3.6.2 (R Project for Statistical Computing), run in RStudio version 1.2.5033. RESULTS: A total of fifteen studies, comprising 6506 cases and 6576 controls, were enrolled in this meta-analysis. Significant associations were observed between miR-146a rs2910164 polymorphism and the risk of lung cancer based on overall pooled subjects under the allele, heterozygous, homozygous, dominant, and recessive genetic models (C vs. G: OR = 1.27, 95% CI: 1.12-1.44; GC vs. GG: OR = 1.23, 95% CI: 1.03-1.46; CC vs. GG: OR = 1.51, 95% CI: 1.18-1.93; GC + CC vs. GG: OR = 1.33, 95% CI: 1.10-1.61; CC vs. GG + GC: OR = 1.32, 95% CI: 1.13-1.53). Ethnicity-based subgroup analyses revealed no statistically significant differences in Asians using heterozygous and dominant genetic models. CONCLUSION: miR-146a rs2910164 G > C polymorphism may be a risk factor of lung cancer. Asian populations exhibiting heterozygous and dominant genotypes need to be further investigated to validate our findings.


Assuntos
Neoplasias Pulmonares/genética , MicroRNAs/genética , Polimorfismo de Nucleotídeo Único , Grupo com Ancestrais do Continente Asiático/genética , Grupo com Ancestrais do Continente Europeu/genética , Genes Dominantes , Heterozigoto , Humanos , Neoplasias Pulmonares/etnologia
3.
Gene ; 757: 144945, 2020 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-32649979

RESUMO

HOX genes are important regulatory genes patterning head formation, including development of the ear. Microtia is a congenital ear anomaly characterized by lacking all or part of the structures of the outer ear. To date, only four HOXA2 mutations were reported in families with autosomal-recessive or dominant microtia, with or without hearing impairment. More identified mutations are needed to confirm the correlation between genotype and phenotype. Here, we collect two Chinese families with non-syndromic bilateral microtia. Next generation sequencing identified two heterozygous nonsense HOXA2 mutations, one in each family. One mutation (c.637A > T, p.Lys213*) is newly reported, while the other one (c.703C > T,p.Gln235*) is consistent with a previous report. In mouse, Hoxa2 can bind to a long-range enhancer and regulate expression of the Hmx1 gene, which is a crucial transcription factor in eye and ear development. Using dual luciferase reporter assays, we showed that both HOXA2 mutations have impaired activation of the long-range enhancer of HMX1. In the present study, we report the first two bilateral non-syndromic microtia cases with HOXA2 mutations of Chinese origin and identify a novel mutation. Our results also provide molecular insights about how these nonsense HOXA2 mutations could affect the activation of its downstream target HMX1 by interacting with the long-range enhancer.


Assuntos
Microtia Congênita/genética , Proteínas de Homeodomínio/genética , Mutação com Perda de Função , Células Cultivadas , Microtia Congênita/patologia , Feminino , Genes Dominantes , Células HEK293 , Proteínas de Homeodomínio/metabolismo , Humanos , Masculino , Linhagem , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
4.
Anticancer Res ; 40(5): 2687-2694, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32366413

RESUMO

BACKGROUND/AIM: Transforming growth factor-ß (TGF-ß) plays dual suppressive and oncogenic roles in mammary carcinogenesis. MATERIALS AND METHODS: To analyze whether TGF-ß exerts suppressive or oncogenic actions on mammary carcinogenesis, transgenic mice overexpressing a dominant-negative mutant type II TGF-ß receptor (TßRII-DNR) driven by the mouse mammary tumor virus (MMTV) promoter were treated with a low dose of urethane, a carcinogen present in fermented food products and alcoholic beverages. RESULTS: Lobular proliferative lesions, showing high ß-casein expression, developed in the mammary glands of TßRII-DNR+/+ mice aged >61 weeks. Compared with wild-type mice, TßRII-DNR+/+ mice administered with urethane showed significant increases in dysplastic hyperplasias and adenocarcinomas of the mammary glands. CONCLUSION: The functional decline of TGF-ß signaling in mammary glands led to a high susceptibility to urethane-induced mammary carcinogenesis. TGF-ß signaling may act as a tumor suppressor during mammary tumor development.


