Cardiovascular System/pathology* ; Constriction, Pathologic/genetics* ; Point Mutation ; Neurofibromin 1/genetics* ; Animals ; Mice

To establish a rabbit model of proprotein convertase subtilisin/kexin type9 () point mutation with CRISPR/Cas9 gene editing technique. According to the PubMed gene protein data, the PCSK9 protein functional regions of human and rabbit were analyzed by Blast. The 386S (Ser) amino acid functional region of human gene was homologous to the 485S of rabbit gene. Three small guide RNAs and one single-stranded donor oligonucleotide were designed according to the 485S base substitution position and sequence analysis of rabbit gene. The synthetic small guide RNAs, Cas9 mRNA and single-stranded donor oligonucleotide were co-injected into the cytoplasm of rabbit fertilized eggs and the embryos were transferred into the pregnant rabbits. PCR, TA cloning and off-target analysis were performed on the F0 rabbits to identify whether the PCSK9 mutation was successful. Fifteen F0 rabbits were obtained. The sequencing results showed that one of them was PCSK9 point mutation homozygote and two of them were PCSK9 point mutation heterozygotes, and the mutation could be stably inherited. The rabbit model of PCSK9 point mutation was successfully constructed by CRISPR/Cas9 technique, which provides an animal model for exploring the molecular mechanism of impaired PCSK9 function and developing reliable and effective diagnosis and treatment measures.
Animals ; CRISPR-Cas Systems/genetics* ; Mutation ; Point Mutation ; Proprotein Convertase 9/metabolism* ; Rabbits

The rearrangement of the gene encoding the transcription factor ETS-related gene (ERG) is thought to play a key role in the development of prostate cancer. However, the studies on the ERG mutations have been rarely reported in non-small cell lung carcinoma (NSCLC). Here, we reported genetic features regarding a case of a 68-year-old male patient who presented the primary synchronous multiple tumor lesions in the separated lungs. The patient was hospitalized due to the presence of tumor lesions at the right and left lungs revealed by a chest computerized tomography (CT) scan. After conducting lobectomies at the both lungs, the tumor nodules were all removed, and the histological analysis suggested adenocarcinoma at the both tumor lesions. The patient was diagnosed with synchronous multiple primary lung cancer (SMPLC) based on Martini-Melamed criteria and American College of Chest Physicians practice guidelines. An exome analysis of 315 genes in the two tumor lesions and a non-tumor lesion was conducted by using Illumina Nextseq500 platform from each tumor region to decipher a potential evolutional progress of SMPLC. Single or pair-end reads were first mapped to a human genome reference and filtered based on the mapping quality score. The read depth was ≥ 1 000× and the depth of coverage was 95%. The data revealed a discordant epidermal growth factor receptor (EGFR) from the separate lungs; additionally, a high frequency of point mutation on exon 9 H310P of the ERG gene was detected at the both sites of the tumor lesions. This case showed that a potential role of the molecular features analysis from each tumor lesion might contribute to the understanding of the evolutional development of SMPLC. This study suggests that the same environment may contribute certain gene(s) mutations in the same sites in the early stages of polyclonal tumor origins; meanwhile the extensive studies on these genes may help us understand the evolution and progress of tumor clones.
Adenocarcinoma ; Aged ; Carcinoma, Non-Small-Cell Lung ; Humans ; Lung Neoplasms/genetics* ; Male ; Neoplasms, Multiple Primary/genetics* ; Point Mutation ; Transcriptional Regulator ERG

OBJECTIVE: To explore the natural mutations in Spike protein (S protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the changes of affinity between virus and associated receptors or drug molecules before and after the mutation based on whole length sequencing results. METHODS: In the study, the bioinformatics analysis of all the published sequences of SARS-CoV-2 was conducted and thus the high frequency mutation sites were affirmed. Taking advantages of PolyPhen-2, the functional influence of each mutation in S protein was prospected. The 3D homologous modelling was performed by SWISS-MODEL to establish mutated S protein structural model, in which the protein-docking was then implemented with angiotensin-converting enzyme 2 (ACE2), dipeptidyl peptidase-4 (DPP4) and aminopeptidase N (APN) by ZDOCK, and the combining capacity of each mutated S protein evaluated by FiPD. Finally, the binding ability between mutated S proteins and anti-virus drugs were prospected and evaluated through AutoDock-Chimera 1.14. RESULTS: The mutations in specific region of S protein had greater tendency to destroy the S protein function by analysis of mutated S protein structure. Protein-receptor docking analysis between naturally mutated S protein and host receptors showed that, in the case of spontaneous mutation, the binding ability of S protein to ACE2 tended to be weakened, while the binding ability of DPP4 tended to be enhanced, and there was no significant change in the binding ability of APN. According to the computational simulation results of affinity binding between small molecular drugs and S protein, the affinity of aplaviroc with S protein was significantly higher than that of other small molecule drug candidates. CONCLUSION The region from 400-1 100 amino acid in S protein of SARS-CoV-2 is the mutation sensitive part during natural state, which was more potential to mutate than other part in S protein during natural state. The mutated SARS-CoV-2 might tend to target human cells with DPP4 as a new receptor rather than keep ACE2 as its unique receptor for human infection. At the same time, aplaviroc, which was used for the treatment of human immunodeficiency virus (HIV) infection, may become a new promising treatment for SARS-CoV-2 and could be a potential choice for the development of SARS-CoV-2 drugs.
Antiviral Agents ; COVID-19 ; Humans ; Peptidyl-Dipeptidase A/genetics* ; Point Mutation ; SARS-CoV-2 ; Spike Glycoprotein, Coronavirus/genetics*

