Genetic screening of liver cancer:State of the art

Liver cancer,primarily hepatocellular carcinoma,remains a global health challenge with rising incidence and limited therapeutic options.Genetic factors play a pivotal role in the development and progression of liver cancer.This state-of-the-art paper provides a comprehensive review of the current landscape of genetic screening strategies for liver cancer.We discuss the genetic underpinnings of liver cancer,emphasizing the critical role of risk-associated genetic variants,somatic mutations,and epigenetic alterations.We also explore the intricate interplay between environmental factors and genetics,highlighting how genetic screening can aid in risk stratification and early detection via using liquid biopsy,and advancements in high-throughput sequencing technologies.By synthesizing the latest research findings,we aim to provide a comprehensive overview of the state-of-the-art genetic screening methods for liver cancer,shedding light on their potential to revolutionize early detection,risk assessment,and targeted therapies in the fight against this devastating disease.

Embryo quality and chromosomal abnormality in embryos from couples undergoing assisted reproductive technology using preimplantation genetic screening

Objective:To detect common chromosomal aneuploidy variations in embryos from couples undergoing assisted reproductive technology and preimplantation genetic screening and their possible associations with embryo quality.Methods:In this study,359 embryos from 62 couples were screened for chromosomes 13,21,18,X,and Y by fluorescence insitu hybridization.For biopsy of blastomere,a laser was used to remove a significantly smaller portion of the zona pellucida.One blastomere was gently biopsied by an aspiration pipette through the hole.After biopsy,the embryo was immediately returned to the embryo scope until transfer.Embryo integrity and blastocyst formation were assessed on day 5.Results:Totally,282 embryos from 62 couples were evaluated.The chromosomes were normal in 199(70.57%)embryos and abnormal in 83(29.43%)embryos.There was no significant association between the quality of embryos and numerical chromosomal abnormality(P=0.67).Conclusions:Embryo quality is not significantly correlated with its genetic status.Hence,the quality of embryos determined by morphological parameters is not an appropriate method for choosing embryos without these abnormalities.

Generation of a human haploid neural stem cell line for genome-wide genetic screening

BACKGROUND Haploid embryonic stem cells(haESCs)have been established in many species.Differentiated haploid cell line types in mammals are lacking due to spontaneous diploidization during differentiation that compromises lineage-specific screens.AIM To derive human haploid neural stem cells(haNSCs)to carry out lineage-specific screens.METHODS Human haNSCs were differentiated from human extended haESCs with the help of Y27632(ROCK signaling pathway inhibitor)and a series of cytokines to reduce diploidization.Neuronal differentiation of haNSCs was performed to examine their neural differentiation potency.Global gene expression analysis was conducted to compare haNSCs with diploid NSCs and haESCs.Fluorescence activated cell sorting was performed to assess the diploidization rate of extended haESCs and haNSCs.Genetic manipulation and screening were utilized to evaluate the significance of human haNSCs as genetic screening tools.RESULTS Human haESCs in extended pluripotent culture medium showed more compact and smaller colonies,a higher efficiency in neural differentiation,a higher cell survival ratio and higher stability in haploidy maintenance.These characteristics effectively facilitated the derivation of human haNSCs.These human haNSCs can be generated by differentiation and maintain haploidy and multipotency to neurons and glia in the long term in vitro.After PiggyBac transfection,there were multiple insertion sites in the human haNSCs’genome,and the insertion sites were evenly spread across all chromosomes.In addition,after the cells were treated with manganese,we were able to generate a list of manganese-induced toxicity genes,demonstrating their utility as genetic screening tools.CONCLUSION This is the first report of a generated human haploid somatic cell line with a complete genome,proliferative ability and neural differentiation potential that provides cell resources for recessive inheritance and drug targeted screening.