Assuntos
Carcinogênese/patologia , Genes Dominantes , Neoplasias Mamárias Animais/genética , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Neoplasias Pulmonares/patologia , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Animais/induzido quimicamente , Neoplasias Mamárias Animais/patologia , Camundongos Transgênicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II , Transgenes , Uretana
5.
Am J Hum Genet ; 106(6): 734-747, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32386559

RESUMO

The calcium-sensing receptor (CaSR) regulates serum calcium concentrations. CASR loss- or gain-of-function mutations cause familial hypocalciuric hypercalcemia type 1 (FHH1) or autosomal-dominant hypocalcemia type 1 (ADH1), respectively, but the population prevalence of FHH1 or ADH1 is unknown. Rare CASR variants were identified in whole-exome sequences from 51,289 de-identified individuals in the DiscovEHR cohort derived from a single US healthcare system. We integrated bioinformatics pathogenicity triage, mean serum Ca concentrations, and mode of inheritance to identify potential FHH1 or ADH1 variants, and we used a Sequence Kernel Association Test (SKAT) to identify rare variant-associated diseases. We identified predicted heterozygous loss-of-function CASR variants (6 different nonsense/frameshift variants and 12 different missense variants) in 38 unrelated individuals, 21 of whom were hypercalcemic. Missense CASR variants were identified in two unrelated hypocalcemic individuals. Functional studies showed that all hypercalcemia-associated missense variants impaired heterologous expression, plasma membrane targeting, and/or signaling, whereas hypocalcemia-associated missense variants increased expression, plasma membrane targeting, and/or signaling. Thus, 38 individuals with a genetic diagnosis of FHH1 and two individuals with a genetic diagnosis of ADH1 were identified in the 51,289 cohort, giving a prevalence in this population of 74.1 per 100,000 for FHH1 and 3.9 per 100,000 for ADH1. SKAT combining all nonsense, frameshift, and missense loss-of-function variants revealed associations with cardiovascular, neurological, and other diseases. In conclusion, FHH1 is a common cause of hypercalcemia, with prevalence similar to that of primary hyperparathyroidism, and is associated with altered disease risks, whereas ADH1 is a major cause of non-surgical hypoparathyroidism.


Assuntos
Assistência à Saúde/estatística & dados numéricos , Hipercalcemia/congênito , Adulto , Idoso , Idoso de 80 Anos ou mais , Cálcio/sangue , Estudos de Coortes , Feminino , Genes Dominantes/genética , Heterozigoto , Humanos , Hipercalcemia/genética , Masculino , Pessoa de Meia-Idade , Mutação , Fenótipo , Prevalência , Receptores de Detecção de Cálcio/genética , Estados Unidos
6.
Am J Hum Genet ; 106(6): 893-904, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32386558

RESUMO

Kinesin-2 enables ciliary assembly and maintenance as an anterograde intraflagellar transport (IFT) motor. Molecular motor activity is driven by a heterotrimeric complex comprised of KIF3A and KIF3B or KIF3C plus one non-motor subunit, KIFAP3. Using exome sequencing, we identified heterozygous KIF3B variants in two unrelated families with hallmark ciliopathy phenotypes. In the first family, the proband presents with hepatic fibrosis, retinitis pigmentosa, and postaxial polydactyly; he harbors a de novo c.748G>C (p.Glu250Gln) variant affecting the kinesin motor domain encoded by KIF3B. The second family is a six-generation pedigree affected predominantly by retinitis pigmentosa. Affected individuals carry a heterozygous c.1568T>C (p.Leu523Pro) KIF3B variant segregating in an autosomal-dominant pattern. We observed a significant increase in primary cilia length in vitro in the context of either of the two mutations while variant KIF3B proteins retained stability indistinguishable from wild type. Furthermore, we tested the effects of KIF3B mutant mRNA expression in the developing zebrafish retina. In the presence of either missense variant, rhodopsin was sequestered to the photoreceptor rod inner segment layer with a concomitant increase in photoreceptor cilia length. Notably, impaired rhodopsin trafficking is also characteristic of recessive KIF3B models as exemplified by an early-onset, autosomal-recessive, progressive retinal degeneration in Bengal cats; we identified a c.1000G>A (p.Ala334Thr) KIF3B variant by genome-wide association study and whole-genome sequencing. Together, our genetic, cell-based, and in vivo modeling data delineate an autosomal-dominant syndromic retinal ciliopathy in humans and suggest that multiple KIF3B pathomechanisms can impair kinesin-driven ciliary transport in the photoreceptor.


