High-resolution Melting Curve Analysis Research Articles

Molecular Characterization of Mycoplasma Synoviae Isolated From the Respiratory System and Joints of Chickens with Special Reference of vlhA Gene

The present work was designed to make sequencing of M. synoviae isolates for identification of nucleotide differences in the M. synoviae vlhA gene which isolated from the respiratory system and Joint from chickens. A total of 224 chicken samples (109 respiratory samples, 115 joint samples) collected from birds from different flocks showing respiratory manifestation and arthritis were examined. Identification of M. synoviae by PCR Using primers corresponding to the single copy preserved 5´ end for vlhA genetic factor, amplicons for 350~400 bp then, generated which 6 and 26 out of 224 samples were positive samples for M. synoviae from culture and tissue by PCR respectively. High-resolution melting curve analysis (HRM) for amplicons using SYBR green fluorescent dye of 9 selected isolates (5 respiratory isolates and 4 arthirtic isolates) showed that different Melting temperature curves were ranged between 84.6-85.1°C for isolates MS from the respiratory samples and other melting temperature curves ranged between 73.6-74.6°Cfor isolates Ms from the arthirtic samples. The results proved that, there was a broad concordance among nucleotide sequence of all isolates which isolated from the respiratory system except in one sample which observed the point mutations and the frame-shift mutation in some nucleotide differ than other respiratory isolates in addition there is complete concordance between nucleotide sequence of all isolates which isolated from joint. However, in comparison the Respiratory M. synoviae isolates with M. synoviae joint isolates, there was clear variation in nucleotide sequence which was confirm the result of HRM. Compared to vaccine MS-H strain arrangement, all isolates show clear variation from (MsH) vaccine strain. This study proved a difference between M. synoviae isolated from the respiratory system and Joint and live commercial vaccine (MsH) strain. Also, these informations showed that variations in the vlhA gene sequence could be introduced into the expressed vlhA gene amino acid codons and effective in pathogenesis rate in herds.

Molecular diagnosis in type I epithelial ovarian cancer.

The term epithelial ovarian cancer (EOC) refers to a heterogeneous group of tumors, including serous, mucinous, endometrioid and clear cell carcinomas, each characterized by specific molecular background and clinical outcome. A growing body of evidence suggests that molecular pathogenesis of ovarian cancer involves two general pathways. The first pathway results in transformation of normal ovarian tissue to borderline tumors, which may further progress to low-grade serous, mucinous, endometrioid and clear cell carcinomas. Tumors from this group are characterized by slow proliferation, and approximately 55% 5-year survival rate. Type I tumors often harbor somatic mutations in protein kinase genes, as well as in genes for other signaling molecules. Both BRAF and KRAS mutations lead to a constitutive activation of their downstream target, mitogen-activated protein kinase. Identification of molecular profile may be crucial for the diagnosis of ovarian tumors, choice of adjuvant targeted therapy after primary cytoreductive treatment, or management of recurrence in patients with advanced type I epithelial ovarian neoplasms. Point mutations in cancer cells can be detected with many various methods. KRAS, NRAS and BRAF mutational status can be determined by Sanger sequencing (still considered a gold standard), as well as using numerous various techniques, such as allele-specific PCR, single nucleotide primer extension assays, pyrosequencing, real-time PCR, high-resolution melting curve analysis, amplification refractory mutation system, strip or chip assay combining PCR followed by hybridization to a KRAS or NRAS-specific probe, next-generation sequencing (NGS), and matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Application of direct sequencing as a routine method for cytological diagnosis used in a hospital setting requires expensive equipment and implementation of complicated procedures. Another factor limiting application of this method in everyday clinical practice are long analytical times. This stimulated search for a simple, rapid, specific and sensitive methodology to detect point mutations. Recently, some new molecular assays for the detection of BRAF, KRAS and NRAS mutations have become available. These are fully-automated molecular diagnostic systems for quantitative allele-specific RT-PCR-based analyses. Using this instrument, even pathologists from less experienced laboratories can easily integrate morphological findings with molecular data being crucial for further diagnostic and therapeutic decisions.