PURPOSE: With the emergence of next-generation sequencing (NGS) technology, profiling a wide range of genomic alterations has become a possibility resulting in improved implementation of targeted cancer therapy. In Asian populations, the prevalence and spectrum of clinically actionable genetic alterations has not yet been determined because of a lack of studies examining high-throughput cancer genomic data. MATERIALS AND METHODS: To address this issue, 1,071 tumor samples were collected from five major cancer institutes in Korea and analyzed using targeted NGS at a centralized laboratory. Samples were either fresh frozen or formalin-fixed, paraffin embedded (FFPE) and the quality and yield of extracted genomic DNA was assessed. In order to estimate the effect of sample condition on the quality of sequencing results, tissue preparation method, specimen type (resected or biopsied) and tissue storage time were compared. RESULTS: We detected 7,360 non-synonymous point mutations, 1,164 small insertions and deletions, 3,173 copy number alterations, and 462 structural variants. Fifty-four percent of tumors had one or more clinically relevant genetic mutation. The distribution of actionable variants was variable among different genes. Fresh frozen tissues, surgically resected specimens, and recently obtained specimens generated superior sequencing results over FFPE tissues, biopsied specimens, and tissues with long storage duration. CONCLUSION: In order to overcome, challenges involved in bringing NGS testing into routine clinical use, a centralized laboratory model was designed that could improve the NGS workflows, provide appropriate turnaround times and control costs with goal of enabling precision medicine.
Academies and Institutes ; Asian Continental Ancestry Group ; DNA ; Humans ; Korea ; Methods ; Paraffin ; Point Mutation ; Precision Medicine ; Prevalence

PURPOSE: Hypoallergenic recombinant Der p 2 has been produced by various genetic manipulations, but mutation of a naturally polymorphic amino acid residue known to affect IgE binding has not been studied. This study aimed to determine the effect of a point mutation (S47W) of residue 47 of Der p 2 on its structure and immunoglobulin (Ig) E binding. Its ability to induce pro-inflammatory responses and to induce blocking IgG antibody was also determined. METHODS: S47 of recombinant Der p 2.0110, one of the predominant variants in Bangkok, was mutated to W (S47W). S47W secreted from Pichia pastoris was examined for secondary structure and for the formation of a hydrophobic cavity by 8-Anilino-1-naphthalenesulfonic acid (ANS) staining. Monoclonal and human IgE-antibody binding was determined by enzyme-linked immunosorbent assay. Allergen-induced degranulation by human epsilon receptor expressed-rat basophil was determined. Stimulation of the pro-inflammatory cytokine interleukin (IL)-8 release from human bronchial epithelial (BEAS2B) cells and inhibition of IgE binding to the wild type allergen by S47W-induced IgG were determined. RESULTS: S47W reduced secondary structure and failed to bind the hydrophobic ANS ligand as well as a monoclonal antibody known to be dependent on the nature of the side chain of residue 114 in an adjacent loop. It could also not stimulate IL-8 release from BEAS2B cells. IgE from house dust mite (HDM)-allergic Thais bound S47W with 100-fold weaker avidity, whereas IgE of HDM-allergic Australians did not. S47W still induced basophil degranulation, although requiring higher concentrations for some subjects. Anti-S47W antiserum-immunized mice blocked the binding of human IgE to wild type Der p 2. CONCLUSIONS: The mutant S47W had altered structure and reduced ability to stimulate pro-inflammatory responses and to bind IgE, but retained its ability to induce blocking antibodies. It thus represents a hypoallergen produced by a single mutation of a non-solvent-accessible amino acid.
Animals ; Antibodies, Blocking ; Asian Continental Ancestry Group ; Basophils ; Dust* ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunoglobulin E ; Immunoglobulin G ; Immunoglobulins ; Interleukin-8 ; Interleukins ; Mice ; Pichia ; Point Mutation ; Pyroglyphidae*