Assuntos
Ciliopatias/genética , Ciliopatias/patologia , Genes Dominantes/genética , Cinesina/genética , Mutação , Retina/patologia , Sequência de Aminoácidos , Animais , Gatos , Pré-Escolar , Cílios/patologia , Feminino , Estudo de Associação Genômica Ampla , Heterozigoto , Humanos , Cinesina/química , Cinesina/metabolismo , Larva , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Células Fotorreceptoras/metabolismo , Retina/citologia , Retina/crescimento & desenvolvimento , Retina/metabolismo , Rodopsina/metabolismo , Adulto Jovem , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento
7.
Hum Genet ; 139(11): 1391-1401, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32440726

RESUMO

Cone-rod dystrophy (CORD) is an inherited retinal degenerative disease characterized by progressive loss of cone and rod photoreceptors. Although several genes have been reported to cause autosomal dominant CORD (adCORD), the genetic causes of adCORD have not been fully elucidated. Here, we identified the ATP1A3 gene, encoding the α3 subunit of Na+, K+-ATPase, as a novel gene associated with adCORD. Using whole-exome sequencing (WES), we found a candidate mutation of ATP1A3 that co-segregated with the disease in an analysis of two affected patients and one healthy relative in an adCORD family. According to our RNA-seq data, we demonstrated that the Atp1a3 mRNA level was extremely high in the murine retina. Overexpression of mutant ATP1A3 in vitro led to a reduced oxygen consumption rate (OCR), reflecting the limited mitochondrial reserve capacity. Furthermore, we generated transgenic mice expressing the ATP1A3 cDNA with patient variant and found decreased electroretinogram (ERG) responses. Moreover, the mutant ATP1A3 is highly expressed in photoreceptor inner segment, where mitochondria are enriched. These results suggest that the ATP1A3 mutation is a new genetic cause responsible for adCORD and indicate that ATP1A3 plays an important role in retinal function.


Assuntos
Distrofias de Cones e Bastonetes/genética , Genes Dominantes/genética , Mutação/genética , ATPase Trocadora de Sódio-Potássio/genética , Adulto , Animais , Linhagem Celular Tumoral , Eletrorretinografia/métodos , Feminino , Células HeLa , Humanos , Masculino , Camundongos , Linhagem , Fenótipo , Retina/patologia , Células Fotorreceptoras Retinianas Cones/patologia , Degeneração Retiniana/genética , Células Fotorreceptoras Retinianas Bastonetes/patologia , Retinite Pigmentosa/genética , Acuidade Visual , Sequenciamento Completo do Exoma/métodos , Adulto Jovem
8.
PLoS Genet ; 16(5): e1008639, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32453731

RESUMO

Hypertrophic cardiomyopathy (HCM) is characterized by thickening of the ventricular muscle without dilation and is often associated with dominant pathogenic variants in cardiac sarcomeric protein genes. Here, we report a family with two infants diagnosed with infantile-onset HCM and mitral valve dysplasia that led to death before one year of age. Using exome sequencing, we discovered that one of the affected children had a homozygous frameshift variant in Myosin light chain 2 (MYL2:NM_000432.3:c.431_432delCT: p.Pro144Argfs*57;MYL2-fs), which alters the last 20 amino acids of the protein and is predicted to impact the most C-terminal of the three EF-hand domains in MYL2. The parents are unaffected heterozygous carriers of the variant and the variant is absent in control cohorts from gnomAD. The absence of the phenotype in carriers and the infantile presentation of severe HCM is in contrast to HCM associated with dominant MYL2 variants. Immunohistochemical analysis of the ventricular muscle of the deceased patient with the MYL2-fs variant showed a marked reduction of MYL2 expression compared to an unaffected control. In vitro overexpression studies further indicate that the MYL2-fs variant is actively degraded. In contrast, an HCM-associated missense variant (MYL2:p.Gly162Arg) and three other MYL2 stop-gain variants (p.E22*, p.K62*, p.E97*) that result in loss of the EF domains are stably expressed but show impaired localization. The degradation of the MYL2-fs can be rescued by inhibiting the cell's proteasome function supporting a post-translational effect of the variant. In vivo rescue experiments with a Drosophila MYL2-homolog (Mlc2) knockdown model indicate that neither the MYL2-fs nor the MYL2:p.Gly162Arg variant supports normal cardiac function. The tools that we have generated provide a rapid screening platform for functional assessment of variants of unknown significance in MYL2. Our study supports an autosomal recessive model of inheritance for MYL2 loss-of-function variants in infantile HCM and highlights the variant-specific molecular differences found in MYL2-associated cardiomyopathy.


