Exclusion of known gene for enamel development in two Brazilian families with amelogenesis imperfecta

Amelogenesis imperfecta (AI) is a genetically heterogeneous group of diseases that result in defective development of tooth enamel. Mutations in several enamel proteins and proteinases have been associated with AI. The object of this study was to evaluate evidence of etiology for the six major candidate gene loci in two Brazilian families with AI. Genomic DNA was obtained from family members and all exons and exon-intron boundaries of the ENAM, AMBN, AMELX, MMP20, KLK4 and Amelotin gene were amplified and sequenced. Each family was also evaluated for linkage to chromosome regions known to contain genes important in enamel development. The present study indicates that the AI in these two families is not caused by any of the known loci for AI or any of the major candidate genes proposed in the literature. These findings indicate extensive genetic heterogeneity for non-syndromic AI

Germ line gain of function with SOS1 mutation in hereditary gingival fibromatosis

Made available in DSpace on 2019-09-12T16:53:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2007Office of Intramural Research (OIR) NIH HHSMutation of human SOS1 is responsible for hereditary gingival fibromatosis type 1, a benign overgrowth condition of the gingiva. Here, we investigated molecular mechanisms responsible for the increased rate of cell proliferation in gingival fibroblasts caused by mutant SOS1 in vitro. Using ectopic expression of wild-type and mutant SOS1 constructs, we found that truncated SOS1 could localize to the plasma membrane, without growth factor stimuli, leading to sustained activation of Ras/MAPK signaling. Additionally, we observed an increase in the magnitude and duration of ERK signaling in hereditary gingival fibromatosis gingival fibroblasts that was associated with phosphorylation of retinoblastoma tumor suppressor protein and the up-regulation of cell cycle regulators, including cyclins C, D, and E and the E2F/DP transcription factors. These factors promote cell cycle progression from G1 to S phase, and their up-regulation may underlie the increased gingival fibroblast proliferation observed. Selective depletion of wild-type and mutant SOS1 through small interfering RNA demonstrates the link between mutation of SOS1, ERK signaling, cell proliferation rate, and the expression levels of Egr-1 and proliferating cell nuclear antigen. These findings elucidate the mechanisms for gingival overgrowth mediated by SOS1 gene mutation in humans.NIH, NIDCR, Sect Human & Craniofacial Genet, Bethesda, MD 20892 USA; NHGRI, NIH, Bethesda, MD 20892 USA; Universidade de Taubaté (Unitau), Dept Dent, Periodont Res & Grad Studies Div, BR-12020 Sao Paulo, Brazi

Multiplexed CRISPR/Cas9-mediated knockout of 19 Fanconi anemia pathway genes in zebrafish revealed their roles in growth, sexual development and fertility.

Fanconi Anemia (FA) is a genomic instability syndrome resulting in aplastic anemia, developmental abnormalities, and predisposition to hematological and other solid organ malignancies. Mutations in genes that encode proteins of the FA pathway fail to orchestrate the repair of DNA damage caused by DNA interstrand crosslinks. Zebrafish harbor homologs for nearly all known FA genes. We used multiplexed CRISPR/Cas9-mediated mutagenesis to generate loss-of-function mutants for 17 FA genes: fanca, fancb, fancc, fancd1/brca2, fancd2, fance, fancf, fancg, fanci, fancj/brip1, fancl, fancm, fancn/palb2, fanco/rad51c, fancp/slx4, fancq/ercc4, fanct/ube2t, and two genes encoding FA-associated proteins: faap100 and faap24. We selected two indel mutations predicted to cause premature truncations for all but two of the genes, and a total of 36 mutant lines were generated for 19 genes. Generating two independent mutant lines for each gene was important to validate their phenotypic consequences. RT-PCR from homozygous mutant fish confirmed the presence of transcripts with indels in all genes. Interestingly, 4 of the indel mutations led to aberrant splicing, which may produce a different protein than predicted from the genomic sequence. Analysis of RNA is thus critical in proper evaluation of the consequences of the mutations introduced in zebrafish genome. We used fluorescent reporter assay, and western blots to confirm loss-of-function for several mutants. Additionally, we developed a DEB treatment assay by evaluating morphological changes in embryos and confirmed that homozygous mutants from all the FA genes that could be tested (11/17), displayed hypersensitivity and thus were indeed null alleles. Our multiplexing strategy helped us to evaluate 11 multiple gene knockout combinations without additional breeding. Homozygous zebrafish for all 19 single and 11 multi-gene knockouts were adult viable, indicating FA genes in zebrafish are generally not essential for early development. None of the mutant fish displayed gross developmental abnormalities except for fancp-/- fish, which were significantly smaller in length than their wildtype clutch mates. Complete female-to-male sex reversal was observed in knockouts for 12/17 FA genes, while partial sex reversal was seen for the other five gene knockouts. All adult females were fertile, and among the adult males, all were fertile except for the fancd1 mutants and one of the fancj mutants. We report here generation and characterization of zebrafish knockout mutants for 17 FA disease-causing genes, providing an integral resource for understanding the pathophysiology associated with the disrupted FA pathway