Real-time PCR and high-resolution melt analysis methods for detection of pathogenic species of Brucella

Abstract Background: It is of great importance to quickly and accurately detect Brucella abortus and Brucella melitensis from clinical and non-clinical samples because of their high prevalence and high risk in causing brucellosis, a life-threating infectious disease affecting both humans and animals. Methods: The current study describes a new method for the detection of brucellosis in clinical samples using real-time polymerase chain reaction (PCR) and high-resolution melt (HRM) curve analysis. This study was conducted on 70 human and 55 animal isolates with more than 1/80 serum antibody titers. Additionally, the accuracy and specificity of the methods were compared. Results: The mean range [cycles threshold±standard deviation (CT±SD)] for the amplified samples was 15.39–25.15 by real-time PCR. The melting peak range (°C) ±SD for B. abortus and B. melitensis was 90.10±0.4 and 89.70±0.4, respectively, and 10 was reported on peak height. Conclusions: The results of HRM analysis can be used for species differentiation and bacterial genotyping according to nucleotide polymorphism. This molecular method could help in diagnosing Brucella quickly and precisely. Quick recognition of Brucella species could decrease its prevalence among humans and animals and mitigate economic loss.

Asymptomatic infections with highly polymorphic Chlamydia suis are ubiquitous in pigs

BackgroundChlamydia suis is an important, globally distributed, highly prevalent and diverse obligate intracellular pathogen infecting pigs. To investigate the prevalence and genetic diversity of C. suis in China, 2,137 nasal, conjunctival, and rectal swabs as well as whole blood and lung samples of pigs were collected in 19 regions from ten provinces of China in this study.ResultsWe report an overall positivity of 62.4% (1,334/2,137) of C. suis following screening by Chlamydia spp. 23S rRNA-based FRET-PCR and high-resolution melting curve analysis and confirmatory sequencing. For C. suis-positive samples, 33.3 % of whole blood and 62.5% of rectal swabs were found to be positive for the C. suis tetR(C) gene, while 13.3% of whole blood and 87.0% of rectal swabs were positive for the C. suis tet(C) gene. Phylogenetic comparison of partial C. suis ompA gene sequences revealed significant genetic diversity in the C. suis strains. This genetic diversity was confirmed by C. suis-specific multilocus sequence typing (MLST), which identified 26 novel sequence types among 27 examined strains. Tanglegrams based on MLST and ompA sequences provided evidence of C. suis recombination amongst the strains analyzed.ConclusionsGenetically highly diverse C. suis strains are exceedingly prevalent in pigs. As it stands, the potential pathogenic effect of C. suis on pig health and production of C. suis remains unclear and will be the subject of further investigations. Further study is also required to address the transmission of C. suis between pigs and the risk of 'spill-over' and 'spill-back' of infections to wild animals and humans.

Assessment of gene copy number variation of Scots pine thaumatin-like protein gene using real-time PCR based methods

The importance and impact of gene copy number variations (CNVs) as a source of polymorphism in the human and other genomes is being increasingly recognized. Less information is available about CNVs in forest tree species, mainly due to the relative lack of genomic resources. In this study, several methods—quantitative polymerase chain reaction, comparative high-resolution melting curve analysis (C-HRM), and digital polymerase chain reaction (dPCR)—were used to investigate CNV of the Scots pine thaumatin-like protein gene (PsTLP). The obtained results were supported by transcriptome analysis of a single Pinus sylvestris individual and publically available pine genome sequences. Although estimations of gene copy number (CN) varied, depending on the region of the PsTLP gene investigated and the endogenous control utilized, our results revealed the existence of copy number variations of the PsTLP gene between Scots pine individuals. Of 23 individuals analyzed, two had an increased calculated relative CN regardless of the analyzed gene region and endogenous control used, while several samples had increased copy numbers of regions of the PsTLP gene. C-HRM results were highly correlated with qPCR data (R 2 TLP3′ = 0.88; R 2 TLPc = 0.92), but interpretation of gene CN from C-HRM results proved to be difficult. The results from selected samples analyzed by digital PCR also were highly correlated with qPCR results (R 2 = 0.90).