Assuntos
Miosinas Cardíacas/genética , Cardiomiopatia Hipertrófica/genética , Família , Mutação da Fase de Leitura , Cadeias Leves de Miosina/genética , Adulto , Animais , Animais Geneticamente Modificados , Cardiomiopatia Hipertrófica/classificação , Cardiomiopatia Hipertrófica/congênito , Cardiomiopatia Hipertrófica/patologia , Células Cultivadas , Consanguinidade , Drosophila , Evolução Fatal , Feminino , Genes Dominantes , Genes Recessivos , Heterozigoto , Humanos , Lactente , Morte do Lactente , Recém-Nascido , Masculino , Linhagem , Fenótipo , Irmãos
9.
Nucleic Acids Res ; 48(11): 6068-6080, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32374842

RESUMO

We have previously found that UV-induced DNA damage causes hyperphosphorylation of the carboxy terminal domain (CTD) of RNA polymerase II (RNAPII), inhibition of transcriptional elongation and changes in alternative splicing (AS) due to kinetic coupling between transcription and splicing. In an unbiased search for protein kinases involved in the AS response to DNA damage, we have identified glycogen synthase kinase 3 (GSK-3) as an unforeseen participant. Unlike Cdk9 inhibition, GSK-3 inhibition only prevents CTD hyperphosphorylation triggered by UV but not basal phosphorylation. This effect is not due to differential degradation of the phospho-CTD isoforms and can be reproduced, at the AS level, by overexpression of a kinase-dead GSK-3 dominant negative mutant. GSK-3 inhibition abrogates both the reduction in RNAPII elongation and changes in AS elicited by UV. We show that GSK-3 phosphorylates the CTD in vitro, but preferentially when the substrate is previously phosphorylated, consistently with the requirement of a priming phosphorylation reported for GSK-3 efficacy. In line with a role for GSK-3 in the response to DNA damage, GSK-3 inhibition prevents UV-induced apoptosis. In summary, we uncover a novel role for a widely studied kinase in key steps of eukaryotic transcription and pre-mRNA processing.


Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas Quinases/metabolismo , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Processamento Alternativo/genética , Processamento Alternativo/efeitos da radiação , Apoptose/efeitos da radiação , Dano ao DNA/efeitos da radiação , Fluorescência , Genes Dominantes , Genes Reporter , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/genética , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fosforilação/efeitos da radiação , Proteínas Quinases/genética , Transcrição Genética/efeitos da radiação , Raios Ultravioleta
10.
Mol Genet Genomics ; 295(4): 843-853, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32227305

RESUMO

Genome-wide association studies (GWAS) have revealed that the genetic contribution to certain complex diseases is well-described by Fisher's infinitesimal model in which a vast number of polymorphisms each confer a small effect. Under Fisher's model, variants have additive effects both across loci and within loci. However, the latter assumption is at odds with the common observation of dominant or recessive rare alleles responsible for monogenic disorders. Here, we searched for evidence of non-additive (dominant or recessive) effects for GWAS variants known to confer susceptibility to the highly heritable quantitative trait, refractive error. Of 146 GWAS variants examined in a discovery sample of 228,423 individuals whose refractive error phenotype was inferred from their age-of-onset of spectacle wear, only 8 had even nominal evidence (p < 0.05) of non-additive effects. In a replication sample of 73,577 individuals who underwent direct assessment of refractive error, 1 of these 8 variants had robust independent evidence of non-additive effects (rs7829127 within ZMAT4, p = 4.76E-05) while a further 2 had suggestive evidence (rs35337422 in RD3L, p = 7.21E-03 and rs12193446 in LAMA2, p = 2.57E-02). Accounting for non-additive effects had minimal impact on the accuracy of a polygenic risk score for refractive error (R2 = 6.04% vs. 6.01%). Our findings demonstrate that very few GWAS variants for refractive error show evidence of a departure from an additive mode of action and that accounting for non-additive risk variants offers little scope to improve the accuracy of polygenic risk scores for myopia.


Assuntos
Estudo de Associação Genômica Ampla , Miopia/genética , Característica Quantitativa Herdável , Erros de Refração/genética , Adulto , Idoso , Bancos de Espécimes Biológicos , Feminino , Genes Dominantes/genética , Predisposição Genética para Doença , Variação Genética/genética , Humanos , Laminina/genética , Masculino , Pessoa de Meia-Idade , Herança Multifatorial/genética , Miopia/patologia , Polimorfismo de Nucleotídeo Único/genética , Erros de Refração/patologia
11.
Hum Genet ; 139(8): 1107-1117, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32270270

RESUMO

Extensive studies have been conducted on the analysis of genome function, especially on the expression quantitative trait loci (eQTL). These studies offered promising results for characterization of the functional sequencing variation and understanding of the basic processes of gene regulation. Parent of origin effect (POE) is an important epigenetic phenomenon describing that the expression of certain genes depends on their allelic parent-of-origin and it is known to play important roles in human complex diseases. However, traditional eQTL mapping approaches do not allow for the detection of imprinting, or they focus on modeling the additive genetic effect thereby ignoring the estimation of the dominance genetic effect. In this study, we proposed a statistical framework to test the additive and dominance genetic effects of the candidate eQTLs along with detection of the POE with a functional model and an orthogonal model for RNA-seq data. We demonstrated the desirable power and preserved Type I errors of the methods in most scenarios, especially the orthogonal model with un-biased estimation of the genetic effects and over-dispersion of the RNA-seq data. The application to a HapMap project trio dataset validated existing imprinting genes and discovered two novel imprinting genes with potential dominance genetic effect and RB1 and IGF1R genes. This study provides new insights into the next generation statistical modeling of eQTL mapping for better understanding of the genetic architecture underlying the mechanisms of gene expression regulation.


