Mendelian gene identification through mouse embryo viability screening

The diagnostic rate of Mendelian disorders in sequencing studies continues to increase, along with the pace of novel disease gene discovery. However, variant interpretation in novel genes not currently associated with disease is particularly challenging and strategies combining gene functional evidence with approaches that evaluate the phenotypic similarities between patients and model organisms have proven successful. A full spectrum of intolerance to loss-of-function variation has been previously described, providing evidence that gene essentiality should not be considered as a simple and fixed binary property. Here we further dissected this spectrum by assessing the embryonic stage at which homozygous loss-of-function results in lethality in mice from the International Mouse Phenotyping Consortium, classifying the set of lethal genes into one of three windows of lethality: early, mid or late gestation lethal. We studied the correlation between these windows of lethality and various gene features including expression across development, paralogy and constraint metrics together with human disease phenotypes, and found that the members of the early gestation lethal category show distinctive characteristics and a strong enrichment for genes linked with recessive forms of inherited metabolic disease. Based on these findings, we explored a gene similarity approach for novel gene discovery focused on this subset of lethal genes. Finally, we investigated unsolved cases from the 100,000 Genomes Project recruited under this disease category to look for signs of enrichment of biallelic predicted pathogenic variants among early gestation lethal genes and highlight two novel candidates with phenotypic overlap between the patients and the mouse knockout.

Modeling Colicin Operons as Genetic Arms in Plasmid-genome Conflicts

Plasmids are acellular propagating entities that depend on bacteria, as molecular parasites, for propagation. A 'tussle' between bacteria and plasmid ensues; bacteria for riddance of the plasmid and plasmid for persistence within a live host. The conflict between bacterial genome and plasmids allows the emergence of 'genetic arms' such as colicin (Col) operons. Endonuclease Col operons encode three proteins; an endonuclease colicin, an immunity protein, and lysis protein. Col operons are plasmid-maintenance systems; (i) the plasmid-cured cells are killed by the colicins; (ii) damaged cells lyse and release the colicins that eliminate the competitors, and (iii) the released plasmids invade new bacteria. Surprisingly, some bacterial genomes have Col operons. The eco-evolutionary drive and physiological relevance of genomic Col operons are unknown. We investigated plasmidic and genomic Col operons using sequence analyses from an eco-evolutionary perspective. We found 1,248 genomic and plasmidic colicins across 30 bacterial genera. Although 51% of the genomes harbor colicins, the majority of the genomic colicins lacked a functional lysis gene, suggesting the negative selection of lethal genes. The immunity gene of the Col operon protects the plasmid-cured host; eliminating the metabolic burden. We show mutual exclusivity of Col operons on genomes and plasmids. We propose an anti-addiction hypothesis for genomic colicins. Using a stochastic agent-based model, we show that the genomic colicins confer an advantage to the host genome in terms of immunity to the toxin and elimination of plasmid burden. Col operons are 'genetic arms' that regulate the ecological interplay of bacterial genomes and plasmids.

Why Recessive Lethal Alleles Have Not Disappeared?

The article derives the probability for lethal recessive alleles in the case of recessive disadvantage or advantage. It is shown that recessive advantage of a lethal gene can be detected by the ratio of heterozygotes and homozygotes. This demonstrates that higher IQ of certain ethnic groups cannot be explained by recessive advantage of lethal genes. The article shows that lethal genes can survive in the population if some lineages of families have much more children than the average.

RNAi-Mediated Screening of Selected Target Genes Against Culex quinquefasciatus (Diptera: Culicidae)

Culex quinquefasciatus, a member of the Culex pipiens complex, is widespread in Saudi Arabia and other parts of the world. It is a vector for lymphatic filariasis, Rift Valley fever, and West Nile virus. Studies have shown the deleterious effect of RNA interference (RNAi)-mediated knockdown of various lethal genes in model and agricultural pest insects. RNAi was proposed as a tool for mosquito control with a focus on Aedes aegypti and Anopheles gambiae. In this study, we examined the effect of RNAi of selected target genes on both larval mortality and adult emergence of Cx. quinquefasciatus through two delivery methods: soaking and nanoparticles. Ten candidate genes were selected for RNAi based on their known lethal effect in other insects. Disruption of three genes, chitin synthase-1, inhibitor of apoptosis 1, and vacuolar adenosine triphosphatase, resulted in the highest mortality among the selected genes using the two treatment methods. Silencing the other seven genes resulted in a medium to low mortality in both assays. These three genes are also active against a wide range of insects and could be used for RNAi-based mosquito control in the future.