Common and overlapping oncogenic pathways contribute to the evolution of acute myeloid leukemias

In this report we demonstrate that the ability to alter self-renewal in vitro and in vivo is a more generalized property of leukemia-associated oncogenes. We further demonstrate that disparate leukemia-associated oncogenes initiate early common and overlapping transformation and self-renewal gene expression programs to mediate these effects. Furthermore, elements of these programs can be detected in established leukemia stem cells from an animal model and across a large cohort of patients with differing acute myeloid leukemia (AML) subtypes, where they strongly predict for disease biology. Finally, individual genes from the programs are demonstrated to partially phenocopy the leukemia-associated oncogenes and themselves alter self-renewal in committed murine progenitors and generate AML when expressed in murine bone marrow. A total of 253 RNA samples derived from adult AML patients were provided by the German Austrian AML Study Group (AMLSG) [AMLSG trials AML HD98A (ClinicalTrials.gov Identifier: NCT00146120), and AML HD98B (Schlenk et al., 2009)]. Conventional cytogenetic banding, and FLT3, CEBPA and NPM1 mutational analysis were performed as previously described (Schlenk et al., N Engl J Med 2008). Detailed clinical, cytogenetic and molecular cytogenetic information are also provided in Table S3 along with the publication. Following enrichment, all samples contained at least 80% leukemic cells. Gene expression profiling (GEP) was performed as previously described (Bullinger et al., N Engl J Med 2004). Separate filtering and batch centering were performed for the group of 253 Samples (relative to GSE16432). Filtered data presented as a supplementary file at the foot of this record

Common and overlapping oncogenic pathways contribute to the evolution of acute myeloid leukemias

In this report we demonstrate that the ability to alter self-renewal in vitro and in vivo is a more generalized property of leukemia-associated oncogenes. We further demonstrate that disparate leukemia-associated oncogenes initiate early common and overlapping transformation and self-renewal gene expression programs to mediate these effects. Furthermore, elements of these programs can be detected in established leukemia stem cells from an animal model and across a large cohort of patients with differing acute myeloid leukemia (AML) subtypes, where they strongly predict for disease biology. Finally, individual genes from the programs are demonstrated to partially phenocopy the leukemia-associated oncogenes and themselves alter self-renewal in committed murine progenitors and generate AML when expressed in murine bone marrow. A total of 253 RNA samples derived from adult AML patients were provided by the German Austrian AML Study Group (AMLSG) [AMLSG trials AML HD98A (ClinicalTrials.gov Identifier: NCT00146120), and AML HD98B (Schlenk et al., 2009)]. Conventional cytogenetic banding, and FLT3, CEBPA and NPM1 mutational analysis were performed as previously described (Schlenk et al., N Engl J Med 2008). Detailed clinical, cytogenetic and molecular cytogenetic information are also provided in Table S3 along with the publication. Following enrichment, all samples contained at least 80% leukemic cells. Gene expression profiling (GEP) was performed as previously described (Bullinger et al., N Engl J Med 2004). Separate filtering and batch centering were performed for the group of 253 Samples (relative to GSE16432). Filtered data presented as a supplementary file at the foot of this record

Common and Overlapping Oncogenic Pathways Contribute to the Evolution of Acute Myeloid Leukemias

Fusion oncogenes in acute myeloid leukemia (AML) promote self-renewal from committed progenitors, thereby linking transformation and self-renewal pathways. Like most cancers, AML is a genetically and biologically heterogeneous disease, but it is unclear whether transformation results from common or overlapping genetic programs acting downstream of multiple mutations or by the engagement of unique genetic programs acting cooperatively downstream of individual mutations. This distinction is important, because the involvement of common programs would imply the existence of common molecular targets to treat AML, no matter which oncogenes are involved. Here we show that the ability to promote self-renewal is a generalized property of leukemia-associated oncogenes. Disparate oncogenes initiated overlapping transformation and self-renewal gene expression programs, the common elements of which were defined in established leukemic stem cells from an animal model as well as from a large cohort of patients with differing AML subtypes, where they strongly predicted pathobiological character. Notably, individual genes commonly activated in these programs could partially phenocopy the self-renewal function of leukemia-associated oncogenes in committed murine progenitors. Furthermore, they could generate AML following expression in murine bone marrow. In summary, our findings reveal the operation of common programs of self-renewal and transformation downstream of leukemia-associated oncogenes, suggesting that mechanistically common therapeutic approaches to AML are likely to be possible, regardless of the identity of the driver oncogene involved. Cancer Res; 71(12); 4117-29. (C)2011 AACR