Assuntos
Regulação da Expressão Gênica/genética , Impressão Genômica/genética , Modelos Genéticos , Modelos Estatísticos , Polimorfismo de Nucleotídeo Único/genética , Locos de Características Quantitativas/genética , Alelos , Criança , Simulação por Computador , Família , Feminino , Genes Dominantes/genética , Genótipo , Projeto HapMap , Humanos , Masculino , Pais , RNA-Seq
12.
Ann Hematol ; 99(4): 715-727, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32112123

RESUMO

Hereditary xerocytosis (HX), also known as dehydrated stomatocytosis (DHSt) is a dominantly inherited genetic disorder exhibiting red cell membrane dehydration caused by the loss of the monovalent cation K+ and water. Variants in mechanosensitive Piezo ionic channels of the PIEZO1 gene are the primary cause of HX. We have utilized high throughput and highly precise next-generation sequencing (NGS) to make a diagnosis and examine the genotype-phenotype relationship in inflexible HX cases. Seven unrelated patients with unexplained hemolytic anemia were scrutinized with a panel probing 8000 genes related to congenital anemia. Targeted next-generation sequencing identified 8 missense variants in the PIEZO1 gene in 7 unrelated Indian patients. Three of the 8 variants are novel (c.1795G > C, c.2915G > A, c.7372 T > C) and the remaining five (c.4082A > G, c.6829C > A, c.7374C > G, c.7381G > A, c.7483_7488dup) are previously reported. The variants have been validated by Sanger sequencing. One patient with autosomal dominant mutation (c.7372 T > C) is associated with iron refractory iron deficiency anemia. Of the 7 patients, one has HX in combination with a novel homozygous variant (c.994G > A) in the PKLR gene causing PK deficiency resulting in severe clinical manifestations with phenotypic variability. In silico prediction using bioinformatics tools were used to study the possible damaging effects of the novel variants. Structural-functional analysis of the novel variants was investigated by molecular modeling software (PyMOL and Swiss PDB). These results encompass the heterogeneous behavior of mechano-sensitive Piezo1 protein observed in HX patients in India. Moreover, NGS imparted a subtle, economical, and quick tool for understanding the genetic cause of undiagnosed cases of congenital hemolytic anemia. NGS grants a potential technology integrating clinical history together with molecular report profiting in such patients and their families.


Assuntos
Anemia Hemolítica Congênita/genética , Hidropisia Fetal/genética , Canais Iônicos/genética , Mutação de Sentido Incorreto , Adolescente , Sequência de Aminoácidos , Anemia Hemolítica Congênita/sangue , Anemia Hemolítica Congênita/complicações , Anemia Hemolítica Congênita/etnologia , Anemia Ferropriva/genética , Animais , Criança , Pré-Escolar , Simulação por Computador , Feminino , Genes Dominantes , Estudos de Associação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hidropisia Fetal/sangue , Hidropisia Fetal/etnologia , Índia , Canais Iônicos/química , Canais Iônicos/fisiologia , Sobrecarga de Ferro/etiologia , Masculino , Camundongos , Modelos Moleculares , Conformação Proteica , Piruvato Quinase/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade
13.
BMC Med Genet ; 21(1): 45, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32122354

RESUMO

BACKGROUND: Charcot-Marie-Tooth disease (CMT) is one of the most commonly inherited neurological disorders. A growing number of genes, involved in glial and neuronal functions, have been associated with different subtypes of CMT leading to improved diagnostics and understanding of pathophysiological mechanisms. However, some patients and families remain genetically unsolved. METHODS: We report on a German family including four affected members over three generations with a CMT phenotype accompanied by cognitive deficits, predominantly with regard to visual abilities and episodic memory. RESULTS: A comprehensive clinical characterization followed by a sequential diagnostic approach disclosed a heterozygous rare SEPT9 missense variant c.1406 T > C, p.(Val469Ala), that segregates with disease. SEPT9 has been linked to various intracellular functions, such as cytokinesis and membrane trafficking. Interestingly, SEPT9-mutations are known to cause hereditary neuralgic amyotrophy (HNA), a recurrent focal peripheral neuropathy. CONCLUSION: We, for the first time, present a SEPT9 variant associated to a CMT phenotype and suggest SEPT9 as new sufficient candidate gene in CMT.


