Specific Point Mutations Research Articles

Activated H-ras gene mutations in transitional cell carcinoma of urinary bladder in a Kashmiri population.

The primary aim of the study was to evaluate the incidence of H-ras specific point mutations among a group of Kashmiri patients diagnosed with bladder cancer. We also explored the correlation of clinic-pathological status of the illness with these mutations. The DNA samples of both tumor and normal tissue were evaluated for the occurrence of H-ras activating mutations in exon 1 and 2 by PCR-SCCP and DNA sequencing. In addition, blood was also collected from all the cases to rule out any germ-line mutation. Point mutations of activated H-ras identified in bladder cancer patients were 14.5% (7 of 48), including four transversions (two G-->T and two A-->T) and three transitions (A-->G). Of the mutations, 71.4% were detected in codon 61 and 28.6% in codon 12. The pattern of mutation in the study showed a significant association with smoking in bladder tumors (P < 0.05). No correlation was found between tumor grade and/or stage and the presence of H-ras mutation. Activation of H-ras by mutation plays a less frequent role than other genetic events in the development of the most transitional cell tumors of the bladder in Kashmiri population.

Electroporation of mice zygotes with dual guide RNA/Cas9 complexes for simple and efficient cloning-free genome editing

In this report, we present an improved protocol for CRISPR/Cas9 genome editing in mice. The procedure consists in the electroporation of intact mouse zygotes with ribonucleoprotein complexes prepared in vitro from recombinant Cas9 nuclease and synthetic dual guide RNA. This simple cloning-free method proves to be extremely efficient for the generation of indels and small deletions by non-homologous end joining, and for the generation of specific point mutations by homology-directed repair. The procedure, which avoids DNA construction, in vitro transcription and oocyte microinjection, greatly simplifies genome editing in mice.

Efficient oligo nucleotide mediated CRISPR-Cas9 gene editing in Aspergilli

CRISPR-Cas9 technologies are revolutionizing fungal gene editing. Here we show that survival of specific Cas9/sgRNA mediated DNA double strand breaks (DSBs) depends on the non-homologous end-joining, NHEJ, DNA repair pathway and we use this observation to develop a tool, TAPE, to assess protospacer efficiency in Aspergillus nidulans. Moreover, we show that in NHEJ deficient strains, highly efficient marker-free gene targeting can be performed. Indeed, we show that even single-stranded oligo nucleotides efficiently work as repair templates of specific Cas9/sgRNA induced DNA DSBs in A. nidulans, A. niger, and in A. oryzae indicating that this type of repair may be wide-spread in filamentous fungi. Importantly, we demonstrate that by using single-stranded oligo nucleotides for CRISPR-Cas9 mediated gene editing it is possible to introduce specific point mutations as well gene deletions at efficiencies approaching 100%. The efficiency of the system invites for multiplexing and we have designed a vector system with the capacity of delivering Cas9 and multiple sgRNAs based on polymerase III promoters and tRNA spacers. We show that it is possible to introduce two point mutations and one gene insertion in one transformation experiment with a very high efficiency. Our system is compatible with future high-throughput gene-editing experiments.

A Simple PCR-based Strategy for the Introduction of Point Mutations in the Yeast Saccharomyces cerevisiae via CRISPR/Cas9.

The methods currently employed for in vivo site-directed mutagenesis in yeast are laborious and/or inefficient. Recent developments of the CRISPR-based approaches hold great promise for genome editing, but its application in the yeast S. cerevisiae remains a time-consuming affair. The rate-limiting step in CRISPR-mediated genetic engineering in yeast is the incorporation of the guide sequences, which target Cas9 to relevant chromosomal locus, into the relevant yeast vectors. Here we present a PCR-based strategy to introduce specific point mutation into the yeast CDC48 gene via CRISPR. Our method eliminates the need for special dam- strain and markedly shortens the elaborate multi-step cloning process, leading to significant savings in time, labor and cost.