Mutations of candidate tumor suppressor genes at chromosome 3p in intrahepatic cholangiocarcinoma

The genetic status of candidate tumor suppressor genes (TSGs) at chromosome 3p has not yet been elucidated in intrahepatic cholangiocarcinoma (iCCA). Herein, we retrospectively investigated 32 fresh iCCA case samples from a single medical institution to clarify mutations of 11 TSGs by next-generation sequencing. Validation of the mutations was performed on the MassARRAY platform or by high-resolution melting curve analysis. We then integrated the gene mutations into copy number alterations at chromosome 3p that had been generated in a previous study using the same fresh iCCA samples, and correlated the integration results with the clinicopathologic features. Nine of the 32 (28.1%) iCCA patients had gene mutations at chromosome 3p, totaling 11 mutations across five genes. Those included five (15.6%) BAP1 mutations, two each (6.3%) of CACNA2D3 and RASSF1 mutations, and one each (3.1%) of ATG7 and PLCD1 mutations. Six (18.8%) cases had concurrent loss of chromosome 3p and gene mutations. Patients with TSG mutations had shorter disease-free and survival times than those without the mutations. Our data showed that iCCA patients with TSG mutations at chromosome 3p faced an adverse prognosis. BAP1 was the common target of mutational inactivation and may be a principal driver of 3p21 losses.

Association of interleukin-28B polymorphisms with platelet count and liver function recovery after liver transplant.

The present genome-wide association study investigated the relationship of interleukin 28B (IL-28B) genetic variants with HBV susceptibility and prognosis of HBV-infected patients. This study aims to examine the role of IL-28B polymorphisms on transplant etiologies and the liver function recovery in Chinese liver transplant recipients.A total of 231 liver transplant recipients were enrolled in the study. The transplant etiologies included progressive HBV hepatitis, HBV-related liver cirrhosis (LC), HBV-related hepatocellular carcinoma (HCC), and non-HBV-related disease. All recipients were in stable condition before transplantation. Three single nucleotide polymorphisms (SNPs) of IL-28B (rs12979860, rs12980275, rs8099917) of recipients were analyzed by high-resolution melting (HRM) curve analysis. Liver function, blood cell count, and coagulation function were regularly tested before and for next 5 years after transplantation.No significant association was found between IL-28B gene polymorphisms and transplant etiologies. Peripheral platelet count in the third and fourth days after transplantation were significantly higher in recipients carrying IL-28B rs12979860 T allele, or rs8099917 C allele (P < .016666667), while there were no significant differences between these variants and International Normalized Ratio (INR) levels. In addition, gamma-glutamyltransferase (GGT) levels in recipients with rs12980275 G allele were higher than those in the wide-type recipients before transplantation (P < .016666667, respectively); nevertheless, no influence of these variants on GGT recovery was observed after transplantation.Genetic variations of IL-28B might impact on liver function recovery by influencing peripheral platelet counts and reducing liver inflammation, but have weak association with transplant etiologies.

Differentiation of minute virus of mice and mouse parvovirus by high resolution melting curve analysis

Murine parvovirus is one of the most prevalent infectious pathogens in mouse colonies. A specific primer pair targeting the VP2 gene of minute virus of mice (MVM) and mouse parvovirus (MPV) was utilized for high resolution melting (HRM) analysis. The resulting melting curves could distinguish these two virus strains and there was no detectable amplification of the other mouse pathogens which included rat parvovirus (KRV), ectromelia virus (ECT), mouse adenovirus (MAD), mouse cytomegalovirus (MCMV), polyoma virus (Poly), Helicobactor hepaticus (H. hepaticus) and Salmonella typhimurium (S. typhimurium). The detection limit of the standard was 10 copies/μL. This study showed that the PCR-HRM assay could be an alternative useful method with high specificity and sensitivity for differentiating murine parvovirus strains MVM and MPV.

