High-resolution Melting Curve Research Articles

Two Leishmania species separation targeting the ITS-rDNA and Cyt b genes by developing and evaluating HRM- qPCR.

Incidence of Cutaneous Leishmaniasis as an infectious and neglected disease is increasing, for the diagnosis of which several traditional methods and conventional PCR techniques have been developed, employing different genes for species identification. Leishmania parasites were sampled, DNA was extracted, and new specific and sensitive primers were designed. Two ITS-rDNA and Cyt b genes were targeted by qPCR using the High- Resolution Melting method to identify Leishmania parasites. The standard curves were drawn, compared, and identified by high-resolution melting curve analysis. Melting temperature and Cycle of Threshold of ITS-rDNA was higher than Cyt b but Cyt b was more sensitive than ITS-rDNA when Leishmania major and Leishmania tropica were analyzed and evaluated. By aligning melt curves, normalizing fluorescence curves, and difference plotting melt curves, each Leishmania species was distinguished easily. L. major and L. tropica were separated at 83.6 °C and 84.7 °C, respectively, with less than 0.9 °C of temperature difference. Developing sensitivity and specificity of real-time PCR based on EvaGreen could detect DNA concentration to less than one pmol. Precise identification of Leishmania parasites is crucial for strategies of disease control. Real-time PCR using EvaGreen provides rapid, highly sensitive, and specific detection of parasite's DNA. The modified High-Resolution Melting could determine unique curves and was able to detect single nucleotide polymorphisms according to small differences in the nucleotide content of Leishmania parasites.

One-shot high-resolution melting curve analysis for KRAS point-mutation discrimination on a digital microfluidics platform.

Single-nucleotide polymorphism (SNP) plays a critical role in personalized medicine, forensics, pharmacogenetics, and disease diagnostics. Among different existing SNP genotyping techniques, melting curve analysis (MCA) becomes increasingly popular due to its high accuracy and straightforward procedures in extracting the melting temperature (Tm). Yet, its study on existing digital microfluidic (DMF) platforms has intrinsic limitations due to the temperature inhomogeneity within a thickened droplet during the on-chip rapid heating process. Although the utilization of an on-chip thermostat can regulate and monitor the dynamic melting process in real time, the limited Tm accuracy resulting from the insufficient system response time to accommodate the fast-melting evolution still poses a great challenge for precise MCA with high throughput. This work proposes a one-shot MCA on a DMF platform. The tailoring of a functional substrate with hierarchical micro/nano structure enables high-resolution patterning of pL-scale droplets. Specifically, the hydrothermal and photocatalysis treatment allows the functional substrate to exhibit a superwettability contrast of >170°, facilitating passive isolation of the pL-scale DNA sample into highly-resolved pL droplets above the 200 μm superhydrophilic patterns. This high-resolution MCA technique can successfully discriminate KRAS gene targets with single-nucleotide mutations in 3 seconds. The high accuracy and consistency in the acquired Tm when compared with off-chip results demonstrate its opportunities for near-patient diagnostics, precision medicines, genetic counseling, and prevention strategies on DMF platforms.

Анализ результатов многолетнего массового скрининга мутаций в генах BRCA1/2 у больных с различными типами злокачественных новообразований