A new point mutation in β2-tubulin confers resistance to carbendazim in Fusarium asiaticum

Resistance to benzimidazole fungicides in many phytopathogenic fungi is caused by specific point mutations in the β-tubulin gene (β-tubulin). However, the mutated locus and genotype of β-tubulin differ among phytopathogenic fungi. To validate the point mutation in Fusarium asiaticum β2-tubulin that confers resistance to carbendazim and to analyze the molecular interaction between carbendazim and F. asiaticum β2-tubulin. In this study, a new point mutation (GAG→GCG, E198A) at codon 198 of β2-tubulin in a wild-type F. asiaticum strain was constructed by site-directed mutagenesis followed by a split marker strategy. The site-directed mutants were verified and exhibited a high level of resistance to carbendazim. In the absence of fungicide treatment, the biological characteristics did not differ between the site-directed mutants and the wild-type strain. Molecular docking between carbendazim and β2-tubulin was carried out using the Surflex-Dock program in Sybyl X-2.0 version and the results indicated that the E198A mutation altered the configuration of β2-tubulin, resulting in the change of the bonding sites and docking scores. We concluded that the point mutation of F. asiaticum β2-tubulin conferring carbendazim resistance may not always be the bonding site for carbendazim.

Chondroblastoma

Chondroblastomas are very rare benign primary bone tumors occurring preferentially in the epiphyses or apophyses of long bones in children and adolescents. In most cases the typical histological and imaging findings lead to acorrect diagnosis that may be substantiated by demonstrating the highly specific point mutation in the H3F3B gene (p.K36M), either by sequencing or immunohistochemistry. Recurrences occur in 5-15% of cases, postsurgical metastatic deposits to the lungs are very rare (<1%). Histologically "malignant" chondroblastomas have been reported as single case reports. The treatment of choice is athorough curettage, also in the case of local relapses.

Nuclear Excluded Autism-Associated Phosphatase and Tensin Homolog Mutations Dysregulate Neuronal Growth

BackgroundPhosphatase and tensin homolog (PTEN) negatively regulates downstream protein kinase B signaling, resulting in decreased cellular growth and proliferation. PTEN is mutated in a subset of children with autism spectrum disorder (ASD); however, the mechanism by which specific point mutations alter PTEN function is largely unknown. Here, we assessed how ASD-associated single-nucleotide variations in PTEN (ASD-PTEN) affect function. MethodsWe used viral-mediated molecular substitution of human PTEN into Pten knockout mouse neurons and assessed neuronal morphology to determine the functional impact of ASD-PTEN. We employed molecular cloning to examine how PTEN’s stability, subcellular localization, and catalytic activity affect neuronal growth. ResultsWe identified a set of ASD-PTEN mutations displaying altered lipid phosphatase function and subcellular localization. We demonstrated that wild-type PTEN can rescue the neuronal hypertrophy, while PTEN H93R, F241S, D252G, W274L, N276S, and D326N failed to rescue this hypertrophy. A subset of these mutations lacked nuclear localization, prompting us to examine the role of nuclear PTEN in regulating neuronal growth. We found that nuclear PTEN alone is sufficient to regulate soma size. Furthermore, forced localization of the D252G and W274L mutations into the nucleus partially restores regulation of soma size. ConclusionsASD-PTEN mutations display decreased stability, catalytic activity, and/or altered subcellular localization. Mutations lacking nuclear localization uncover a novel mechanism whereby lipid phosphatase activity in the nucleus can regulate mammalian target of rapamycin signaling and neuronal growth.

Role of insertion sequence Aba-1 and AdeS in reduced tigecycline susceptibility in MDR-Acinetobacter baumannii clinical isolates from Cairo, Egypt

Infections caused by multidrug resistant (MDR) Acinetobacter baumannii (A. baumannii) especially in intensive care units have limited therapeutic options. Overexpression of the adeABC efflux pump may be caused either by the ISAba-1 insertion or by specific point mutations in adeR and adeS, therefore, plays a major role in conferring MDR-A. baumannii. We aimed in our study to monitor the tigecycline (TGC) susceptibility and to study the role of ISAba-1 and the adeS regulator within the AdeABC efflux pump among MDR-A. baumannii clinical isolates. MDR-A. baumannii (63) isolated from ICU patients were identified by detection of OXA-51-like gene. TGC MIC was determined by E-test and broth microdilution. PCR analysis of adeR, adeS, adeB and ISAba1 genes were done with further sequencing of adeS gene. Reduced susceptibility to TGC (MIC: 3–4 mg/L) was noticed in 6/63 (9.5%) MDR-A. baumannii isolates, ISAba-1 was detected in three isolates that two of which showed amino acid substitutions in the adeS operon. We concluded that the amino acids mutations in the adeS gene in presence of insertion ISAba-1 may play a role in conferring reduced TGC susceptibility of MDR-A. baumannii.

