High-throughput screening of mouse gene knockouts identifies established and novel skeletal phenotypes

Screening gene function in vivo is a powerful approach to discover novel drug targets. We present high-throughput screening （HTS） data for 3 762 distinct global gene knockout （KO） mouse lines with viable adult homozygous mice generated using either gene-trap or homologous recombination technologies. Bone mass was determined from DEXA scans of male and female mice at 14 weeks of age and by microCT analyses of bones from male mice at 16 weeks of age. Wild-type （WT） cagemates/littermates were examined for each gene KO. Lethality was observed in an additional 850 KO lines. Since primary HTS are susceptible to false positive findings, additional cohorts of mice from KO lines with intriguing HTS bone data were examined. Aging, ovariectomy, histomorphometry and bone strength studies were performed and possible non-skeletal phenotypes were explored. Together, these screens identified multiple genes affecting bone mass： 23 previously reported genes （Calcr, Cebpb, Crtap, Dcstamp, Dkkl, Duoxa2, Enppl, Fgf23, Kissl/Kisslr, Kl （Klotho）, Lrp5, Mstn, Neol, Npr2, Ostml, Postn, Sfrp4, S1c30a5, Sic39a13, Sost, Sumf1, Src, Wnt10b）, five novel genes extensively characterized （Cldn18, Fam20c, Lrrkl, Sgpll, Wnt16）, five novel genes with preliminary characterization （Agpat2, RassfS, Slc10a7, Stc26a7, Slc30a10） and three novel undisclosed genes coding for potential osteoporosis drug targets.

Bioinformatics Analysis Raises Candidate Genes in Blood for Early Screening of Parkinson's Disease

Parkinson＇s disease （PD） is a typical degenerative disease, which is characterized by the most obvious symptoms of movement dysfunction, including shaking, rigidity, slowness of movement and difficulty in walking and gait. This disease can not be clearly identified through laboratory tests at present, thus application of high-throughput technique in studying the expression profiles of PD helps to find the genetic markers for its early diagnosis. Studies on expression profiles of neurodegenerative diseases have revealed the novel genes and pathways involved in the progress of illness. In this study, the expression profiles of PD in blood were compared, showing that 181 differentially expressed genes （DEG） exhibit a similar expression trend both in patients and in normal controls.

Screening of Genes with Unique Mutations of Microcus

Yersinia pestis is the causative agent of bubonic and pneumonic plagues. Strains of Y. pestis are classified into four biovars： antiqua, mediaevalis, orientalis, and microtus[11. There are two microtus-related plague loci in China： the Microtus brandti plague focus in the Xilin Gol Grassland （focus L） and the Microtus fuscus plague focus in the Ojnghai-Tibet Plateau （focus M）.

Research progress and application of the CRISPR/Cas9 gene-editing technology based on hepatocellular carcinoma

Hepatocellular carcinoma(HCC)is now a common cause of cancer death,with no obvious change in patient survival over the past few years.Although the traditional therapeutic modalities for HCC patients mainly involved in surgery,chemotherapy,and radiotherapy,which have achieved admirable achievements,challenges are still existed,such as drug resistance and toxicity.The emerging gene therapy of clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9-based(CRISPR/Cas9),as an alternative to traditional treatment methods,has attracted considerable attention for eradicating resistant malignant tumors and regulating multiple crucial events of target gene-editing.Recently,advances in CRISPR/Cas9-based anti-drugs are presented at the intersection of science,such as chemistry,materials science,tumor biology,and genetics.In this review,the principle as well as statues of CRISPR/Cas9 technique were introduced first to show its feasibility.Additionally,the emphasis was placed on the applications of CRISPR/Cas9 technology in therapeutic HCC.Further,a broad overview of non-viral delivery systems for the CRISPR/Cas9-based anti-drugs in HCC treatment was summarized to delineate their design,action mechanisms,and anticancer applications.Finally,the limitations and prospects of current studies were also discussed,and we hope to provide comprehensively theoretical basis for the designing of anti-drugs.

A screening analysis of the GJB2 c.176 del 16 mutation responsible for hereditary deafness in a Chinese family

Objective:To determine whether a new-born child from a family carrying a deafness gene needs cochlear implantation to avoid dysphonia by screening and sequencing a deafness-related gene.Results:Both screening and sequencing results confirmed that the new born child had a normal GJB2 gene despite the fact that she has a brother suffering from hearing loss triggered by an allelic GJB2 c.176 del 16 mutation.We cloned the GJB2 genes derived from their respective blood genomic DNA into GFP fused plasmids and transfected those plasmids into the 293 T cell line to test for gene function.While the mutated GJB2gene(GJB2 c.176 del 16) of her deaf brother was found to be unable to form the gap junction structure between two adjacent cells,the baby girl’s GJB2 gene ran into no such problems.Conclusion:The screening and sequencing as well as the GJB2 gene function tests invariably showed results consistent with the ABR tested hearing phenotype,which means that the child,with a normal wild type GJB2 gene,does not need early intervention to prevent her from developing hearing loss and dysphonia at a later stage in life.