Assuntos
Doença de Charcot-Marie-Tooth/genética , Polimorfismo de Nucleotídeo Único , Septinas/genética , Adulto , Alanina/genética , Substituição de Aminoácidos/genética , Doença de Charcot-Marie-Tooth/diagnóstico , Família , Feminino , Frequência do Gene , Genes Dominantes , Alemanha , Humanos , Imagem por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Linhagem , Valina/genética , Adulto Jovem
14.
Hum Genet ; 139(4): 531-543, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32030560

RESUMO

We present a comprehensive clinically oriented workflow for large-insert genome sequencing (liGS)-based nucleotide level resolution and interpretation of de novo (dn) apparently balanced chromosomal abnormalities (BCA) in prenatal diagnosis (PND). Retrospective or concomitant with conventional PND and liGS, molecular and newly developed clinically inspired bioinformatic tools (TAD-GConTool and CNV-ConTool) are applied to analyze and assess the functional and phenotypic outcome of dn structural variants (dnSVs). Retrospective analysis of four phenotype-associated dnSVs identified during conventional PND precisely reveal the genomic elements disrupted by the translocation breakpoints. Identification of autosomal dominant disease due to the disruption of ANKS1B and WDR26 by t(12;17)(q23.1;q21.33)dn and t(1;3)(q24.11;p25.3)dn breakpoints, respectively, substantiated the proposed workflow. We then applied this workflow to two ongoing prenatal cases with apparently balanced dnBCAs: 46,XX,t(16;17)(q24;q21.3)dn referred for increased risk on combined first trimester screening and 46,XY,t(2;19)(p13;q13.1)dn referred due to a previous trisomy 21 pregnancy. Translocation breakpoints in the t(16;17) involve ANKRD11 and WNT3 and disruption of ANKRD11 resulted in KBG syndrome confirmed in postnatal follow-up. Breakpoints in the t(2;19) are within ATP6V1B1 and the 3' UTR of CEP89, and are not interpreted to cause disease. Genotype-phenotype correlation confirms the causative role of WDR26 in the Skraban-Deardorff and 1q41q42 microdeletion phenocopy syndromes, and that disruption of ANKS1B causes ANKS1B haploinsufficiency syndrome. In sum, we show that an liGS-based approach can be realized in PND care providing additional information concerning clinical outcomes of dnBCAs in patients with such rearrangements.


Assuntos
Anormalidades Múltiplas , Doenças do Desenvolvimento Ósseo , Transtornos Cromossômicos , Cromossomos Humanos/genética , Facies , Genes Dominantes , Deficiência Intelectual , Diagnóstico Pré-Natal , Anormalidades Dentárias , Translocação Genética , Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/genética , Adolescente , Adulto , Doenças do Desenvolvimento Ósseo/diagnóstico , Doenças do Desenvolvimento Ósseo/genética , Transtornos Cromossômicos/diagnóstico , Transtornos Cromossômicos/genética , Feminino , Humanos , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , Masculino , Gravidez , Anormalidades Dentárias/diagnóstico , Anormalidades Dentárias/genética , Fluxo de Trabalho
16.
Am J Pathol ; 190(3): 554-562, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31953038

RESUMO

BCL-2-associated athanogene 3 (BAG3) is a co-chaperone to heat shock proteins important in degrading misfolded proteins through chaperone-assisted selective autophagy. The recurrent dominant BAG3-P209L mutation results in a severe childhood-onset myofibrillar myopathy (MFM) associated with progressive muscle weakness, cardiomyopathy, and respiratory failure. Because a homozygous knock-in (KI) strain for the mP215L mutation homologous to the human P209L mutation did not have a gross phenotype, compound heterozygote knockout (KO) and KI mP215L mice were generated to establish whether further reduction in BAG3 expression would lead to a phenotype. The KI/KO mice have a significant decrease in voluntary movement compared with wild-type and KI/KI mice in the open field starting at 7 months. The KI/KI and KI/KO mice both have significantly smaller muscle fiber cross-sectional area. However, only the KI/KO mice have clear skeletal muscle histologic changes in MFM. As in patient muscle, there are increased levels of BAG3-interacting proteins, such as p62, heat shock protein B8, and αB-crystallin. The KI/KO mP215L strain is the first murine model of BAG3 myopathy that resembles the human skeletal muscle pathologic features. The results support the hypothesis that the pathologic development of MFM requires a significant decrease in BAG3 protein level and not only a gain of function caused by the dominant missense mutation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/genética , Miopatias Congênitas Estruturais/patologia , Animais , Cardiomiopatias/genética , Cardiomiopatias/patologia , Modelos Animais de Doenças , Genes Dominantes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Fibras Musculares Esqueléticas/patologia , Músculo Esquelético/patologia , Mutação , Miopatias Congênitas Estruturais/genética , Fenótipo
17.
Am J Cardiol ; 125(6): 880-886, 2020 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-31932084