ID: 1036 FAM134B, a new player in human colorectal cancer pathogenesis

Background: FAM134B (family with sequence similarity 134, member B) is a relatively new player in many human diseases including cancers. In colorectal carcinomas, FAM134B plays important role in the pathogenesis and associated with biological aggressiveness of the disease. However, expression pattern, the frequency of mutations, methylation status in colon cancer cells and tissue samples has never been studied. Also, the functional roles of FAM134B in cell have never been studied in colorectal cancer.&#x0D; Objectives: To investigate FAM134B promoter methylation, mutations in tissues samples from patients with colorectal cancer and cell lines. Also, promoter methylation, expression and functional roles of FAM134B in colon cancer were studied.&#x0D; Methods: Methylation and mutations in FAM134B sequence in cancer tissues (n=126) and matched non-cancer samples was studied by high-resolution melt curve analysis followed by Sanger sequencing. FAM134B expression was studied and quantified in cell lines and cancer tissues samples using immunofluorescence, immunocytochemistry, Western blot and real time PCR. In vitro functional assays and mouse xenotransplantation model were performed to unveil the molecular roles of FAM134B in colon cancer pathogenesis followed by shRNA-mediated silencing in cells.&#x0D; Results: We noted that 46.5% (41/88) patients with colorectal cancer were identified as FAM134B mutations positive. Thirty-one novel pathogenic mutations were detected and these mutations were associated with gender of the patients, presence of metachronous cancer, size, T staging, presence of distant metastases and positivity of microsatellite instability (MSI) in the cancer (p &lt; 0.05). Majority of cancer tissues had shown promoter hyper-methylation and were correlated with reduced mRNA and protein expression in both cancer samples and cells. FAM134B expression in cancer cells derived from advanced stages (stage III; SW48 and stage IV; HCT116) of colon cancer was significantly (p&lt;0.01) reduced when compared to non-neoplastic colon cells (FHC) and cancer cells derived from stage II colon cancer (SW480). Expression of FAM134B mRNA in cancer tissues was noted significantly (p&lt;0.001) downregulated when compared to that of non-cancer tissues samples. FAM134B suppression significantly (p&lt;0.05) increased the proliferation of colon cancer cells, remarkably increased (3452% ; p&lt;0.05) the clonogenic, migration capacity, and increases the proportion of cells in S phase of cell cycle (p&lt;0.01). Xenotransplantation model showed that larger and higher grade tumors were formed in mice treated with FAM134B knockdown cells.&#x0D; Conclusion: Reduced expression in cancer samples, in vitro and in vivo functional studies implied that FAM134B acts as a cancer inhibitor in colon cancer play important roles in colorectal carcinogenesis.

Identification of a new genetic marker in Mycoplasma synoviae vaccine strain MS-H and development of a strategy using polymerase chain reaction and high-resolution melting curve analysis for differentiating MS-H from field strains

Mycoplasma synoviae (MS) is an economically important avian pathogen worldwide, causing subclinical respiratory tract infection and infectious synovitis in chickens and turkeys. A temperature-sensitive (ts+) live attenuated vaccine MS-H, derived from the Australian field strain 86079/7NS, is now widely used in many countries to control the disease induced by MS. Differentiation of MS-H vaccine from field strains is crucial for monitoring vaccination programs in commercial poultry. Comparison of genomic sequences of MS-H and its parent strain revealed an adenine deletion at nucleotide position 468 of the MS-H oppF-1 gene. This mutation was shown to be unique to MS-H in further comparative analyses of oppF-1 genes of MS-H re-isolates and field strains from Australia and other countries. Based on this single nucleotide, a combination of nested PCR and high-resolution melting (HRM) curve analysis was used to evaluate its potential for use in differentiation of MS-H from field strains. The mean genotype confidence percentages of 99.27 and 48.20 for MS-H and field strains, respectively, demonstrated the high discriminative power of the newly developed assay (oppF PCR-HRM). A set of 13 tracheal swab samples collected from MS-H vaccinated specific pathogen free birds and commercial chicken flocks infected with MS were tested using the oppF PCR-HRM test and results were totally consistent with those obtained using vlhA genotyping. The nested-PCR HRM method established in this study proved to be a rapid, simple and cost effective tool for discriminating the MS-H vaccine strain from Australian and international strains in pure cultures and on tracheal swabs.

Evaluation of a Role for NPY and NPY2R in the Pathogenesis of Obesity by Mutation and Copy Number Variation Analysis in Obese Children and Adolescents.

Neuropeptide Y (NPY) and its G protein-coupled NPY Y2 Receptor (NPY2R) are highly expressed in orexigenic NPY/Agouti-related peptide neurons within the arcuate nucleus, a major integrator of appetite control in the hypothalamus. As NPY and NPY2R are interesting candidate genes for obesity, we hypothesized that a genetic variation in these genes might be implicated in the pathogenesis of obesity. In the first part of this study, we performed a mutation analysis of the coding region of NPY and NPY2R with high-resolution melting curve analysis. For the highly conserved NPY gene, an extended population of 436 obese children and adolescents was screened, while for NPY2R, a smaller subset of 306 patients was used. A control population of 300 healthy individuals was screened for NPY2R to determine the general prevalence of the variants found among patients. Direct sequencing was performed for samples with melting patterns deviating from wild-type. In the second part of this study, Multiplex Amplicon Quantification (MAQ) analysis was performed in 308 obese children and adolescents to detect copy number variation (CNV) in the NPY2R region. Mutation analysis of the NPY gene led to the identification of one common missense variant (L7P; MAF 0.04), while the screening of the NPY2R gene resulted in the identification of one rare missense variant F87I in the patient population. In our CNV analysis, we could not identify copy number variation in the NPY2R region among obese children and adolescents. In summary, this study clearly indicates that genetic variation in NPY and NPY2R is at low frequency and thus does not make a major contribution to the obese phenotype in the general population.