Prioritization of risk genes in colorectal cancer by integrative analysis of multi-omics data and gene networks

Genome-wide association studies(GWASs)have identified over 140 colorectal cancer(CRC)-associated loci;however,target genes at the majority of loci and underlying molecular mechanisms are poorly understood.Here,we utilized a Bayesian approach,integrative risk gene selector(iRIGS),to prioritize risk genes at CRC GWAS loci by integrating multi-omics data.As a result,a total of 105 high-confidence risk genes(HRGs)were identified,which exhibited strong gene dependencies for CRC and enrichment in the biological processes implicated in CRC.Among the 105 HRGs,CEBPB,located at the 20q13.13 locus,acted as a transcription factor playing critical roles in cancer.Our subsequent assays indicated the tumor promoter function of CEBPB that facilitated CRC cell proliferation by regulating multiple oncogenic pathways such as MAPK,PI3K-Akt,and Ras signaling.Next,by integrating a fine-mapping analysis and three independent case-control studies in Chinese populations consisting of 8,039 cases and 12,775 controls,we elucidated that rs1810503,a putative functional variant regulating CEBPB,was associated with CRC risk(OR=0.90,95%CI=0.86–0.93,P=1.07×10^(−7)).The association between rs1810503 and CRC risk was further validated in three additional multi-ancestry populations consisting of 24,254 cases and 58,741 controls.Mechanistically,the rs1810503 A to T allele change weakened the enhancer activity in an allele-specific manner to decrease CEBPB expression via longrange promoter-enhancer interactions,mediated by the transcription factor,REST,and thus decreased CRC risk.In summary,our study provides a genetic resource and a generalizable strategy for CRC etiology investigation,and highlights the biological implications of CEBPB in CRC tumorigenesis,shedding new light on the etiology of CRC.

A magnetic nanoparticles-based method for DNA extraction from the saliva of stroke patients

C677T polymorphism in the methylenetetrahydrofolate reductase （MTHFR） gene is a risk factor for stroke, suggesting that widespread detection could help to prevent stroke. DNA from 70 stroke pa- tients and 70 healthy controls was extracted from saliva using a magnetic nanoparticles-based method and from blood using conventional methods. Real-time PCR results revealed that the C677T polymorphism was genotyped by PCR using DNA extracted from both saliva and blood samples. The genotype results were confirmed by gene sequencing, and results for saliva and blood samples were consistent. The mutation TT genotype frequency was significantly higher in the stroke group than in controls. Homocysteine levels were significantly higher than controls in both TT genotype groups. Therefore, this noninvasive magnetic nanoparticles-based method using saliva samples could be used to screen for the MTHFR C677T polymorphism in target populations.

Screening and validation of reference genes for qPCR analysis in gonads and embryos of Takifugu bimaculatus

Suitable reference genes are one of the necessary conditions for obtaining reliable results by real-time fluorescence quantitative PCR(qPCR).In this study,the expression of the 10 common candidate reference genes(18s rRNA,rps27,cnbp,rpl7,ube2,hsp-at,gapdh,β-actin,rpl13a,1-ef1a)at different developmental stages of gonad and embryo of Takifugu bimaculatus were analyzed by qPCR.And the expression stability of these reference genes is analyzed by GeNorm,NormFinder and Bestkeeper softwares.The results showed that the expression stability of 1-ef1a was the highest(p<0.05)combined with the results of three analysis softwares,and the optimal number of reference genes was 3,which were 1-ef1a,hsp-at and rps27.Based on this result,we analyzed the expression of objective genes(foxl2 and dmrt1)at different developmental stages(early,medium-term and late)of gonads and embryos(eye vesicle stage,motility stage,heart anlage stage,retinal pigmentation stage,newly hatched larva)of T.bimaculatus by qPCR.Among which the highest expression level of foxl2 was in the middle stage of ovary(p<0.05),and dmrt1 was expressed in the early stage of testis with the highest level(p<0.05).The expression levels of foxl2 and dmrt1 in the early stages of embryonic development were low or even almost non-existent,and had a slight increase in the late embryonic development(newly hatched larva).The expression profile of objective genes are consistent with their roles in the development of gonads and embryos,which indicates that 1-ef1a,hsp-at and rps27 are the suitable reference gene combination for detecting the expression of objective genes by qPCR in gonads and embryos of T.bimaculatus.This research also provides the reliability data for screening the reference genes by qPCR in fish.