Background: germline mutations in the BRCA1 and BRCA2 genes are associated with a high risk of developing cancer of various localizations. Currently, the determination of BRCA status in such patients is important for choosing surgical tactics and determining indications for the administration of multiple chemotherapy drugs. Aim: to evaluate the results of mass genetic screening for mutations in the BRCA1 and BRCA2 genes in patients with various types of malignant neoplasms (MN). Patients and Methods: the results of genetic screening of 5043 patients who are reviewed in the study. The patients had the following diagnoses: breast cancer (BC, n=4216), ovarian cancer (OC, n=481), multiple primary malignant neoplasms (n=174), pancreatic cancer (n=96) and prostate cancer (n=75). Real-time PCR-based genetic testing for eight recurrent BRCA1/2 gene mutations specific for the Russian population was performed for all patients. The study of the entire coding sequence of the BRCA1/2 genes was carried out in 655 patients with negative screening results for mutations using high-resolution melting curve analysis, Sanger sequencing, and massively parallel sequencing. Results and Discussion: among 5043 cancer patients, the total frequency of BRCA mutations was 8%. In patients with BC the recurrent variants accounted for 6.7%, bilateral BC — 13.5%, OC — 17%, multiple primary malignant neoplasms (MPMN) — 13.1%, pancreatic cancer — 3.1%, while in patients with prostate cancer no mutations were found. The frequency of the c.5266dup mutation in the BRCA1 gene was 5.63%. Forty nine patients (0.97%) were carriers of another mutation — p.C61G, which is specific for the Slavic populations. Other variants were found much less frequently (0.1–0.38%). Thus, screening for common mutations helped to determine the prevalence of each of the variants and proved a high frequency of c.5266dup and p.C61G mutations. The analysis of the coding sequence of the BRCA1/2 genes in 655 patients revealed pathogenic mutations (class 5) in 13% of cases. The prevalence of rare variants in patients with BC was 11.1%, bilateral BC — 26.5%, OC — 14.9%, МPМN — 31%, pancreatic cancer — 2.3%, and prostate cancer — 2.5%. In the BRCA1 gene, 41 unique clinically significant mutations were found, and four of them were repeated in unrelated patients. In the BRCA2 gene, 29 unique clinically significant mutations were found, five of which were repeated. Moreover, 19 missense mutations were detected which, according to the databases, were categorized as variants with unknown clinical significance (class 3). They were analyzed using seven pathogenicity prediction algorithms. As a result, five mutations were interpreted by the most algorithms as likely pathogenic or pathogenic (class 4/5), five mutations were re-assessed as variants with unknown significance, and nine variants were identified as clinically insignificant. Conclusion: thus, the Russian population is characterized by the presence of multiple recurrent mutations, while in patients with different cancer types the prevalence of c.5266dup and p.C61G is significantly higher than the prevalence of other variants. Analysis of recurrent variants in the BRCA1/2 genes enables identification of approx. 80% of carriers of the pathogenic mutations. The use of the panel kit which includes such variants is considered as an inexpensive and sensitive algorithm for the first-step screening of the BRCA1/2 genes. A follow-on investigation of all coding gene regions (sequencing) is feasible for the studied patient groups and can significantly increase the detectability of pathogenic mutations in the BRCA1/2 genes. KEYWORDS: BRCA1 mutations, BRCA2 mutations, breast cancer, ovarian cancer, malignant neoplasms. FOR CITATION: Stroganova A.M., Pospekhova N.I., Golovina D.A. et al. Review of the results of mass screening for the BRCA1/2 gene mutations in patients with different types of malignant neoplasms. Russian Medical Inquiry. 2022;6(6):297–308 (in Russ.). DOI: 10.32364/2587-6821- 2022-6-6-297-308.

Identification of a novel bacterium Scardovia wiggsiae in high caries risk adolescence: A metagenomic and melt curve analysis.

Background:Mutans streptococci which comprise only 2% have long been presumed to be the specific pathogen responsible for caries. A novel caries associated bacterium namely Scardovia wiggsiae is recognized to be ecologically competitive in active caries lesions. The actual pathogen needs to be identified, so as to specifically target and reduce the prevalence of caries in a given community.Aim:The aim of the study is to evaluate the presence of S. wiggsiae in combination with other bacteria in caries risk adolescence.Methods:Sixty adolescent subjects were screened. Phase I-to determine the prevalence of S. wiggsiae in saliva, plaque and dentinal caries samples of low and high caries risk individuals (n = 30 each) using polymerase chain reaction (PCR); Phase-II-to identify its presence by 16SrRNA metagenomic analysis and quantitatively evaluate the cariogenic pathogen using high-resolution melt curve analysis and real-time PCR.Results:Highest prevalence of S. wiggsiae was observed in dentinal caries followed by plaque and saliva samples of high caries risk individuals under PCR analysis. Metagenomic analysis showed two-fold statistically increased presence of Lactobacilli and Bifidobacteriaceae (S. wiggsiae) in dentinal samples compared to plaque samples (P = 0.05). Mutans streptococcus recorded the minimum.Conclusion:Scardovia wiggsiae is identified as one of the predominant microorganism.