Multiple genetic variants associated with posttransplantation diabetes mellitus in Chinese Han populations.

Posttransplantation diabetes mellitus (PTDM) is a major complication after solid organ transplantation. This study is to investigate the association of nine genetic variant factors and PTDM in Chinese Han patients. HLA-DP (rs3077, rs9277535), HLA-DQ (rs7453920), signal transducer and activator of transcription 4 (STAT4) (rs7574865), IL-28B (rs12979860, rs8099917, and rs12980275), and IL-18 (rs1946518 and rs187238) were investigated in 260 liver transplant recipients (PTDM vs non-PTDM) by high-resolution melting curve analysis. Serum interleukin (IL)-1β, IL-6, IL-8, IL-17, interferon-γ, inducible protein-10, monocyte chemoattractant protein-1, and macrophage inflammatory protein-1b were analyzed by a Bio-Plex suspension array system (Bio-Plex Multiplex Immunoassays, Bio-Rad, Hercules, CA, USA). Signal transducer and activator of transcription 4 (rs7574865) T allele and IL-18 (rs1946518) A allele increase the risk for insulin resistance and PTDM. Recipients with STAT4 (rs7574865) T allele are associated with an increased concentration of IL-1β, interferon-γ, monocyte chemoattractant protein, and macrophage inflammatory protein-1b. The genetic variants of STAT4 (rs7574865) and IL-18 (rs1946518) may be new important markers for PTDM.

Epigenetic discrimination of identical twins from blood under the forensic scenario

Monozygotic (MZ) twins share the same STR profile, demonstrating a practical problem in forensic casework. DNA methylation has provided a suitable resource for MZ twin differentiation; however, studies addressing the forensic feasibility are lacking. Here, we investigated epigenetic MZ twin differentiation from blood under the forensic scenario comprising i) the discovery of candidate markers in reference-type blood DNA via genome-wide analysis, ii) the technical validation of candidate markers in reference-type blood DNA using a suitable targeted method, and iii) the analysis of the validated markers in trace-type DNA. Genome-wide methylation analysis in blood DNA from 10 MZ twin pairs resulted in 19–111 twin-differentially methylated sites (tDMSs) per pair with >0.3 twin-to-twin differences. Considering all top three candidate tDMSs across all pairs in the technical validation based on methylation-specific qPCR, 67.85% generated >0.1 twin-to-twin differences. Of the validated tDMSs, 68.4% showed >0.1 twin-to-twin differences with qPCR in trace-type DNA across 8 pairs. Using an updated marker selection strategy, 8 additional candidate tDMSs were obtained for an example MZ pair, of which 7 showed >0.1 twin-to-twin differences in both reference- and trace-type DNA. Lastly, we introduce a high-resolution melting curve analysis of the entire fragment that can complement the proposed approach. Overall, our study demonstrates the general feasibility of epigenetic twin differentiation in the forensic context and highlights that the number of informative tDMSs in the final trace DNA analysis is crucial, as some candidate markers identified in reference DNA were shown not informative in the trace DNA due to various, including technical, reasons. Future studies will need to address the optimal number of epigenetic markers required for reliable identification of MZ twin individuals including statistical considerations.

Topical equol preparation improves structural and molecular skin parameters.