RESUMO

Autosomal dominant hypercholesterolemia results from mutations affecting the low-density lipoprotein receptor pathway, including proprotein convertase subtilisin/kexin type 9 (PCSK9) gain-of-function mutations (GoFm) and apolipoprotein B (APOB) loss-of-function mutations (LoFm). This study examined the long-term efficacy and safety of alirocumab in patients with PCSK9 GoFm and APOB LoFm who participated in the open-label extension to a Phase 2 double-blind study (NCT01604824). Of the 23 patients who completed the 14-week double-blind period and 8-week follow-up, 21 opted to continue in the open-label extension (PCSK9 GoFm, n = 15; APOB LoFm, n = 6). Patients received alirocumab 150 mg every 2 weeks from week 32 up to 3 years for PCSK9 GoFm and 2 years for APOB LoFm. Mean duration of alirocumab exposure was 129 weeks (median: 144 weeks). After initiation of alirocumab treatment, low-density lipoprotein cholesterol (LDL-C) decreased in both groups. At week 80, mean percent reduction in LDL-C from baseline was 58.0% and 47.1% for PCSK9 GoFm and APOB LoFm groups, respectively. Treatment-emergent adverse events were reported in 19 patients (90.5%); no patients discontinued treatment due to treatment-emergent adverse events. In patients with autosomal dominant hypercholesterolemia and elevated LDL-C levels despite receiving maximally tolerated lipid-lowering therapies, alirocumab 150 mg every 2 weeks resulted in clinically meaningful reductions in LDL-C, sustained through to 3 years and 2 years for patients with PCSK9 GoFm and APOB LoFm, respectively. Alirocumab was generally well tolerated with no unexpected safety concerns.


Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Apolipoproteína B-100/genética , Mutação com Ganho de Função/genética , Hiperlipoproteinemia Tipo II/tratamento farmacológico , Hiperlipoproteinemia Tipo II/genética , Mutação com Perda de Função/genética , Pró-Proteína Convertase 9/genética , Adulto , Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Monoclonais Humanizados/farmacocinética , Aberrações Cromossômicas , Método Duplo-Cego , Feminino , Genes Dominantes , Genótipo , Humanos , Hiperlipoproteinemia Tipo II/sangue , Assistência de Longa Duração , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento
18.
J Hum Genet ; 65(4): 397-410, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31911611

RESUMO

Molecular diagnosis of rare inherited palmoplantar keratoderma (PPK) is still challenging. We investigated at the clinical and genetic level a consanguineous Tunisian family presenting an autosomal dominant atypical form of transgrediens and progrediens PPK to better characterize this ultrarare disease and to identify its molecular etiology. Whole-exome sequencing (WES), filtering strategies, and bioinformatics analysis have been achieved. Clinical investigation and follow up over 13 years of this Tunisian family with three siblings formerly diagnosed as an autosomal recessive form of Mal de Melela-like conducted us to reconsider its initial phenotype. Indeed, the three patients presented clinical features that overlap both Mal de Meleda and progressive symmetric erythrokeratoderma (PSEK). The mode of inheritance was also reconsidered, since the mother, initially classified as unaffected, exhibited a similar expression of the disease. WES analysis showed the absence of potentially functional rare variants in known PPKs or PSEK-related genes. Results revealed a novel heterozygous nonsynonymous variant in cadherin-12 gene (CDH12, NM_004061, c.1655C > A, p.Thr552Asn) in all affected family members. This variant is absent in dbSNP and in 50 in-house control exomes. In addition, in silico analysis of the mutated 3D domain structure predicted that this variant would result in cadherin-12 protein destabilization and thermal instability. Functional annotation and biological network construction data provide further supporting evidence for the potential role of CDH12 in the maintenance of skin integrity. Taken together, these results suggest that CDH12 gene is a potential candidate gene for an atypical presentation of an autosomal dominant form of transgrediens and progrediens PPK.