Optimization and development of high-resolution melting curve analysis (HRMA) assay for detection of New Delhi metallo-β-lactamase (NDM) producing Pseudomonas aeruginosa.

New Delhi metallo-β-lactamase-1 (NDM-1) producing Pseudomonas aeruginosa strain detection plays a vital role in confirming bacterial disease diagnosis and following the source of an outbreak for public health. However, the standard method for NDM-1 determination, which relies on the features of the colony of the bacteria cultured from the patient's specimen, is time-consuming and lacks accuracy and sensitivity. This study aimed to standardize a high-resolution melting curve analysis (HRMA) assay to detect NDM producing P. aeruginosa. For optimization and development of the HRMA method, a reference strain of P. aeruginosa was used. For evaluating the broad range PCR data, ABI Step One-Plus Manager Software version 3.2 and Precision Melt Analysis Software 3.02 (Applied Biosystems) were used.Based on the results, expected results were obtained for all tested strains, with high analytical sensitivity and specificity. Temperature melting analyses of the HRMA time PCR assays showed the Tm at 89.57 °C, 76.92 °C and 82.97 °C for N-1, N-2 and N-3 genes, respectively. Also, melting point temperatures of the blaVIM, blaSPM and blaSIM amplicons for isolates identified as MBL strains were 84.56 °C, 85.35 °C and 86.62 °C, respectively. The amplification results using negative control genomes as templates were negative, showing the specificity of the designed assays. Our study's data indicated that the sensitivity and specificity of the HRMA method are linked to the primer length and the fluorescent dye. We can further identify antibiotic resistance in NDMproducing P. aeruginosa by software analysis and melting curve analysis.

High-Resolution Melting Curve Analysis to Predict Extended Blood Group Phenotypes among Thai Donors and Patients

Background: High-resolution melting (HRM) analysis is an alternative method for red cell genotyping. Differences in melting curves between homozygous and heterozygous genotypes can predict phenotypes in blood group systems based on single-nucleotide polymorphisms. This study aimed to implement HRM analysis to predict additional extended blood group phenotypes in Thai donor and patient populations. Methods: Blood samples obtained from 300 unrelated Thai blood donors and 23 patients with chronic transfusions were included. HRM analysis was developed and validated in genotyping of KEL*01 and KEL*02, JK*01 and JK*02, FY*01, FY*02, and FY*02 N.01, DI*01 and DI*02, GYPB*03 and GYPB*04, RHCE*E and RHCE*e, and DO*01 and DO*02. Then genotyping results from HRM and polymerase chain reaction with sequence-specific primer (PCR-SSP) and phenotyping results were compared. Results: The validated genotyping results in known DNA controls by HRM analysis agreed with DNA sequencing. The genotyping results among 300 donors in 15 alleles by HRM analysis were in complete concordance with those obtained by serological testing and PCR-SSP. The sensitivity and specificity of the HRM assay were both 100%. Among patients, 13 had alloantibodies that possessed predicted antigen-negative phenotypes corresponding to those antibody specificities, and the highest probability of genotyped-matched donors was given to the remaining patients. Conclusions: We developed and implemented the HRM analysis assay for red cell genotyping to predict extended blood group antigens in Thai donor and patient populations. The data from this study may help inform about and support transfusion care of Thai patients to reduce the risk of alloimmunisation.

WITHDRAWN: Exploring in-silico prediction for the development of a RT-qPCR-high resolution melting assay for the broad detection of emaraviruses

