RESUMO
Site-specific gene addition can allow stable transgene expression for gene therapy. When possible, this is preferred over the use of promiscuously integrating vectors, which are sometimes associated with clonal expansion and oncogenesis. Site-specific endonucleases that can induce high rates of targeted genome editing are finding increasing applications in biological discovery and gene therapy. However, two safety concerns persist: endonuclease-associated adverse effects, both on-target and off-target; and oncogene activation caused by promoter integration, even without nucleases. Here we perform recombinant adeno-associated virus (rAAV)-mediated promoterless gene targeting without nucleases and demonstrate amelioration of the bleeding diathesis in haemophilia B mice. In particular, we target a promoterless human coagulation factor IX (F9) gene to the liver-expressed mouse albumin (Alb) locus. F9 is targeted, along with a preceding 2A-peptide coding sequence, to be integrated just upstream to the Alb stop codon. While F9 is fused to Alb at the DNA and RNA levels, two separate proteins are synthesized by way of ribosomal skipping. Thus, F9 expression is linked to robust hepatic albumin expression without disrupting it. We injected an AAV8-F9 vector into neonatal and adult mice and achieved on-target integration into â¼0.5% of the albumin alleles in hepatocytes. We established that F9 was produced only from on-target integration, and ribosomal skipping was highly efficient. Stable F9 plasma levels at 7-20% of normal were obtained, and treated F9-deficient mice had normal coagulation times. In conclusion, transgene integration as a 2A-fusion to a highly expressed endogenous gene may obviate the requirement for nucleases and/or vector-borne promoters. This method may allow for safe and efficacious gene targeting in both infants and adults by greatly diminishing off-target effects while still providing therapeutic levels of expression from integration.
Assuntos
Fator IX/genética , Fator IX/metabolismo , Marcação de Genes/métodos , Hemofilia B/genética , Alelos , Animais , Códon de Terminação/genética , Dependovirus/genética , Dependovirus/fisiologia , Modelos Animais de Doenças , Endonucleases , Feminino , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Ribossomos/metabolismo , Albumina Sérica/genética , Transgenes/genéticaRESUMO
AIMS AND BACKGROUND: Mutations in the TARDBP gene, which encodes the TAR DNA binding protein (TDP-43), have been described in individuals with familial and sporadic amyotrophic lateral sclerosis (ALS). We screened the TARDBP gene in 285 French sporadic ALS patients to assess the frequency of TARDBP mutations in ALS. RESULTS: Six individuals had potentially deleterious mutations of which three were novel including a Y374X truncating mutation and P363A and A382P missense mutations. This suggests that TARDBP mutations may predispose to ALS in approximately 2% of the individuals followed in this study. CONCLUSION: Our findings, combined with those from other collections, brings the total number of mutations in unrelated ALS patients to 17, further suggesting that mutations in the TARDBP gene have an important role in the pathogenesis of ALS.
Assuntos
Esclerose Lateral Amiotrófica/genética , Proteínas de Ligação a DNA/genética , Mutação , Esclerose Lateral Amiotrófica/metabolismo , Sequência de Bases , Estudos de Coortes , Análise Mutacional de DNA , Proteínas de Ligação a DNA/metabolismo , Feminino , Frequência do Gene , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência MolecularRESUMO
Mice in which lung epithelial cells can be induced to express an oncogenic Kras(G12D) develop lung adenocarcinomas in a manner analogous to humans. A myriad of genetic changes accompany lung adenocarcinomas, many of which are poorly understood. To get a comprehensive understanding of both the transcriptional and post-transcriptional changes that accompany lung adenocarcinomas, we took an omics approach in profiling both the coding genes and the non-coding small RNAs in an induced mouse model of lung adenocarcinoma. RNAseq transcriptome analysis of Kras(G12D) tumors from F1 hybrid mice revealed features specific to tumor samples. This includes the repression of a network of GTPase-related genes (Prkg1, Gnao1 and Rgs9) in tumor samples and an enrichment of Apobec1-mediated cytosine to uridine RNA editing. Furthermore, analysis of known single-nucleotide polymorphisms revealed not only a change in expression of Cd22 but also that its expression became allele specific in tumors. The most salient finding, however, came from small RNA sequencing of the tumor samples, which revealed that a cluster of â¼53 microRNAs and mRNAs at the Dlk1-Dio3 locus on mouse chromosome 12qF1 was markedly and consistently increased in tumors. Activation of this locus occurred specifically in sorted tumor-originating cancer cells. Interestingly, the 12qF1 RNAs were repressed in cultured Kras(G12D) tumor cells but reactivated when transplanted in vivo. These microRNAs have been implicated in stem cell pleuripotency and proteins targeted by these microRNAs are involved in key pathways in cancer as well as embryogenesis. Taken together, our results strongly imply that these microRNAs represent key targets in unraveling the mechanism of lung oncogenesis.