Assuntos
Caderinas , Transtornos Cromossômicos , Eritroceratodermia Variável , Genes Dominantes , Mutação de Sentido Incorreto , Adulto , Idoso , Caderinas/química , Caderinas/genética , Transtornos Cromossômicos/genética , Transtornos Cromossômicos/patologia , Simulação por Computador , Eritroceratodermia Variável/genética , Eritroceratodermia Variável/patologia , Feminino , Humanos , Masculino , Domínios Proteicos , Pele/patologia , Sequenciamento Completo do Exoma
19.
J Virol ; 94(7)2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-31915278

RESUMO

One step of the life cycle common to all rotaviruses (RV) studied so far is the formation of viroplasms, membrane-less cytosolic inclusions providing a microenvironment for early morphogenesis and RNA replication. Viroplasm-like structures (VLS) are simplified viroplasm models consisting of complexes of nonstructural protein 5 (NSP5) with the RV core shell VP2 or NSP2. We identified and characterized the domains required for NSP5-VP2 interaction and VLS formation. VP2 mutations L124A, V865A, and I878A impaired both NSP5 hyperphosphorylation and NSP5/VP2 VLS formation. Moreover, NSP5-VP2 interaction does not depend on NSP5 hyperphosphorylation. The NSP5 tail region is required for VP2 interaction. Notably, VP2 L124A expression acts as a dominant-negative element by disrupting the formation of either VLS or viroplasms and blocking RNA synthesis. In silico analyses revealed that VP2 L124, V865, and I878 are conserved among RV species A to H. Detailed knowledge of the protein interaction interface required for viroplasm formation may facilitate the design of broad-spectrum antivirals to block RV replication.IMPORTANCE Alternative treatments to combat rotavirus infection are a requirement for susceptible communities where vaccines cannot be applied. This demand is urgent for newborn infants, immunocompromised patients, adults traveling to high-risk regions, and even for the livestock industry. Aside from structural and physiological divergences among RV species studied before now, all replicate within cytosolic inclusions termed viroplasms. These inclusions are composed of viral and cellular proteins and viral RNA. Viroplasm-like structures (VLS), composed of RV protein NSP5 with either NSP2 or VP2, are models for investigating viroplasms. In this study, we identified a conserved amino acid in the VP2 protein, L124, necessary for its interaction with NSP5 and the formation of both VLSs and viroplasms. As RV vaccines cover a narrow range of viral strains, the identification of VP2 L124 residue lays the foundations for the design of drugs that specifically block NSP5-VP2 interaction as a broad-spectrum RV antiviral.


Assuntos
Proteínas do Capsídeo/química , Citosol/virologia , Rotavirus/fisiologia , Proteínas não Estruturais Virais/química , Proteínas Virais/química , Animais , Proteínas do Capsídeo/genética , Chlorocebus aethiops , Simulação por Computador , Genes Dominantes , Cobaias , Células HEK293 , Humanos , Macaca mulatta , Camundongos , Mutação , Fosforilação , Ligação Proteica , Domínios Proteicos , RNA Viral/biossíntese , Proteínas não Estruturais Virais/genética , Proteínas Virais/genética , Replicação Viral
20.
FASEB J ; 34(2): 2105-2125, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31908021

RESUMO

How receptor tyrosine kinase (RTK) growth signaling is controlled physiologically is incompletely understood. We have previously provided evidence that the survival and mitotic activities of vascular endothelial cell growth factor receptor-2 (VEGFR2) signaling are dependent on C3a/C5a receptor (C3ar1/C5ar1) and IL-6 receptor (IL-6R)-gp130 joint signaling in a physically interactive platform. Herein, we document that the platelet derived and epidermal growth factor receptors (PDGFR and EGFR) are regulated by the same interconnection and clarify the mechanism underlying the dependence. We show that the joint signaling is required to overcome dominant restraint on RTK function by the combined repression of tonically activated PHLPP, SOCS1/SOCS3, and CK2/Fyn dependent PTEN. Signaling studies showed that augmented PI-3KÉ£ activation is the process that overcomes the multilevel growth restraint. Live-cell flow cytometry and single-particle tracking indicated that blockade of C3ar1/C5ar1 or IL-6R signaling suppresses RTK growth factor binding and RTK complex formation. C3ar1/C5ar1 blockade abrogated growth signaling of four additional RTKs. Active relief of dominant growth repression via joint C3ar1/C5ar1 and IL-6R joint signaling thus enables RTK mitotic/survival signaling.


Assuntos
Células Endoteliais/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Receptor da Anafilatoxina C5a/metabolismo , Receptores de Complemento/metabolismo , Receptores de Interleucina-6/metabolismo , Transdução de Sinais , Proteína 1 Supressora da Sinalização de Citocina/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Linhagem Celular , Células Endoteliais/citologia , Genes Dominantes , Camundongos , Camundongos Knockout , PTEN Fosfo-Hidrolase/genética , Fosfoproteínas Fosfatases/genética , Receptor da Anafilatoxina C5a/genética , Receptores de Complemento/genética , Receptores de Interleucina-6/genética , Proteína 1 Supressora da Sinalização de Citocina/genética , Proteína 3 Supressora da Sinalização de Citocinas/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética
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