High-resolution melting (HRM) has shown to be reliable for the detection, discrimination, and diagnosis of several diseases of plants, animals, and humans. The aim of this research was to explore the ability to predict HRM outputs when coupled to reverse transcription quantitative polymerase chain reaction (RT-qPCR). This research used the species in the Emaravirus genus as model to framework the development of genus-specific RT-qPCR-HRM assays. A pair of degenerate genus-specific primers were designed for use in endpoint RT-PCR and RT-qPCR-HRM detection of emaraviruses. Eleven species of RNA viruses infecting economically important crops are classified within the genus Emaravirus, family Fimoviridae. There are at least fifteen other non-classified species that may be added. Some of these viruses are spreading rapidly and cause economically important diseases on several crops, raising a need for a sensitive diagnostic technique for taxonomic and quarantine purposes. RT-PCR and RT-qPCR-HRM were able to detect seven emaravirus species in-vitro with sensitivity up to one fg of cDNA. Specific parameters for prediction in-silico of the melting temperatures of each expected emaravirus amplicon are provided and compared to the data obtained in-vitro. A very distinct isolate of the High Plains wheat mosaic virus was also detected. The prediction in-silico of fluorescence of high-resolution DNA melting curves of predicted RT-PCR products using uMeltSM speeded the design and development of RT-qPCR-HRM assay. This approach avoided rounds of HRM tests in-vitro when searching for the optimal regions that provides accurate diagnosis. The resultant assay provided sensitive detection and reliable diagnosis for potentially any emaravirus, including new species or strains.

Prognostic relevance of combined IDH1 and NPM1 mutations in the intermediate cytogenetic de novo acute myeloid leukemia.

Despite the great advance in treatment, cytogenetically normal Acute myeloid leukemia (CN-AML) is still a challenging entity. The discovery of IDH1 mutation in AML together with the frequent co-mutations; NPM1 and FLT3-ITD throughs a new insight into the pathogenesis and outcome of CN-AML. Recently, there has been an increasing number of recurring mutations in other genes for which the forecasting effect is still required. Despite the large number of risk variables established, there are relatively few prognostic indicators that can help in treatment decisions in AML patients. This study aimed at recording the frequency of IDH1 and NPM1 mutations in newly diagnosed AML and, dual clinicopathological significance. IDH1 and NPM1 mutations were analyzed using High-Resolution Melting curve analysis PCR in 78 newly diagnosed AML patients; 30 pediatric and 48 adult AML patients. IDH1 mutation was detected in 6 out of the 48 adult AML cases (12.5%) and all of them had intermediate cytogenetic prognostic stratification. 5/6 mutant IDH1 patients showed NPM1 co-mutation (P-value= 0.008). Mutant IDH1 patients showed significant resistance to induction therapy (P-value <0.001) and even those who achieved complete remission were relapsed later. Within the intermediate cytogenetic group, the IDH1 mutated patients had short overall survival (HR 12.9, 95% CI (3.1- 53.45) and event-free survival (HR 15.7, 95% CI (2.99-82.72) and P-value <0.001). IDH1 mutation is closely linked to the intermediate cytogenetic stratified group and in particular old age patients and has a great impact on their survival.

Low-Cost Platform for Multiplexed Electrochemical Melting Curve Analysis.

Detection and identification of single nucleotide polymorphisms (SNPs) have garnered increasing interest in the past decade, finding potential application in detection of antibiotic resistance, advanced forensic science, as well as clinical diagnostics and prognostics, moving toward the realization of personalized medicine. Many different techniques have been developed for genotyping SNPs, and ideally these techniques should be rapid, easy-to-use, cost-effective, flexible, scalable, easily automated, and requiring minimal end-user intervention. While high-resolution melting curve analysis has been widely used for the detection of SNPs, fluorescence detection does not meet many of the desired requirements, and electrochemical detection is an attractive alternative due to its high sensitivity, simplicity, cost-effectiveness, and compatibility with microfabrication. Herein, we describe the multiplexed electrochemical melting curve analysis of duplex surfaces tethered to electrodes of an array. In this approach, thiolated probes designed to hybridize to a DNA sequence containing the SNP to be interrogated are immobilized on gold electrodes. Asymmetric PCR using a ferrocene-labeled forward primer is used to generate this single-stranded redox-labeled PCR amplicon. Following hybridization with the probe immobilized on the electrode surface, the electrode array is exposed to a controlled ramping of temperature, with concomitant constant washing of the electrode array surface while simultaneously carrying out voltammetric measurements. The optimum position of the site complementary to the SNP site in the immobilized probe to achieve maximum differentiation in melting temperature between wild-type and single base mismatch, thus facilitating allelic discrimination, was determined and applied to the detection of a cardiomyopathy associated SNP.

Association Between CLOCK Gene Variants and Restless Legs Syndrome in Koreans.