CADASIL: A Case Report with an Atypical Presentation and Novel Mutation

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy CADASIL is a small vessel disease that commonly presents with ischemic episodes cognitive deficits migraine with aura and psychiatric disturbances It is caused by mutations in the NOTCH gene of which several specific point mutations have been identified We report a year old female with a confirmed CADASIL diagnosis an atypical presentation and an unreported NOTCH mutation She presented with stroke like symptoms and MRI was initially read as several subacute infarcts throughout the brain She denied any history of headaches A review of her imaging suggested CADASIL as a more likely diagnosis She underwent NOTCH testing which showed a pathogenic p Arg Cys mutation and a previously unreported p Pro Leu mutation

Analysis of Point Mutations in Clinical Samples of Urothelial Carcinoma.

In the last two decades specific point mutations in oncogenes have been identified in urinary bladder cancers. Identification of these mutations in clinical samples (e.g., urine or tumor tissue) can be of use for diagnostic or prognostic purposes. In this chapter we describe how mutations in multiple oncogenes can be identified with a simple assay.

Terbinafine Resistance of Trichophyton Clinical Isolates Caused by Specific Point Mutations in the Squalene Epoxidase Gene.

Terbinafine is one of the allylamine antifungal agents whose target is squalene epoxidase (SQLE). This agent has been extensively used in the therapy of dermatophyte infections. The incidence of patients with tinea pedis or unguium tolerant to terbinafine treatment prompted us to screen the terbinafine resistance of all Trichophyton clinical isolates from the laboratory of the Centre Hospitalier Universitaire Vaudois collected over a 3-year period and to identify their mechanism of resistance. Among 2,056 tested isolates, 17 (≈1%) showed reduced terbinafine susceptibility, and all of these were found to harbor SQLE gene alleles with different single point mutations, leading to single amino acid substitutions at one of four positions (Leu393, Phe397, Phe415, and His440) of the SQLE protein. Point mutations leading to the corresponding amino acid substitutions were introduced into the endogenous SQLE gene of a terbinafine-sensitive Arthroderma vanbreuseghemii (formerly Trichophyton mentagrophytes) strain. All of the generated A. vanbreuseghemii transformants expressing mutated SQLE proteins exhibited obvious terbinafine-resistant phenotypes compared to the phenotypes of the parent strain and of transformants expressing wild-type SQLE proteins. Nearly identical phenotypes were also observed in A. vanbreuseghemii transformants expressing mutant forms of Trichophyton rubrum SQLE proteins. Considering that the genome size of dermatophytes is about 22 Mb, the frequency of terbinafine-resistant clinical isolates was strikingly high. Increased exposure to antifungal drugs could favor the generation of resistant strains.

Baculoviral delivery of CRISPR/Cas9 facilitates efficient genome editing in human cells.

The CRISPR/Cas9 system is a highly effective tool for genome editing. Key to robust genome editing is the efficient delivery of the CRISPR/Cas9 machinery. Viral delivery systems are efficient vehicles for the transduction of foreign genes but commonly used viral vectors suffer from a limited capacity in the genetic information they can carry. Baculovirus however is capable of carrying large exogenous DNA fragments. Here we investigate the use of baculoviral vectors as a delivery vehicle for CRISPR/Cas9 based genome-editing tools. We demonstrate transduction of a panel of cell lines with Cas9 and an sgRNA sequence, which results in efficient knockout of all four targeted subunits of the chromosomal passenger complex (CPC). We further show that introduction of a homology directed repair template into the same CRISPR/Cas9 baculovirus facilitates introduction of specific point mutations and endogenous gene tags. Tagging of the CPC recruitment factor Haspin with the fluorescent reporter YFP allowed us to study its native localization as well as recruitment to the cohesin subunit Pds5B.

Target-Site Resistance to Fenoxaprop-P-ethyl in Keng Stiffgrass (Sclerochloa kengiana) from China

Keng stiffgrass is a serious farmland grass weed distributed globally in winter wheat fields and rice–wheat double-cropping areas. The intensive use of acetyl-CoA carboxylase (ACCase)-inhibiting herbicides has led to the evolution of resistance in a growing number of grass weeds. In this study, whole-plant pot bioassay experiments were conducted to establish that a Keng stiffgrass population from eastern China, JYJD-2, has evolved high-level resistance to fenoxaprop-P-ethyl and moderate resistance to quizalofop-P-ethyl and pinoxaden. Using the derived cleaved amplified polymorphic sequence method, a tryptophan-to-cysteine mutation at codon position 1999 (W1999C) was detected in the ACCase gene of the resistant population JYJD-2. Of the 100 JYJD-2 plants tested, we found 47 heterozygous resistant and 53 homozygous sensitive individuals. In vitro ACCase assays revealed that the IC50value of the ACCase activity of the resistant population JYJD-2 was 6.48-fold higher than that of the susceptible population JYJD-1. To the best of our knowledge, this is the first report of the occurrence of W1999C mutation in the ACCase gene of fenoxaprop-P-ethyl–resistant Keng stiffgrass. This study confirmed the resistance of Keng stiffgrass to the ACCase inhibitor fenoxaprop-P-ethyl, cross-resistance to other ACCase inhibitors, and the resistance being conferred by specific ACCase point mutations at amino acid position 1999.