Equol has been shown to improve skin health and regeneration, due to its antioxidative, phytoestrogenic and epigenetic characteristics. The effects of a topical intervention on skin structure, telomere length and epigenetic markers in skin cells were analysed. Sixty-four participants were divided in four groups and three of them treated topically with the following: emulsion with Equol powder (Isoflavandiol-E-55-RS®); emulsion with microencapsulated Equol (Vesisorb® Isoflavandiol-E-55-RS®) and an emulsion with lecithin (Vesisorb® placebo). A control group of 6 volunteers did not receive any intervention. The active compound was a 0.5% equol-racemate. For 58 participants, all samples were collected. Structural analysis, molecular analysis and questionnaires were performed at the start of the study and after 8 weeks of intervention, twice a day. Structural skin parameters were analysed by Visioscan® VC 98 and Cutometer® dual MPA 580. Molecular analyses from epidermal cells collected by skin stripping of the forehead included telomere length and LINE-1 methylation, following DNA extraction, bisulfite conversion and qPCR as well as high-resolution melting curve analysis. Effects of nutrition and lifestyle habits were evaluated with a standardized food and lifestyle questionnaire. The surface analysis showed significant improvements in skin roughness, skin texture and skin smoothness after both interventions. Cutometer® dual MPA 580 measurement revealed improvement of skin firmness and elasticity parameters for both preparations. A decrease in mean LINE-1 methylation (n.s.) and telomere length (sign. P < 0.05) was observed in the sample group with age. In the treated groups, significantly longer telomeres were observed after intervention. Whether changes in telomere length reflect changes in the regulation of telomerase, epigenetic interactions or turnover of keratinocytes needs further research. Stability and availability of preparations in skin seems to be high as not many significant differences in the activity of pure or encapsulated substances were seen. The results of this study indicate that equol has beneficial effects on structural as well as molecular skin parameters and encourages further investigations to decipher the epigenetic regulation of skin ageing and interactions of equol.

Role of rs13117307 single nuclear polymorphism in the risk of uterine cervical cancer from Polish population and its impact on exocyst complex component 1 expression

We evaluated the role of NM_001024924.1:c.1330+1646C>T (rs13117307) single nucleotide polymorphism (SNP), situated in the intronic region of exocyst complex component 1 (EXCO1), in the development and spreading of cervical squamous cell carcinoma (SCC). Utilizing high resolution melting curve analysis, we analyzed this polymorphism in patients with cervical SCC (n=485) and controls (n=509) in the Polish Caucasian population. Logistic regression analysis was used to adjust for age, parity, oral contraceptive use, tobacco smoking, and menopausal status. The influence of this polymorphism on the expression of EXCO1 was assessed by reverse transcription and real-time quantitative PCR analysis. For all patients with SCC, the p trend value calculated for rs13117307 was statistically significant (ptrend=0.0158). The adjusted odds ratio (OR) for T/T vs. C/C was 1.434 (95 % CI 1.105–1.861, p=0.007). We also found a significant contribution of rs13117307 to tumor stages III, IV and grade of differentiation G3. Other contributors are parity, oral contraceptive use, smoking, and women of postmenopausal age. We observed significant upregulation of EXCO1 transcript levels in the non-cancerous cervical tissues in carriers of the T/T vs. C/C (p=0.016), as well as an increase in the EXCO1 transcript levels in the cervical SCC tissue in carriers of the T/T vs. C/C (p=0.029) and for T/T vs C/T (p=0.0032). The rs13117307 SNP variants may upregulate the transcription of EXCO1, as well as the risk of development and spreading of cervical SCC.

Real-time PCR followed by high-resolution melting curve analysis: A rapid and pragmatic approach for screening of multidrug-resistant extrapulmonary tuberculosis

IntroductionMultidrug resistance (MDR) in extrapulmonary tuberculosis (EPTB) is a diagnostic challenge in an endemic country like India. Timely detection of MDR-TB can contribute to a better patient outcome. ObjectiveTo perform real-time PCR (qPCR) using rpoB, mpb64 and IS6110 gene on a variety of EPTB samples and to compare the performance of different gene targets. All qPCR positive samples were subjected to high resolution melt-curve analysis (HRM analysis) for rpoB and katG gene to evaluate its potential for MDR screening among different sample types. MethodsReal-time PCR using rpoB, mpb64 and IS6110 genes was carried out on 200 cases of study group and 100 cases of non-TB control group. The study group consisted of 100 culture-confirmed and 100 clinically suspected cases of EPTB. Phenotypic drug susceptibility testing (DST) for culture isolates was performed by the 1% indirect agar proportion method. DNA extracted from all qPCR positive samples was subjected to rpoB and katG HRM analysis for screening of MDR. Sequencing was used to confirm the results of HRM analysis and the results were also compared with phenotypic DST in all culture positive cases. ResultsThe sensitivity of qPCR using rpoB, mpb64 and IS6110 was 86.5%, 86.5% and 76.5%, respectively. All isolates from the control group were negative by all the three targets, giving a specificity of 100%. HRM analysis detected MDR in 22/200 (11%) isolates. 3/200 (1.5%) had mono-rifampicin resistance while 8/200 (4%) had mono-isoniazid resistance. HRM analysis identified an additional 4 MDR cases directly from the samples which were negative by culture. On sequencing, mutations were observed at codon 531 (60%); 533 (16%); 516 (12%) and 526 (12%) of the rpoB gene and at codon 315 (100%) of the katG gene. There was 100% concordance in the results of phenotypic DST, HRM analysis and sequencing. ConclusionThe HRM analysis can play a promising role in the reliable and rapid screening of EPTB samples for detection of MDR.