Androgen receptor (AR) is a steroid receptor transcriptional factor for testosterone and dihydrotestosterone consisting of four main domains, the N-terminal domain, DNA-binding domain, hinge region, and ligand-binding domain. AR plays pivotal roles in prostate cancer, especially castration-resistant prostate cancer (CRPC). Androgen deprivation therapy can suppress hormone-naïve prostate cancer, but prostate cancer changes AR and adapts to survive under castration levels of androgen. These mechanisms include AR point mutations, AR overexpression, changes of androgen biosynthesis, constitutively active AR splice variants without ligand binding, and changes of androgen cofactors. Studies of AR in CRPC revealed that AR was still active in CRPC, and it remains as a potential target to treat CRPC. Enzalutamide is a second-generation antiandrogen effective in patients with CRPC before and after taxane-based chemotherapy. However, CRPC is still incurable and can develop drug resistance. Understanding the mechanisms of this resistance can enable new-generation therapies for CRPC. Several promising new AR-targeted therapies have been developed. Apalutamide is a new Food and Drug Administration-approved androgen agonist binding to the ligand-binding domain, and clinical trials of other new AR-targeted agents binding to the ligand-binding domain or N-terminal domain are underway. This review focuses on the functions of AR in prostate cancer and the development of CRPC and promising new agents against CRPC.
Androgen Antagonists ; Castration ; Dihydrotestosterone ; Drug Resistance ; Drug Therapy ; Humans ; Point Mutation ; Prostate ; Prostatic Neoplasms ; Receptors, Androgen ; Receptors, Steroid ; Testosterone

Breast Diseases ; diagnostic imaging ; metabolism ; Breast Neoplasms ; diagnostic imaging ; metabolism ; Class I Phosphatidylinositol 3-Kinases ; genetics ; Female ; Humans ; Middle Aged ; Point Mutation

Alport syndrome (ATS) is an inherited glomerular disease caused by mutations in one of the type IV collagen novel chains (α3, α4, and α5). ATS is characterized by persistent microscopic hematuria that starts during infancy, eventually leading to either progressive nephritis or end-stage renal disease. There are 3 known genetic forms of ATS, namely X-linked ATS, autosomal recessive ATS, and autosomal dominant ATS. About 80% of patients with ATS have X-linked ATS, which is caused by mutations in the type IV collagen α5 chain gene, COL4A5. Although an 80% mutation detection rate is observed in men with X-linked ATS, some difficulties do exist in the genetic diagnosis of ATS. Most mutations are point mutations without hotspots in the COL4A3, COL4A4, and COL4A5 genes. Further, there are insufficient data on the detection of COL4A3 and COL4A4 mutations for their comparison between patients with autosomal recessive or dominant ATS. Therefore, diagnosis of ATS in female patients with no apparent family history can be challenging. Therefore, in this study, we used whole-exome sequencing (WES) to identify mutations in type IV collagen in 2 girls with glomerular basement membrane structural changes suspected to be associated with ATS; these patients had no relevant family history. Our results revealed de novo c.4688G>A (p.Arg1563Gln) and c.2714G>A (p.Gly905Asp) mutations in COL4A5. Therefore, we suggest that WES is an effective approach to obtain genetic information in ATS, particularly in female patients without a relevant family history, to detect unexpected DNA variations.
Child ; Collagen Type IV ; Diagnosis ; DNA ; Exome ; Female ; Glomerular Basement Membrane ; Hematuria ; Humans ; Kidney Failure, Chronic ; Korea ; Male ; Nephritis ; Nephritis, Hereditary ; Point Mutation

Thyroid nodules are the most common endocrine tumor. Ultrasonography and fine-needle aspiration (FNA) are currently accurate diagnostic tools for evaluating thyroid nodules. However, 10–30% of FNA specimens are cytologically indeterminate. Making an accurate diagnosis between benign and malignant nodules is important so that patients with malignant nodule receive proper treatment and patients with benign nodule can avoid unnecessary treatment. Several genetic changes such as point mutations of the BRAF or RAS and rearrangements of the RET/PTC1, RET/PTC3, PAX8/PPARY are used to adjust to indeterminate FNA. Such a mutational analysis has an excellent positive predictive value (PPV), but there is a weakness in the low negative predictive value (NPV). Gene-expression classifier (GEC) has been found helpful in identify nodules that are benign rather than malignant. GEC has an excellent NPV, but there is a weakness of low PPV. Multiplatform mutational and miRNA test (MPT) and next-generation sequencing assay (NGS) are being studied to compensate for these weaknesses. Molecular tests appear to be a good solution for improving the accuracy of indeterminate FNA cytology specimens and support the clinical management decisions in patients with indeterminate cytologic nodules, but further prospective multicenter trials are required for validation of reported findings and need evaluation of cost-effectiveness. This paper will review recently available molecular diagnostic tools of thyroid nodule.
Biopsy, Fine-Needle ; Diagnosis ; Humans ; MicroRNAs ; Multicenter Studies as Topic ; Pathology, Molecular ; Point Mutation ; Prospective Studies ; Thyroid Gland ; Thyroid Neoplasms ; Thyroid Nodule ; Ultrasonography