Abstract 55: Disruption of PCSK9 Using a Targeted Base Editing Strategy

Proprotein convertase subtilisin/kexin type 9 (PCSK9) increases blood low-density lipoprotein (LDL) cholesterol by acting as an LDL receptor antagonist, thereby impairing LDL particle clearance. Since genetic disruption of PCSK9 is linked to reduced risk of coronary heart disease (CHD), our recent work has sought to permanently knock out the gene by using new genome editing technology. Recently reported “base editors” introduce point mutations at specific locations in the genome without the need for DNA double-strand breaks and, thus, with a lowered incidence of off-target effects. These base editors build on the CRISPR-Cas9 system by tethering an RNA-editing domain to a nickase version of Cas9, allowing for specific CT and/or GA base alterations. In this study, we used the “BE3” base editor (which uses the APOBEC-1 RNA-editing domain) to specifically target codons encoding tryptophans (TGG) or glutamines (CAG or CAA) to introduce nonsense mutations (producing stop codons TAG, TGA, or TAA) into human PCSK9 . The number of testable targets was increased by introducing specific point mutations into the BE3 construct (D1135V, R1335Q, T1337R in the Cas9 portion of BE3) to alter the protospacer adjacent motif (PAM) from NGG to NGA. Using HEK293 cells, we individually targeted a number of codons spanning the first seven exons in PCSK9 and identified several efficient targets. Most notably, the codons encoding glutamine 278 and glutamine 302 could be altered to stop codons in ~50% of alleles, as determined by the CEL-I nuclease mismatch assay and Sanger sequencing. We then targeted glutamine 278 in human induced pluripotent stem cells (iPSCs) and demonstrated base editing to introduce nonsense mutations into PCSK9 , albeit at a lower efficiency than in HEK293 cells. As a next step towards translation to human patients, we are targeting Pcsk9 in the mouse liver in vivo with BE3. Base editing may prove to be an efficient, safer strategy than standard CRISPR-Cas9 genome editing and holds promise as a strategy for the prevention of CHD.

Nicotinic alpha 7 receptor expression and modulation of the lung epithelial response to lipopolysaccharide.

Nicotine modulates multiple inflammatory responses in the lung through the nicotinic acetylcholine receptor subtype alpha7 (α7). Previously we reported that α7 modulates both the hematopoietic and epithelium responses in the lung to the bacterial inflammogen, lipopolysaccharide (LPS). Here we apply immunohistochemistry, flow cytometry and RNA-Seq analysis of isolated distal lung epithelium to further define α7-expression and function in this tissue. Mouse lines were used that co-express a bicistronic tau-green fluorescent protein (tGFP) as a reporter of α7 (α7G) expression and that harbor an α7 with a specific point mutation (α7E260A:G) that selectively uncouples it from cell calcium-signaling mechanisms. The tGFP reporter reveals strong cell-specific α7-expression by alveolar macrophages (AM), Club cells and ATII cells. Ciliated cells do not express detectible tGFP, but their numbers decrease by one-third in the α7E260A:G lung compared to controls. Transcriptional comparisons (RNA-Seq) between α7G and α7E260A:G enriched lung epithelium 24 hours after challenge with either intra-nasal (i.n.) saline or LPS reveals a robust α7-genotype impact on both the stasis and inflammatory response of this tissue. Overall the α7E260A:G lung epithelium exhibits reduced inflammatory cytokine/chemokine expression to i.n. LPS. Transcripts specific to Club cells (e.g., CC10, secretoglobins and Muc5b) or to ATII cells (e.g., surfactant proteins) were constitutively decreased in in the α7E260A:G lung, but they were strongly induced in response to i.n. LPS. Protein analysis applying immunohistochemistry and ELISA also revealed α7-associated differences suggested by RNA-Seq including altered mucin protein 5b (Muc5b) accumulation in the α7E260A:G bronchia, that in some cases appeared to form airway plugs, and a substantial increase in extracellular matrix deposits around α7E260A:G airway bronchia linings that was not seen in controls. Our results show that α7 is an important modulator of normal gene expression stasis and the response to an inhaled inflammogen in the distal lung epithelium. Further, when normal α7 signaling is disrupted, changes in lung gene expression resemble those associated with long-term lung pathologies seen in humans who use inhaled nicotine products.