Gateway-Compatible CRISPR-Cas9 Vectors and a Rapid Detection by High-Resolution Melting Curve Analysis

CRISPR-Cas9 system rapidly became an indispensable tool in plant biology to perform targeted mutagenesis. A CRISPR-Cas9-mediated double strand break followed by non-homologous end joining (NHEJ) repair most frequently results in a single base pair deletion or insertions (indels), which is hard to detect using methods based on enzymes that detect heteroduplex DNA. In addition, somatic tissues of the T1 generation inevitably contain a mosaic population, in which the portion of cells carrying the mutation can be too small to be detected by the enzyme-based methods. Here we report an optimized experimental protocol for detecting Arabidopsis mutants carrying a CRISPR-Cas9 mediated mutation, using high-resolution melting (HRM) curve analysis. Single-base pair insertion or deletion (indel) can be easily detected using this method. We have also examined the detection limit for the template containing a one bp indel compared to the WT genome. Our results show that <5% of mutant DNA containing one bp indel can be detected using this method. The vector developed in this study can be used with a Gateway technology-compatible derivative of pCUT vectors, with which off-target mutations could not be detected even by a whole genome sequencing.

Abstract 3420: Mutational status, expression and functional behaviors of FAM134B in colorectal cancer

Abstract Background: Family with sequence similarity 134B (FAM134B) is an ER-autophagy regulator and involved in the pathogenesis of neuronal disorders, vascular diseases and carcinomas. In colorectal carcinomas, FAM134B plays important role in the pathogenesis and associated with aggressiveness of the disease. However, the frequency of mutations, expression pattern and functional roles in cell have never been studied in colorectal cancer. Objectives: To investigate FAM134B mutations in tissues samples from patients with colorectal cancer and cell lines. Also, the expression of FAM134B at protein and mRNA levels were examined. In addition, functional roles of FAM134B in colon cancer were studied. Methods: Mutations in FAM134B sequence in eighty-eight cancer tissues and matched non-cancer samples was studied by high-resolution melt curve analysis followed by Sanger sequencing. FAM134B expression was studied and quantified in cell lines and cancer tissues samples using immunofluorescence, immunocytochemistry, Western blot and real-time PCR. In vitro functional assays were performed to unveil the molecular roles of FAM134B in colon cancer pathogenesis followed by shRNA-mediated silencing in cells. Mouse xenotransplantation model was used to confirm the functional behavior of FAM134B in colon cancer. Results: In this study, 46.5% (41/88) patients with colorectal cancer were identified as FAM134B mutations positive. Thirty-one novel pathogenic mutations were detected. Of the 31 mutations, 8 novel frameshift mutations caused nonsense-mediated mRNA decay and associated with gender of the patients, presence of metachronous cancer, size, T staging, presence of distant metastases and positivity of microsatellite instability (MSI) in the cancer (p &amp;lt; 0.05). FAM134B expression in cancer cells derived from advanced stages (stage III; SW48 and stage IV; HCT116) of colon cancer was significantly (p&amp;lt;0.01) reduced when compared to non-neoplastic colon cells (FHC) and cancer cells derived from stage II colon cancer (SW480). Expression of FAM134B mRNA in cancer tissues was noted significantly (p&amp;lt;0.001) downregulated when compared to that of non-cancer tissues samples. FAM134B suppression significantly (p&amp;lt;0.05) increased the proliferation of colon cancer cells, remarkably increased (34-52%; p&amp;lt;0.05) the clonogenic, migration capacity, and increases the proportion of cells in S phase of cell-cycle (p&amp;lt;0.01). Xenotransplantation model showed that larger and higher-grade tumors were formed in mice treated with FAM134B knockdown cells. Conclusion: In vitro and in vivo functional studies implied that FAM134B acts as a cancer inhibitor in colon cancer. Also, FAM134B mutation is common in colorectal cancer and the association of mutation with adverse clinical and pathological parameters are in concur with the tumour suppressive property of the gene. Note: This abstract was not presented at the meeting. Citation Format: Farhadul Islam, Vinod Gopalan, Riajul Wahab, Katherine Ting-wei Lee, Afraa Mamoori, Cu-tai Lu, Robert A Smith, Alfred K-Y Lam. Mutational status, expression and functional behaviors of FAM134B in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3420. doi:10.1158/1538-7445.AM2017-3420