Role of genetic screening in a chinese woman of childbearing age with hypertrophic cardiomyopathy

Objectives The objective of this study was to evaluate the role of genetic screening in a Chinese woman of childbearing age with hypertrophic cardiomyopathy.Methods and Results One 39 year-old woman with presyncope for 10 years was admitted.Both of her father and her son died of sudden death and she strongly desire to get another baby. A series of clinical and laboratory studies were performed. Hypertrophic cardiomyopathy was diagnosed finally and implantable Cardioverter Defibrillator was implanted to prevent sudden cardiac death for her.Genomic DNA was isolated and the most common causal genes for hypertrophic cardiomyopathy were screened.A known pathogenetic heterozygous mutation c.700 g】a(p.Argl86Gln) in TNNI3 was found,which was not found in 100 normal control individuals matched for age,sex and geographical region.Because 50%probability of the pathogenetic mutation is inherited to the offsprings,she will get a healthy baby in vitro fertilization which the egg comes from a healthy female donor to prevent from the inherited HCM.Conclusions We found a pathogenetic TNNI3 mutation in a Chinese woman of childbearing age with hypertrophic cardiomyopathy.The genetic screening definite DNA-based diagnosis and help to design a healthy fertilization in vitro for female with hypertrophic cardiomyopathy.

Screening for genetic loci affecting the active zone formation in C. elegans

Objective To screen and identify genetic loci affecting the active zone formation in C. elegans. Methods A SYD-2：：GFP reporter was constructed and used as an active zone marker for forward genetic screen to identify genetic loci affecting the active zone formation. Results Eight isolated mutant alleles were characterized from 15,000 haploid genomes. The SYD-2：：GFP phenotypes of these mutants are mainly reflected as the changes of number, morphology, distribution of puncta and the gaps appearance. Some mutants also exhibit visible behavioral or physical phenotypes, and aldicarb resistant or sensitive phenotypes. Conclusion These mutants provide the opportunity for further systematic research on the active zone formation and the neurotransmission.

Genetic screening for quality-of-life improvement and post-genetic testing consideration in Saudi Arabia

The Saudi genome project started in 2013 with a great hope to improve medical care and disease prevention.Among the genes are those related to nutrition and fitness that can optimize an individual’s lifestyle.Our aim was to review the knowledge and acceptance of nutrition and fitness genetic testing to enhance the quality of life among the population of Saudi Arabia.For the study an electronic questionnaire consisting of 27 questions was prepared,and it was answered by 302 respondents.The respondents’demographics showed about 50% of respondents were aged 18-25 years and about 50% of respondents were aged 26-60 years.More than 50% of respondents were interested in having a genetic test to enhance their health,while 40% were interested in having a genetic test to enhance their fitness.Less than 50% of respondents had an understanding of the effects of coffee,macronutrition and micronutrition,elements,and enzyme activity.These results represented a contribution to the discussion on the relevance of genetic testing validity and acceptance among the population of Saudi Arabia.The results might help in producing specific guidelines on genetic testing and genomic analysis and help in the implementation of fitness and future health plans in cooperation with Saudi genome projects.Future study will focus on population structure and genetic frequency related to specific diets or fitness.

Genetic risk stratification of inflammatory bowel disease-associated venous thromboembolism:An Asian perspective

The utilisation of polygenic scoring models may enhance the clinician’s ability to risk stratify an inflammatory bowel disease patient’s individual risk for venous thromboembolism(VTE)and guide the appropriate usage of VTE thromboprophylaxis,yet there is a need to validate such models in ethnically diverse populations.

The Applications of CRISPR Screening in Aging Research

The advancement of Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)gene editing technology has revolutionized the comprehension of human genome,propelling molecular and cellular biology research into unexplored realms and accelerating progress in life sciences and medicine.CRISPR-based gene screening,recognized for its efficiency and practicality,is widely utilized across diverse biological fields.Aging is a multifaceted process governed by a myriad of genetic and epigenetic factors.Unraveling the genes regulating aging holds promise for understanding this intricate phenomenon and devising strategies for its assessment and intervention.This review provides a comprehensive overview of the progress in CRISPR screening and its applications in aging research,while also offering insights into future directions.CRISPR-based genetic-manipulation tools are positioned as indispensable instruments for mitigating aging and managing age-related diseases.