Assuntos
Adenocarcinoma/metabolismo , Regulação Neoplásica da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Iodeto Peroxidase/genética , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/genética , MicroRNAs/metabolismo , Alelos , Animais , Proteínas de Ligação ao Cálcio , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Mapeamento Cromossômico , Feminino , Perfilação da Expressão Gênica , Genes ras/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Iodeto Peroxidase/metabolismo , Perda de Heterozigosidade , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Família Multigênica , Mutação , Polimorfismo de Nucleotídeo Único , RNA/metabolismo , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Regulação para CimaAssuntos
Ataxia/genética , Mapeamento Cromossômico/métodos , Cromossomos Humanos Par 8/genética , Doenças Neurodegenerativas/genética , Tratos Piramidais/patologia , Adulto , Idoso , Ataxia/diagnóstico , Diagnóstico Diferencial , Feminino , Genes Dominantes/genética , Ligação Genética/genética , Humanos , Escore Lod , Masculino , Pessoa de Meia-Idade , Doenças Neurodegenerativas/diagnóstico , Linhagem , Degenerações Espinocerebelares/diagnósticoRESUMO
BACKGROUND: The identification of mutations in the TARDBP and more recently the identification of mutations in the FUS gene as the cause of amyotrophic lateral sclerosis (ALS) is providing the field with new insight about the mechanisms involved in this severe neurodegenerative disease. METHODS: To extend these recent genetic reports, we screened the entire gene in a cohort of 200 patients with ALS. An additional 285 patients with sporadic ALS were screened for variants in exon 15 for which mutations were previously reported. RESULTS: In total, 3 different mutations were identified in 4 different patients, including 1 3-bp deletion in exon 3 of a patient with sporadic ALS and 2 missense mutations in exon 15 of 1 patient with familial ALS and 2 patients with sporadic ALS. CONCLUSIONS: Our study identified sporadic patients with mutations in the FUS gene. The accumulation and description of different genes and mutations helps to develop a more comprehensive picture of the genetic events underlying amyotrophic lateral sclerosis.
Assuntos
Esclerose Lateral Amiotrófica/genética , Mutação , Proteína FUS de Ligação a RNA/genética , Canadá , Cromossomos Humanos Par 16/genética , Estudos de Coortes , Feminino , Predisposição Genética para Doença , Testes Genéticos , Humanos , MasculinoRESUMO
BACKGROUND: Six candidate gene studies report a genetic association of DNA variants within the paraoxonase locus with sporadic amyotrophic lateral sclerosis (ALS). However, several other large studies, including five genome-wide association studies, have not duplicated this finding. METHODS: We conducted a meta-analysis of 10 published studies and one unpublished study of the paraoxonase locus, encompassing 4,037 ALS cases and 4,609 controls, including genome-wide association data from 2,018 ALS cases and 2,425 controls. RESULTS: The combined fixed effects odds ratio (OR) for rs662 (PON1 Q192R) was 1.09 (95% confidence interval [CI], 1.02-1.16, p = 0.01); the genotypic OR for RR homozygotes at Q192R was 1.25 (95% CI, 1.07-1.45, p = 0.0004); the combined OR for rs854560 (PON1 L55M) was 0.97 (95% CI, 0.86-1.10, p = 0.62); the OR for rs10487132 (PON2) was 1.08 (95% CI, 0.92-1.27, p = 0.35). Although the rs662 polymorphism reached a nominal level of significance, no polymorphism was significant after multiple testing correction. In the subanalysis of samples with genome-wide data from which population outliers were removed, rs662 had an OR of 1.06 (95% CI, 0.97-1.16, p = 0.22). CONCLUSIONS: In contrast to previous positive smaller studies, our genetic meta-analysis showed no significant association of amyotrophic lateral sclerosis (ALS) with the PON locus. This is the largest meta-analysis of a candidate gene in ALS to date and the first ALS meta-analysis to include data from whole genome association studies. The findings reinforce the need for much larger and more collaborative investigations of the genetic determinants of ALS.
Assuntos
Esclerose Lateral Amiotrófica/genética , Arildialquilfosfatase/genética , Predisposição Genética para Doença/genética , Polimorfismo de Nucleotídeo Único/genética , Viés , Mapeamento Cromossômico/métodos , Análise Mutacional de DNA/métodos , Análise Mutacional de DNA/estatística & dados numéricos , Interpretação Estatística de Dados , Marcadores Genéticos/genética , Testes Genéticos/métodos , Testes Genéticos/estatística & dados numéricos , Estudo de Associação Genômica Ampla/métodos , Estudo de Associação Genômica Ampla/estatística & dados numéricos , Genótipo , Humanos , Razão de Chances , Reprodutibilidade dos TestesRESUMO
BACKGROUND: The paraoxonase gene cluster on chromosome 7 comprising the PON1-3 genes is an attractive candidate for association in amyotrophic lateral sclerosis (ALS) given the role of paraoxonase genes during the response to oxidative stress and their contribution to the enzymatic break down of nerve toxins. Oxidative stress is considered one of the mechanisms involved in ALS pathogenesis. Evidence for this includes the fact that mutations of SOD1, which normally reduce the production of toxic superoxide anion, account for 12% to 23% of familial cases in ALS. In addition, PON variants were shown to be associated with susceptibility to ALS in several North American and European populations. METHODS: We extended this analysis to examine 20 single nucleotide polymorphisms (SNPs) across the PON gene cluster in a set of patients from France (480 cases, 475 controls), Quebec (159 cases, 95 controls), and Sweden (558 cases, 506 controls). RESULTS: Although individual SNPs were not considered associated on their own, a haplotype of SNPs at the C-terminal portion of PON2 that includes the PON2 C311S amino acid change was significant in the French (p value 0.0075) and Quebec (p value 0.026) populations as well as all three populations combined (p value 1.69 x 10(-6)). Stratification of the samples showed that this variation was pertinent to ALS susceptibility as a whole, and not to a particular subset of patients. CONCLUSIONS: These findings contribute to the increasing weight of evidence that genetic variants in the paraoxonase gene cluster are associated with amyotrophic lateral sclerosis.
Assuntos
Esclerose Lateral Amiotrófica/genética , Arildialquilfosfatase/genética , Família Multigênica , Polimorfismo de Nucleotídeo Único , Feminino , França , Genótipo , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , Quebeque , SuéciaRESUMO
We present phenotypic and genotypic data for an additional family with autosomal dominant sensory ataxia, a disease characterized by gait difficulties associated with diminished sensation in the limbs and areflexia. The same disease haplotype spanning the entire SNAX1 locus is observed in affected members of this second family, enabling the locus to be reduced to a 7.3-cM interval.