The Impact of Diagnostic MRI on the Early Detection of Lethal Genes in Human Genome and to Develop Genomic Medicine to Treat Brain Cancers

Early detection of the onset of diseases are essential if we want to maintain good health. MRI technique provides a three-dimensional image of a microscopic lesion in an organ. Human body is made of 220 different tissues which interact to make an organ and several organs interact to make a human. As a part of medical record, taking MRI of each organ every year and comparing them with each other will identify the appearance of microscopic changes. For example, if you are diagnosed with Brain cancer today, you did not get the cancer yesterday. Abnormal changes are the result of accumulation of harmful mutations over the years predicting the onset of diseases. Once the brain tumor is confirmed, the patient dies within fourteen months. To save the life of the patient, the following three strategies are available and they are Surgery, Radiation and Chemotherapy. This article describes the Chemotherapeutic approach to treat cancers in general and brain cancer, Glioblastoma, in particular. Using rational approach, we designed AZQ (US Patent 4,146,622 & 4,233,215) to treat Glioblastomas. MRI Would identify appearance of microscopic lesions of Glioblastoma and help us start treatment with AZQ long before the disease is confirmed. Using similar rational approach of early diagnosis with MRI, we could design drugs to treat other diseases including cancers.

An efficient i-GONAD method for creating and maintaining lethal mutant mice using an inversion balancer identified from the C3H/HeJJcl strain

As the efficiency of the clustered regularly interspaced short palindromic repeats/Cas system is extremely high, creation and maintenance of homozygous lethal mutants are often difficult. Here, we present an efficient in vivo electroporation method called improved genome editing via oviductal nucleic acid delivery (i-GONAD), wherein one of two alleles in the lethal gene was selectively edited in the presence of a non-targeted B6.C3H-In(6)1J inversion identified from the C3H/HeJJcl strain. This method did not require isolation, culture, transfer, or other in vitro handling of mouse embryos. The edited lethal genes were stably maintained in heterozygotes, as recombination is strongly suppressed within this inversion interval. Using this strategy, we successfully generated the first Tprkb null knockout strain with an embryonic lethal mutation and showed that B6.C3H-In(6)1J can efficiently suppress recombination. As B6.C3H-In(6)1J was tagged with a gene encoding the visible coat color marker, Mitf, the Tprkb mutation could be visually recognized. We listed the stock balancer strains currently available as public bioresources to create these lethal gene knockouts. This method will allow for more efficient experiments for further analysis of lethal mutants.

Why Recessive Lethal Alleles Have Not Disappeared?

The article derives the probability for lethal recessive alleles in the case of recessive disadvantage or advantage. It is shown that the recessive advantage of a lethal gene can be detected by the ratio of heterozygotes and homozygotes. This demonstrates that the higher IQ of certain ethnic groups cannot be explained by the recessive advantage of lethal genes. The article shows that lethal genes can survive in the population if some lineages of families have much more children than the average.

HEREDITARY LOAD IN MEAT AND EGG CHICKENS OF VARIOUS ORIGIN

The aim of the study was to determine the spectrum and frequency of hereditary genetic defects of embryo development in meat and egg hens of different genetic origin, obtained during an experiment to study the effectiveness of crossing roosters of imported meat crosses with meat and egg females of domestic selection. All embryos that died during egg incubation were carefully examined to detect morphopathology. The main research method is pathomorphological analysis of genetic defects of dead embryos. The spectrum and frequency of morphological and anatomical hereditary defects of embryos were established during pathological and anatomical examination of incubation waste. During the autopsy of dead embryos, morphological disorders in the structure of the skeleton were determined, as well as various disproportions of its individual parts. In the meat-egg hens of the original maternal form during three years of monitoring with the highest frequency (2.11–6.06%) revealed such a defect in embryo development as "donald duck". This indicates that the bird is "contaminated" with a "harmful gene" that causes this abnormality. It is worth noting that in groups of chickens that were artificially inseminated with the semen of roosters of meat crosses, this hereditary defect was also recorded. In the descendants of the first and second generations, this anomaly was absent, possibly due to its transition to a heterozygous state. Thus, we can conclude that the allele that expresses this lethal has in its genotype a bird of the local subpopulation "K". Only in meat and egg hens of domestic selection a wide variety of double anomalies was found – four species with a frequency of 1.05–1.12%. Most likely, double abnormalities are the result of homozygosity of dead embryos simultaneously for two lethal genes. Whereas, in chickens of other studied groups, only single anomalies of dead embryos were found, among them "exencephaly", "crossed beak", "shortened beak". Genetic defect of embryos "exencephaly" was found in chickens of most groups. It is obvious that the gene that determines the manifestation of this anomaly is significantly distributed in meat and egg hens of domestic selection and roosters used crosses. And in chickens of the created synthetic population "K-5" only this hereditary defect is revealed. In meat and egg hens of subpopulation "K" (F10) of the original maternal form and in the group of females, which were inseminated with polysperm of meat cocks of the cross "Cobb-500", with a low frequency (0.78–1.12%) a single "crossed beak" anomaly was found among the frozen embryos. This anomaly was not detected in the offspring of the first generation and in groups of chickens inseminated with semen of overripe brood stock. And only in the "cobb" bird of the "K-51" group among the hereditary defects of the dead embryos was this anomaly identified. In meat-egg hens of F10 subpopulation "K" with a low frequency (1.12%) was found a hereditary defect of embryos "shortened beak". Then this anomaly was found only in the "cobb" bird of both the first and second generation. The data show that the hereditary factor that determines this defect, the descendants inherited from the cocks of the cross "Cobb-500". The level of genetic burden in meat and egg hens of the local subpopulation "K", depending on the generation, ranged from 5.26 to 8.99% and was wavy in nature. In the descendants of the first generation it is low – 1.64–4.17%, in the "ross" crossbreeds is much smaller compared to the "cobb". The descendants of the second generation of different origins also had a low hereditary burden – in the range of 4.35–5.88%, which does not exceed the maximum allowable species value of 8.0%. The results of the research showed that crossing meat-egg hens of domestic selection with roosters of imported meat crosses "Cobb-500" and "Ross-308" did not lead to significant "contamination" of genotypes of F1-F2 offspring with lethal genes. This indicates a low share in the heredity of the used offspring of hidden carriers of "defective" genes. This may be due to the high selection of birds of foreign crosses, which does not contribute to the accumulation in its gene pool of lethal genes that cause embryonic developmental abnormalities. In chickens of the created synthetic population "K-5" the level of genetic burden is low (3.03%), which indicates the relative "purity" of this bird from lethal genes.