Generation of a series of mutant lines resistant to imidazolinone by screening an EMS-based mutant library in common wheat

The breeding of herbicide-resistant wheat varieties has helped control weeds in wheat fields economically and effectively.Imidazolinone (IMI) herbicides are popular as they have low toxicity in mammals,are effective at small doses,and exhibit broad-spectrum herbicidal action in the field.Therefore,the isolation and genetic and molecular characterization of IMI-resistant wheat mutants will enhance weed management in wheat fields.In the present study,352 IMI-resistant plants were isolated by genetic screening from a mutant pool prepared by EMS-based random mutagenesis.Cloning of the mutated genes from the IMI-resistant plants indicated that ten taals alleles had been isolated,and mutation in one of three Ta ALS homolog genes conferred IMI resistance,and such a mutation is a dominant trait.Further analysis showed that taals-d exhibited the greatest IMI resistance,whereas taals-b exhibited the weakest resistance to IMI among three homologous taals mutants.In terms of IMI resistance,the taals triple mutant was stronger than the taals double mutants,and the taals double mutants were stronger than the single mutants,indicating a dose-dependent effect of the Ta ALS mutation on IMI resistance in wheat.Biochemical analysis indicated that the mutation in Ta ALS increased the tolerance of Ta ALS to inhibition by IMI.Our work details the genetic and molecular characterization of als wheat mutants,provides a foundation for understanding IMI resistance and breeding wheat varieties with herbicide resistance,and indicates that genetic screening using a mutagenized pool is an effective and important means of breeding crops with additional desired agricultural traits.

A novel genetic screen for leukemia stem cell immortalization genes

Leukemia, like many other cancers, is thought to arise from a small population of stem cells that have the capacity to self-renewal extensively and to initiate, sustain or regenerate the disease. Elimination of the leukemia stem cells （LSCs） will likely be essential, and probably sufficient, for curing this disease.Recent studies have shown that LSCs can be derived from early hematopoietic progenitors as well as more differentiated derivatives; the key feature being these cells have acquired an increased proliferative capacity and the ability to self-renew extensively. Genes that make this possible are attractive drug targets for treating leukemia. In our laboratory we have developed a novel in vitro genetic screen that uses retroviral insertional mutagenesis as a tool for identifying genes that are able to convert both normal hematoooietic progenitors and committed mveloid progenitor cells into cells that resemble LSCs.

A Comparative Analysis of CRISPR Screening Technologies

This paper offers a general review and comparative analysis of various types of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technologies. It evaluates the strengths and weaknesses of these technologies to identify the optimal approach for conducting genetic screens. Through an extensive literature review, this paper examines CRISPR nuclease, CRISPR activation (CRISPRa), and CRISPR interference (CRISPRi) screens. This study concludes that CRISPRa and CRISPRi are more advantageous due to their use of deactivated Cas9 proteins that only over-express or deactivate genes rather than irreversibly breaking genes like CRISPRn. Notably, CRISPRa is unique in its ability to over-express genes, while the other two technologies deactivate genes. Future studies may focus on inducing multiple mutations simultaneously—both gain-of-function and gene knockout—to carry out a more complete screen that can test the combinatorial effect of genes. Likewise, targeting both exons and introns can offer a more thorough understanding of a specific phenotype.

Preparation of single chain variable fragment of MG_7 mAb by phage display technology

AIM: To develop the single chain variable fragment of MG MG(7)murine anti-human gastric cancer monoclonal antibody using the phage display technology for obtaining a tumor-targeting mediator. METHODS: mRNA was isolated from MG MG(7) producing murine hybridoma cell line and converted into cDNA. The variable fragments of heavy and light chain were amplified separately and assembled into ScFv with a specially constructed DNA linker by PCR. The ScFvs DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E. Coli TG1. The transformed cells were infected with M13K07 helper phage to form MG MG(7) recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by means of bacterial colony count and restriction analysis. After two rounds of panning with gastric cancer cell line KATO III of highly expressing MG(7)-binding antigen, the phage clones displaying ScFv of the antibody were selected by ELISA from the enriched phage clones. The antigen-binding affinity of the positive clone was detected by competition ELISA. HB2151 E. Coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the MG(7) ScFv. ELISA assay was used to detect the antigen-binding affinity of the soluble MG(7) ScFv. Finally, the relative molecular mass of soluble MG(7) ScFv was measured by SDS-PAGE. RESULTS: The V(H), V(L) and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 X 10(6) and 8 of 11 random clones were recombinants. Two phage clones could strongly compete with the original MG(7) antibody for binding to the antigen expressed on KATO III cells. Within 2 strong positive phage clones, the soluble MG(7) ScFv from one clone was found to have the binding activity with KATO III cells. SDS-PAGE showed that the relative molecular weight of soluble MG(7) ScFv was 32. CONCLUSION: The MG(7) ScFv was successfully produced by phage antibody technology, which may be useful for broadening the scope of application of the antibody.