Analysis of three alleles of the vestigial gene in Drosophila melanogaster.

The vestigial wing gene in D. melanogaster is a popular locus used to illustrate classical Mendelian inheritance patterns because the short, crinkled-wing phenotype directed by the vg1 allele is easy to identify. Carolina Biological notes that its stock of vestigial wing flies also has flies with more developed, but still under-developed wings, the more common phenotype being a narrow plank-shaped transparent wing. A line of flies was bred that only displayed the “plank-wing” phenotype. Genetic analysis of the mutant allele, vgpl, showed that it was recessive to the wild-type allele vg+, and mutants displayed variable expressivity. PCR products were generated from all three alleles: the products from wild-type DNA covered the whole gene while products from the mutant DNAs covered about 75% of the gene. The presence of retrotransposon 412 was confirmed in vg1 and also found in vgpl; a reported second large insert in the vg1 remains to be amplified. Sanger DNA sequencing of the first 4,812 bp, beginning 428 bp upstream of the transcription start site revealed allele specific point mutations and small insertions, some specific for each of the vg1 and vgpl alleles. The DNA sequence analysis so far confirmed the partial genetic reversion of the vg1 to a new vgpl allele. When complete, a full DNA sequence analysis should suggest nucleotide changes that cause the new “plank wing” phenotype.This work was funded in part by the Research Challenge Fund through a Summer Undergraduate Research Experience Grant from the West Virginia Higher Education Policy Commission Division of Science and Research.

Identifying Key Domains in Endothelial IQGAP1 Critical for Leukocyte Transmigration

Inflammation results in the dilation of local blood vessels, the activation of endothelium by cytokines and the recruitment of circulating leukocytes into areas of tissue damage. The majority of leukocytes undergo transendothelial migration (TEM) by using a unique endothelial cell membrane system referred to as the lateral border recycling compartment (LBRC). IQ‐domain GTPase‐activating protein 1 (IQGAP1) was identified as an LBRC‐associated protein and subsequently found to be essential for TEM. IQGAP1 is a scaffolding protein comprised of six major interaction domains. It has been reported to interact productively with over 135 cellular proteins. In endothelial cells IQGAP1 partially localizes to cell borders and knocking down its expression prevents TEM. However, it is not known which of the many proteins it is known to interact with carries out its role in TEM. Since these proteins bind to specific IQGAP1 domains, as a first step toward understanding the mechanism by which IQGAP1 regulates TEM, we explored which of its six protein interactions domains were critical for its function. We knocked down IQGAP1 in endothelial cells and re‐expressed truncated forms of IQGAP1 with specific regions deleted to identify the regions (and thereby the potential binding partners) that are critical for TEM. We show that the actin‐binding domain and the calmodulin‐binding isoleucine‐glutamine (IQ) domain of IQGAP1 are critical for its function during TEM.Human endothelial cells in which &gt;80% of endogenous IQGAP1 was depleted by shRNA did not support TEM; re‐expression of wild‐type IQGAP1 restored TEM to control levels. Re‐expression of IQGAP1 with a GFP‐tagged construct after knockdown also showed IQGAP1 enrichment around transmigrating leukocytes. Re‐expression of IQGAP1 truncation mutants lacking the N‐terminal actin‐binding domain did not localize to endothelial borders and did not support TEM. Re‐expression of truncated IQGAP1 constructs containing the actin‐binding domain and the IQ domain localized to cell borders and supported TEM. Constructs containing the actin‐binding domain but lacking the IQ domain localized to the cell borders but did not restore TEM.After identifying the IQ domain as having a critical role in TEM, we generated specific point mutations in this domain within the full‐length construct. In particular, the isoleucine (I)‐glutamine (Q) motif after which the IQ domain is named has been identified in the literature as having an essential role in IQGAP1 function and we are currently testing whether they are also important in TEM. Using these constructs in future studies will help identify the role of the IQ domain in TEM and its interacting partner(s). Identifying the binding partners of IQGAP1 that are critical for inflammation will provide clues to its mechanism of action and potential therapeutic targets for anti‐inflammatory therapy.Support or Funding InformationFunded by R01 HL046849, R01 HL064774, Alpha Omega Alpha Carolyn Kuckein Student Research Fellowship

Phenotypic and Molecular Antimicrobial Susceptibility of Helicobacter pylori.