Modeling colicin operons as genetic arms in plasmid-genome conflicts

Plasmids are acellular propagating entities that depend, as molecular parasites, on bacteria for propagation. The conflict between the bacterial genome and the parasitic plasmids allows the emergence of genetic arms such as Colicin (Col) operons. Endonuclease Col operons encode three proteins; an endonuclease colicin (cleaves nucleic acids), an immunity protein (inactivates its cognate colicin), and lysis protein (aids in colicin release via host cell lysis). Col operons are efficient plasmid-maintenance systems; (i) the plasmid cured cells are killed by the colicins; (ii) damaged cells lyse and releases the colicins that eliminate the competitors; and (iii) the released plasmids invade new bacteria. Surprisingly, some bacterial genomes have Col operons. The eco-evolutionary drive and physiological relevance of genomic Col operons are unknown. We investigated plasmidic and genomic Col operons using sequence analyses from an eco-evolutionary perspective. We found 1,248 genomic and plasmidic colicins across 30 bacterial genera. Although 51% of the genomes harbor colicins, the majority of the genomic colicins lacked a functional lysis gene, suggesting the negative selection of lethal genes. The immunity gene of the Col operon protects the cured host thereby eliminating the metabolic burden due to plasmid. We show mutual exclusivity of col operons on genomes and plasmids. We propose anti-addiction hypothesis for genomic colicins. Using a stochastic agent-based model, we show that the genomic colicins confer an advantage to the host genome in terms of immunity to the toxin and elimination of plasmid burden. Col operons are genetic arms that regulate the ecological interplay of bacterial genomes and plasmids.

CRISPR-cas9 Screening Identified Lethal Genes Enriched in Cell Cycle Pathway and of Prognosis Significance in Breast Cancer

BackgroundLethal genes have not been systematically analyzed in breast cancer which may have significant prognostic value. The current study aims to investigate vital genes related to cell viability by analyzing the CRISPR-cas9 screening data, which may provide novel therapeutic target for patients.MethodsGenes differentially expressed between tumor and normal tissue from the Cancer Genome Atlas (TCGA) and genes related to cell viability by CRISPR-cas9 screening from Depmap (Cancer Dependency Map) were overlapped. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analysis was conducted to identify which pathways of overlapped genes were enriched. GSE21653 set was randomized into training and internal validation dataset at a ratio of 3:1, and external validation was performed in GSE20685 set. The least absolute shrinkage and selection operator (LASSO) regression was used to construct a signature to predict recurrence-free survival (RFS) of breast cancer patients. Univariate and multivariate Cox regression were used to evaluate the prognostic value of this signature. Differentially expressed genes (DEGs) between high-risk and low-risk patients were then analyzed to identify the main pathways regulated by this signature. Weighted correlation network analysis (WGCNA) was conducted to recognize modules correlated with high risk. Enrichment analysis was then used to identify pathways regulated by genes shared in the overlapped genes, DEGs, and WGCNA.ResultsA total of 86 oncogenes were upregulated in TCGA database and overlapped with lethal genes in Depmap database, which were enriched in cell cycle pathway. A total of 51 genes were included in the gene signature based on LASSO regression, and the median risk score of 2.36 was used as cut-off to separate low-risk patients from high-risk patients. High-risk patients showed worse RFS compared with low-risk patients in internal training, internal validation, and external validation dataset. Time-dependent receiver operating characteristic curves of 3 and 5 years indicated that risk score was superior to tumor stage, age, and PAM50 in both entire and external validation datasets. Cell cycle was the main different pathway between the high-risk and low-risk groups. Meanwhile, cell cycle was also the main pathway enriched in the 25 genes which were shared among 86 genes, DEGs, and WGCNA.ConclusionCell cycle pathway, identified by CRISPR-cas9 screening, was a key pathway regulating cell viability, which has significant prognostic values and can serve as a new target for breast cancer patient treatment.