Hereditary pancreatitis

Hereditary pancreatitis is an autosomal dominant condition,which results in recurrent attacks of acute pancreatitis,progressing to chronic pancreatitis often at a young age.The majority of patients with hereditary pancreatitis expressone of two mutations (R122H or N29I) in the cationictrypsinogen gene (PRSS1 gene). It has been hypothesisedthat one of these mutations, the R122H mutation causespancreatitis by altering a trypsin recognition site sopreventing deactivation of trypsin within the pancreas andprolonging its action, resulting in autodigestion. Families withthese two mutations have been identified in many countriesand there are also other rarer mutations, which have alsobeen linked to hereditary pancreatitis.Patients with hereditary pancreatitis present in the sameway as those with sporadic pancreatitis but at an earlierage. It is common for patients to remain undiagnosed formany years, particularly ifthey present with non-specificsymptoms. Hereditary pancreatitis should always beconsidered in patients who present with recurrent pancreatitiswith a family history of pancreatic disease. If patients withthe 2 common mutations are compared, those with theR122H mutation are more likely to present at a younger ageand are more likely to require surgical intervention than thosewith N29I. Hereditary pancreatitis carries a 40 % lifetimerisk of pancreatic cancer with those patients aged between50 to 70 being most at risk in whom screening tests maybecome important.

Advances in preimplantation genetic diagnosis/screening

Preimplantation genetic diagnosis(PGD)gives couples who have a high risk of transmitting genetic disorders to their baby the chance to have a healthy offspring through embryo genetic analysis and selection.Preimplantation genetic screening(PGS)is an effective method to select euploid embryos that may prevent repeated implantation failure or miscarriage.However,how and to whom PGS should be provided is a controversial topic.The first successful case of PGD of a human being was reported in 1990,and there have been tremendous improvements in this technology since then.Both embryo biopsy and genetic technologies have been improved dramatically,which increase the accuracy and expand the indications of PGD/PGS.

重谈酵母遗传学

遗传学是研究生物的遗传与变异的科学,是研究基因的结构、功能、变异、传递和表达规律的学科.遗传学发展的早期,遗传学家们研究的对象很广泛,随着时代的发展。

A large-scale functional approach to uncover human genes and pathways in Drosophila

We demonstrate the feasibility of performing a systematic screen for human gene functions in Drosophila by assaying for their ability to induce overexpression phenotypes. Over 1 500 transgenic fly lines corresponding to 236 human genes have been established. In all, 51 lines are capable of eliciting a phenotype suggesting that the human genes are functional. These heterologous genes are functionally relevant as we have found a similar mutant phenotype caused either by a dominant negative mutant form of the human ribosomal protein L8 gene or by RNAi downregulation of the Drosophila RPL8. Significantly, the Drosophila RPL8 mutant can be rescued by wild-type human RPL8. We also provide genetic evidence that Drosophila RPL8 is a new member of the insulin signaling pathway. In summary, the functions of many human genes appear to be highly conserved, and the ability to identify them in Drosophila represents a powerful genetic tool for large-scale analysis of human transcripts in vivo.