Failure to eradicate Helicobacter pylori infection is often a result of antimicrobial resistance, which for clarithromycin is typically mediated by specific point mutations in the 23S rRNA gene. The purpose of this study was to define current patterns of antimicrobial susceptibility in H. pylori isolates derived primarily from the United States and to survey them for the presence of point mutations in the 23S rRNA gene and assess the ability of these mutations to predict phenotypic clarithromycin susceptibility. Antimicrobial susceptibility testing was performed using agar dilution on 413 H. pylori isolates submitted to Mayo Medical Laboratories for susceptibility testing. For a subset of these isolates, a 150-bp segment of the 23S rRNA gene was sequenced. A total of 1,970 MICs were reported over the 4-year study period. The rate of clarithromycin resistance was high (70.4%), and elevated MICs were frequently observed for metronidazole (82.4% of isolates had an MIC of >8 μg/ml) and ciprofloxacin (53.5% of isolates had an MIC of >1 μg/ml). A total of 111 archived H. pylori isolates underwent 23S rRNA gene sequencing; we found 95% concordance between genotypes and phenotypes (P = 0.9802). Resistance to clarithromycin was most commonly due to an A2143G mutation (82%), followed by A2142G (14%) and A2142C (4%) mutations. Clinical H. pylori isolates derived primarily from the United States demonstrated a high rate of clarithromycin resistance and elevated metronidazole and ciprofloxacin MICs. The relative distribution of point mutations at positions 2143 and 2142 in the 23S rRNA gene in clarithromycin-resistant H. pylori was similar to that reported from other parts of the world; these mutations predict phenotypic resistance to clarithromycin.

Specific mutations in KRAS codon 12 are associated with worse overall survival in patients with advanced and recurrent colorectal cancer

Background:Activating mutations in KRAS have been suggested as potential predictive and prognostic biomarkers. However, the prognostic impact of specific point mutations remains less clear. This study assessed the prognostic impact of specific KRAS mutations on survival for patients with colorectal cancer.Methods:Retrospective review of patients KRAS typed for advanced and recurrent colorectal cancer between 2010 and 2015 in a UK Cancer Network.Results:We evaluated the impact of KRAS genotype in 392 patients. Mutated KRAS was detected in 42.9% of tumours. KRAS mutations were more common in moderate vs well-differentiated tumours. On multivariate analysis, primary tumour T stage (HR 2.77 (1.54–4.98), P=0.001), N stage (HR 1.51 (1.01–2.26), P=0.04), curative intent surgery (HR 0.51 (0.34–0.76), P=0.001), tumour grade (HR 0.44 (0.30–0.65), P=0.001) and KRAS mutation (1.54 (1.23–2.12), P=0.005) were all predictive of overall survival. Patients with KRAS codon 12 mutations had worse overall survival (HR 1.76 (95% CI 1.27–2.43), P=0.001). Among the five most common codon 12 mutations, only p.G12C (HR 2.21 (1.15–4.25), P=0.01) and p.G12V (HR 1.69 (1.08–2.62), P=0.02) were predictive of overall survival.Conclusions:For patients with colorectal cancer, p.G12C and p.G12V mutations in codon 12 were independently associated with worse overall survival after diagnosis.

Pinpoint the Positions of Single Nucleotide Polymorphisms by a Nanocluster Dimer.

Single nucleotide polymorphisms (SNPs) are the most fundamental internal causes for many genetic diseases. However, the location information on SNPs in a specific DNA sequence is not well acquired through current SNPs detection methods, except for accurate DNA sequencing. Here we report a fluorescence enhancement phenomenon in the process of two silver nanoclusters (AgNCs) approaching closely to form a nanocluster dimer (NCD). The fluorescence intensity is sensitive to the distance between two AgNCs; therefore, the NCD lights into different fluorescence intensities upon binding SNPs targets with mismatched bases at different positions. Interestingly, the fluorescence intensities of the NCD decrease linearly when the position of single mismatched base moves gradually from the middle point to the end of the target DNA. The NCD is a single probe acting as a universal platform to pinpoint various SNP positions. With this single probe, we cannot only identify the existence of SNPs but also pinpoint the location of a specific single mismatched base in the adjacent positions. This strategy is feasible to detect specific gene point mutations in clinical samples.