Abstract 1243: FAM134B mutation in esophageal squamous cell carcinoma: Its clinical significance and quantification by electrochemical methods

Abstract Aim: The aim of this research was to detect novel sites of FAM134B mutations, copy number variations and their clinicopathological significance in esophageal squamous cell carcinoma (ESCC) patients. Also, this study was intended to develop a simple and inexpensive electrochemical detection method for the analysis of FAM134B mutations using a single-use and disposable screen-printed electrode. Method: Approximately 102 fresh tissue samples of ESCC and matched non-cancer adjacent tissues were recruited. The DNA copy numbers of FAM134B were initially studied by qRT-PCR. The FAM134B mutations were then quantified via high resolution melt curve (HRM) and Sanger sequencing analysis. In order to quantify the level of point mutation or SNPs in FAM134B gene, a new electrochemical method was also developed. The underlying working principle of the method is relied on the base dependent affinity interactions towards gold electrode. Since two DNA sequences with different DNA base compositions (i.e., amplified mutated sequences will be distinctly different than its wild type sequence) will have different adsorption affinity towards an unmodified gold electrode, accurate measurement of adsorbed DNA on the electrode surface will give the measure of point mutation or SNPs present in the DNA sequences. Target DNA sequences were first extracted from clinical samples and then PCR amplified and purified prior to adsorption on a single-use screen-printed gold electrode. The amount of mutation sites on a DNA sequence is quantified by monitoring the Faradaic current generated by the [Fe(CN)6]3-/4- system present in the electrolyte solution. Result: Amplification of FAM134B DNA was noted in 37% of ESCC tissues whereas 35% cases showed loss of FAM134B copies compared to matched non-tumor tissues. Overall, thirty-seven FAM134B mutations were documented in exons 4, 5, 7, 9 as well as introns 2, 4-8 of FAM134B. Also, FAM134B mutations were detected in all the metastatic ESCC cases and in 14% (8/57) of the primary ESCC. Using the new electrochemical method, we were able to detect mutations in 50 ng of target PCR-amplified product within 1 h with high reproducibility (% RSD= &amp;lt;2) and specificity. Conclusion: DNA copy number variations and frequent mutations of FAM134B in metastatic lymph node tissues in ESCC patients indicate its critical role in the pathogenesis of ESCCs. Also the mutation detection via electrochemical methods was successful distinguishing single point mutation in DNA from oesophageal cancer implying its potential application in point mutation detection in clinical diagnostics. Note: This abstract was not presented at the meeting. Citation Format: Md. Hakimul Haque, Vinod Gopalan, Muhammad J. A. Shiddiky, Alfred K. Lam. FAM134B mutation in esophageal squamous cell carcinoma: Its clinical significance and quantification by electrochemical methods [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1243. doi:10.1158/1538-7445.AM2017-1243

High resolution melting curve analysis revealed SNPs in major cannabinoid genes associated with drug and non-drug types of cannabis

ABSTRACTCannabis sativa L. has a long history of cultivation as food, fibre, medicine and recreational drugs. Production of high tetrahydrocannabinol (THC) plants for narcotic use (drug type) is illegal and under control in most countries. In contrast, cultivation of low THC plants (fibre type, also known as ‘industrial hemp’) is promoted in many countries. The determination of C. sativa L. chemotypes is based on the major cannabinoids content, THC, cannabidiol (CBD) and cannabinol (CBN). The THC:CBD content ratio is a candidate marker for differentiation of the fibre and drug type of cannabis. The ability to accurately characterize the cannabinoid chemical phenotype (chemotype) is crucial for the development of specific C. sativa cultivars for pharmacological, hemp fibre or seed end-use. High resolution melting (HRM) curve analysis is used as a rapid and effective mechanism for detection of single-nucleotide polymorphisms in plants. In this report, we developed a HRM protocol for differentiation of drug and non-drug cannabis plants. According to the results, HRM analysis based on single-nucleotide polymorphisms in THCAS gene is an accurate method to differentiate the drug type of cannabis which could be used for the control of legal and illegal cannabis cultivation.