Genetic counseling, prenatal screening and diagnosis of Down syndrome in the second trimester in women of advanced maternal age： a prospective study

Background The incidence of autosomal trisomy in livebirths is strongly dependent on maternal age. Special consideration is given to the provision of prenatal screening and cytogenetic testing to women of advanced maternal age （AMA）. The aim of this study was to evaluate the effectiveness of second trimester prenatal screening and amniocentesis for Down syndrome （DS） and compare the trends of choice of screening and amniocentesis among AMA women. Methods A total of 5404 AMA patients with natural singleton pregnancy were recruited for this prospective study from January 2008 to December 2010. The gestational weeks were from 15 weeks to 20~6 weeks. The patients referred were grouped into a screening group （2107 cases） and an amniocentesis group （3297 cases） by their own decision. The prevalence of DS was compared between the two groups by chi-square test. Choice rates for each maternal age with trends were compared by regression analysis. Results There were 18 cases of fetal DS detected in the screening group with a prevalence of 8.54%o （18/2107）. Twenty- five cases of fetal DS were diagnosed in the amniocentesis group with a prevalence of 7,58%0 （25/3297）. No statistical difference was observed in the prevalence of DS between the screening and amniocentesis group （P=0.928）. The invasive testing rate for DS in the amniocentesis group was 5.54 times higher than that of the screening group （1/131.88 vs. 1/23.78）. With the increase of the maternal age, the choice of amniocentesis increased while the choice of the screening showed an opposite trend. The choice of the AMA women between the screening and amniocentesis was significantly age relevant （P=0.012）. Conclusions The second trimester serum screening age alone to screen for DS. We suggest educating screening and amniocentesis options. in combination with maternal age was more effective than maternal the patients by recommending AMA women be informed of both

A large scale screen for genes （3rd chromosome） related to Wingless signaling pathway

A wing specific F 1 genetic screen was carried out using the powerful Drosophila genetic system, combined with yeast FRT/FLP and GAL4/UAS system. Form the wing phenotypes and germline clone embryonic cuticle phenotypes observed in these mutant alleles, a number of mutant alleles of known or unknown genes were isolated. Among them, fifteen mutant alleles related to Wingless signal transduction were further isolated; the arm of these mutations located were determined, and their location in the chromosome were roughly mapped.

A New Next-Generation Sequencing-Based Assay for Concurrent Preimplantation Genetic Diagnosis of Charcot-Marie-Tooth Disease Type 1A and Aneuploidy Screening

Charcot-Marie-Tooth （CMT） disease is the most common hereditary neuropathy, with a population prevalence of 1 in 2500. CMT disease type 1A （CMT1A）, accounting for ~70% of CMT1 cases and ~ 50% of all CMT cases, is transmitted in an autosomal dominant manner. CMT1A maps to chromo- some 17pl 1.2 and is caused, in the majority of cases, by a 1.4- Mb tandem duplication that includes the peripheral myelin protein22 （PMP22） gene （Li et al., 2013）. The disease usually presents in the first 20 years of age, causing difficulty in walking or running, distal symmetrical muscle weakness and wasting, and sensory loss （van Paassen et al., 2014）.

The Mapping and Characterization of Cruella (Cru), a Novel Allele of Capping Protein α (Cpa), Identified from a Conditional Screen for Negative Regulators of Cell Growth and Cell Division

A Flp/FRT EMS mutagenesis screen was conducted in the eye of Drosophila melanogaster on chromosome 2R to identify negative regulators of cell growth and cell division. In addition to the EMS mutation in the mosaic eye, an ark loss of function allele (ark82) was utilized to block apoptosis in the homozygous mutant cells, setting up a screen for conditional regulators of cell growth and cell division. In the present study, we focus on the characterization and mapping of one mutant that resulted from this screen, Cruella (cru). A cross between flies with the flippase enzyme directed to the developing eye and flies with the mutations cru, ark82, revealed an unusual phenotype that resulted in the homozygous mutant tissue appearing black, in contrast to the expected red. To map the location of this mutation, complementation tests against the Bloomington deficiency kit were conducted. Cru failed to complement previously characterized alleles of capping protein α (cpa). Thus, cpacru is a novel allele of cpa and displays phenotypes similar to previously characterized alleles such as cpa 107E, cpa 69E, and cpascrd . The human homolog, Cap Z, is conserved in humans and serves a similar role in act in filament regulation.