Rationale: Familial recurrence studies provide strong evidence for a genetic component to the predisposition to sporadic, non-syndromic Tetralogy of Fallot (TOF), the most common cyanotic congenital heart disease (CHD) phenotype. Rare genetic variants have been identified as important contributors to the risk of CHD, but relatively small numbers of TOF cases have been studied to date. Objective: We used whole exome sequencing (WES) to assess the prevalence of unique, deleterious variants in the largest cohort of non-syndromic TOF patients reported to date. Methods and Results: 829 TOF patients underwent WES. The presence of unique, deleterious variants was determined; defined by their absence in the Genome Aggregation Database (gnomAD) and a scaled combined annotation-dependent depletion (CADD) score of ≥20. The clustering of variants in two genes, NOTCH1 and FLT4, surpassed thresholds for genome-wide significance (assigned as P<5x10-8) after correction for multiple comparisons. NOTCH1 was most frequently found to harbour unique, deleterious variants. 31 changes were observed in 37 probands (4.5%; 95% confidence interval [CI]:3.2-6.1%) and included seven loss-of-function variants 22 missense variants and two in-frame indels. Sanger-sequencing of the unaffected parents of seven cases identified five de novo variants. Three NOTCH1 variants (p.G200R, p.C607Y and p.N1875S) were subjected to functional evaluation and two showed a reduction in Jagged1-induced NOTCH signalling. FLT4 variants were found in 2.4% (95% CI:1.6-3.8%) of TOF patients, with 21 patients harbouring 22 unique, deleterious variants. The variants identified were distinct to those that cause the congenital lymphoedema syndrome Milroy Disease. In addition to NOTCH1, FLT4 and the well-established TOF gene, TBX1, we identified potential association with variants in several other candidates including RYR1, ZFPM1, CAMTA2, DLX6 and PCM1. Conclusions: The NOTCH1 locus is the most frequent site of genetic variants predisposing to nonsyndromic TOF, followed by FLT4. Together, variants in these genes are found in almost 7% of TOF patients.
Now that the mouse and human genome sequences are complete, biologists need systematic approaches to determine the function of each gene. A powerful way to discover gene function is to determine the consequence of mutations in living organisms. Large-scale production of mouse mutations with the point mutagen N-ethyl-N-nitrosourea (ENU) is a key strategy for analysing the human genome because mouse mutants will reveal functions unique to mammals, and many may model human diseases. To examine genes conserved between human and mouse, we performed a recessive ENU mutagenesis screen that uses a balancer chromosome, inversion chromosome 11 (refs 4, 5). Initially identified in the fruitfly, balancer chromosomes are valuable genetic tools that allow the easy isolation of mutations on selected chromosomes. Here we show the isolation of 230 new recessive mouse mutations, 88 of which are on chromosome 11. This genetic strategy efficiently generates and maps mutations on a single chromosome, even as mutations throughout the genome are discovered. The mutations reveal new defects in haematopoiesis, craniofacial and cardiovascular development, and fertility.
The FKBP-12-rapamycin associated protein (FRAP, also known as mTOR and RAFT-1) is a member of the phosphoinositide kinase related kinase family. FRAP has serine͞threonine kinase activity and mediates the cellular response to mitogens through signaling to p70s6 kinase (p70 s6k ) and 4E-BP1, resulting in an increase in translation of subsets of cellular mRNAs. Translational up-regulation is blocked by inactivation of FRAP signaling by rapamycin, resulting in G 1 cell cycle arrest. Rapamycin is used as an immunosuppressant for kidney transplants and is currently under investigation as an antiproliferative agent in tumors because of its ability to block FRAP activity. Although the role of FRAP has been extensively studied in vitro, characterization of mammalian FRAP function in vivo has been limited to the immune system and tumor models. Here we report the identification of a loss-of-function mutation in the mouse FRAP gene, which illustrates a requirement for FRAP activity in embryonic development. Our studies also determined that rapamycin treatment of the early embryo results in a phenotype indistinguishable from the FRAP mutant, demonstrating that rapamycin has teratogenic activity.