A screening assay for thyroid hormone signaling disruption based on thyroid hormone-response gene expression analysis in the frog Pelophylax nigromaculatus

Amphibian metamorphosis provides a wonderful model to study the thyroid hormone（TH）signaling disrupting activity of environmental chemicals, with Xenopus laevis as the most commonly used species. This study aimed to establish a rapid and sensitive screening assay based on TH-response gene expression analysis using Pelophylax nigromaculatus, a native frog species distributed widely in East Asia, especially in China. To achieve this, five candidate TH-response genes that were sensitive to T3 induction were chosen as molecular markers, and T3 induction was determined as 0.2 nmol/L T3 exposure for 48 hr. The developed assay can detect the agonistic activity of T3 with a lowest observed effective concentration of 0.001 nmol/L and EC50 at around 0.118–1.229 nmol/L, exhibiting comparable or higher sensitivity than previously reported assays. We further validated the efficiency of the developed assay by detecting the TH signaling disrupting activity of tetrabromobisphenol A（TBBPA）, a known TH signaling disruptor. In accordance with previous reports, we found a weak TH agonistic activity for TBBPA in the absence of T3,whereas a TH antagonistic activity was found for TBBPA at higher concentrations in the presence of T3, showing that the P. nigromaculatus assay is effective for detecting TH signaling disrupting activity. Importantly, we observed non-monotonic dose-dependent disrupting activity of TBBPA in the presence of T3, which is difficult to detect with in vitro reporter gene assays. Overall, the developed P. nigromaculatus assay can be used to screen TH signaling disrupting activity of environmental chemicals with high sensitivity.

Autosomal recessive hereditary auditory neuropathy

Objectives: Auditory neuropathy (AN) is a sensorineural hearing disorder characterized by absent or abnormal auditory brainstem responses (ABRs) and normal cochlear outer hair cell function as measured by otoacoustic emissions (OAEs). Many risk factors are thought to be involved in its etiology and pathophysiology. Three Chinese pedigrees with familial AN are presented herein to demonstrate involvement of genetic factors in AN etiology. Methods: Probands of the above - mentioned pedigrees, who had been diagnosed with AN, were evaluated and followed up in the Department of Otolaryngology Head and Neck Surgery, China PLA General Hospital. Their family members were studied and the pedigree diagrams were established. History of illness, physical examination,pure tone audiometry, acoustic reflex, ABRs and transient evoked and distortion- product otoacoustic emissions (TEOAEs and DPOAEs) were obtained from members of these families. DPOAE changes under the influence of contralateral sound stimuli were observed by presenting a set of continuous white noise to the non - recording ear to exam the function of auditory efferent system. Some subjects received vestibular caloric test, computed tomography (CT)scan of the temporal bone and electrocardiography (ECG) to exclude other possible neuropathy disorders. Results: In most affected subjects, hearing loss of various degrees and speech discrimination difficulties started at 10 to16 years of age. Their audiological evaluation showed absence of acoustic reflex and ABRs. As expected in AN, these subjects exhibited near normal cochlear outer hair cell function as shown in TEOAE & DPOAE recordings. Pure- tone audiometry revealed hearing loss ranging from mild to severe in these patients. Autosomal recessive inheritance patterns were observed in the three families. In Pedigree Ⅰ and Ⅱ, two affected brothers were found respectively, while in pedigree Ⅲ, 2 sisters were affected. All the patients were otherwise normal without evidence of peripheral neuropathy at the time of this writing. Conclusions: In this study, patients with feature of non- syndromic hereditary auditory neuropathy were identified in three Chinese families.Pedigree analysis indicates autosomal recessive inheritances in the pedigrees. The observed inheritance and clinical audiologic findings are different from those previously described for non-syndromic low-frequency sensorineural hearing loss. This information should facilitate future molecular candidate genes screening for understanding the mechanism of AN.

Clinical Analysis of a Hypokalemic Salt-losing Tubulopathy Case

Hypokalemia is among the most common electrolyte disorders in clinical practice and severe condition is life-threatening and has received extensive attention in clinical practice. Genetic diagnosis remains the gold standard for the diagnosis and identification of hereditary renal tubular diseases. Below is the report of genotyping and diagnosis of a case of secondary Gitelman syndrome （GS） based on next-generation sequencing.