Mutations of the orphan nuclear receptors, steroidogenic factor 1 (SF-1) and DAX-1, cause complex endocrine phenotypes that include impaired adrenal development and hypogonadotrophic hypogonadism. These similar phenotypes suggest that SF-1 and DAX-1 act in the same pathway(s) of endocrine development. To explore this model, we now compare directly their sites of expression. In mouse embryos, SF-1 expression in the urogenital ridge and brain either preceded or coincided with Dax-1 expression, with coordinate expression thereafter in the adrenal cortex, testis, ovary, hypothalamus, and anterior pituitary. The striking colocalization of SF-1 and Dax-1 supports the model that they are intimately linked in a common pathway of endocrine development. The slightly earlier onset of SF-1 expression and its ability to bind specifically to a conserved sequence in the Dax-1 5'-flanking region suggested that SF-1 may activate Dax-1 expression. However, promoter activity of Dax-1 5'-flanking sequences did not require this potential SF-1-responsive element, and Dax-1 expression was unimpaired in knockout mice lacking SF-1, establishing that SF-1 is not required for Dax-1 gene expression in these settings. Although the precise mechanisms remain to be established and may be multifactorial, our results strongly suggest that these two orphan nuclear receptors interact in a common pathway of endocrine development.
A greater understanding of the causes of human disease can come from identifying characteristics that are specific to disease genes. However, a full understanding of the contribution of essential genes to human disease is lacking, due to the premise that these genes tend to cause developmental abnormalities rather than adult disease. We tested the hypothesis that human orthologs of mouse essential genes are associated with a variety of human diseases, rather than only those related to miscarriage and birth defects. We segregated human disease genes according to whether the knockout phenotype of their mouse ortholog was lethal or viable, defining those with orthologs producing lethal knockouts as essential disease genes. We show that the human orthologs of mouse essential genes are associated with a wide spectrum of diseases affecting diverse physiological systems. Notably, human disease genes with essential mouse orthologs are over-represented among disease genes associated with cancer, suggesting links between adult cellular abnormalities and developmental functions. The proteins encoded by essential genes are highly connected in protein-protein interaction networks, which we find correlates with an over-representation of nuclear proteins amongst essential disease genes. Disease genes associated with essential orthologs also are more likely than those with non-essential orthologs to contribute to disease through an autosomal dominant inheritance pattern, suggesting that these diseases may actually result from semi-dominant mutant alleles. Overall, we have described attributes found in disease genes according to the essentiality status of their mouse orthologs. These findings demonstrate that disease genes do occupy highly connected positions in protein-protein interaction networks, and that due to the complexity of disease-associated alleles, essential genes cannot be ignored as candidates for causing diverse human diseases.
Background CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as “two-donor floxing” method). Results We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. Conclusion We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis , an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models. Electronic supplementary material The online version of this article (10.1186/s13059-019-1776-2) contains supplementary material, which is available to authorized users.
Retroviral insertions that activate proto-oncogenes are a primary cause of tumors in certain strains of mice. The AKXD recombinant inbred mice are predisposed to a variety of leukemias and lymphomas as a result of viral integration. One common insertion site, the ecotropic viral insertion site 3 (Evi3), has been implicated in most B-cell tumors in the AKXD-27 strain. The Evi3 gene encodes a zinc-finger protein with sequence similarity to the Early B-cell Factor-Associated Zinc-finger gene (EBFAZ). We show that the Evi3 gene is overexpressed in several tumors with viral insertions at Evi3, which results in the upregulation of Early B-cell Factor (EBF)-target gene expression, suggesting that Evi3 modulates EBF activity. Reconstitution of primary leukemia cells showed that these tumors express high densities of the Bcell surface proteins CD19 and CD38, which are EBF targets. Using a transactivation assay, we show that the terminal six zinc-fingers of Evi3 are required for modification of EBF activity. This is the first evidence that Evi3 expression in tumors alters the level of EBF target genes, and the first characterization of the Evi3 protein domains required for modulation of EBF activity. Further, these data imply that Evi3 misexpression initiates tumorigenesis by perturbing B-cell development via an interaction with EBF.
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