ObjectivePrevious studies have suggested various causes of restless legs syndrome (RLS), including iron and dopamine concentrations in the brain. Genetic influences have also been reported in many studies. There is also a possibility that circadian clock genes may be involved because symptoms of RLS worsen at night. We investigated whether CLOCK and NPAS2 gene polymorphisms were associated with RLS. MethodsA total of 227 patients with RLS and 229 non-RLS matched controls were assessed according to the International Restless Legs Syndrome Study Group diagnostic criteria. Genotyping was performed using reverse transcription polymerase chain reaction and high-resolution melting curve analyses. ResultsAlthough the genotype distributions of the CLOCK variants (rs1801260 and rs2412646) were not significantly different between patients with RLS and non-RLS controls, the allele frequencies of CLOCK rs1801260 showed marginally significant differences between the two groups (X2=2.98, p=0.085). Furthermore, there was a significant difference in the distribution of CLOCK haplotypes (rs1801260-rs2412646) between patients with RLS and non-RLS controls (p=0.013). The distributions of allelic, genotypic, and haplotypic variants of NPAS2 (rs2305160 and rs6725296) were not significantly different between the two groups. ConclusionOur results suggest that CLOCK variants may be associated with decreased susceptibility to RLS.

Characterization of a 7 bp indel in MARCH1 promoter associated with reproductive traits in Malabari and Attappady Black goats of India

Membrane-associated RING CH-type finger 1(MARCH1) gene belongs to a family member of RING-v type E3 ubiquitin ligases, which consists of 11 known members, some of which targets important aspects of immune response. MARCH1 gene has a regulatory role in immunity and placental health; hence it affects foetal growth and survivability. To understand the role of MARCH1 gene in caprine reproduction, differential gene expression profile was studied in reproductive tissues of goats. Relative abundance of MARCH1 mRNA was significantly (≤0.01) higher in placenta than ovaries, uterus and fallopian tubes. Further, the differential placental gene expression profile was studied in two groups of goats with normal kidding and kid mortality at birth. MARCH1 gene was significantly upregulated in the placenta of goats with early kid mortality than normal kidding. An indel mutation (a 7 bp insertion) located in the promoter region of MARCH1 gene was screened in 282 goats belonging to Malabari and Attappady Black breeds by agarose gel electrophoresis and revealed three genotypes (DD, ID and II). A novel High resolution melt curve analysis protocol was designed for the rapid screening of this indel. Genotype II had higher litter size and average birth weight of kids than ID and DD genotypes. These results suggest the influence of the detected indel in placental health and foetal growth, together the role of MARCH1 in reproduction as well as the potential use of indel in MARCH1 as a molecular marker in future goat breeding programmes.

Detection of MSH2 Gene Methylation in Extramammary Paget's Disease by Methylation-Sensitive High-Resolution Melting Analysis.

Background Extramammary Paget's disease (EMPD) is a rare skin tumor. Hypermethylation in the MSH2 promoter resulting in the downregulation of its protein expression shows a high detection rate in EMPD tumor tissue, which indicates that the methylation of MSH2 may play an important role in the pathogenesis of EMPD. Objective This study aims to establish a rapid analysis strategy based on the methylation-sensitive high-resolution melting curve (MS-HRM) to detect the methylation level of the MSH2 promoter. Methods With the use of universal methylated human DNA products, we established the MS-HRM standard curve to quantitatively detect the methylation level of the MSH2 promoter. Then, all 57 EMPD tumor DNA samples were analyzed. Pyrosequencing assay was also carried out to test the accuracy and efficacy of MS-HRM. Besides, a total of 54 human normal and other cancerous tissues were included in this study to test the reliability and versatility of the MS-HRM standard curve. Results In this study, by using the established MS-HRM, we found that 96.5% (55/57) EMPD tumor samples had varying methylation levels in the MSH2 promoter ranging from 0% to 30%. Then, the methylation data were compared to the results obtained from pyrosequencing, which showed a high correlation between these two techniques by Pearson's correlation (r = 0.9425) and Bland–Altman plots (mean difference = −0.1069) indicating that the methylation levels analyzed by MS-HRM were consistent with DNA pyrosequencing. Furthermore, in 23 normal and 31 other cancerous tissue samples, there were two colorectal cancer (CRC) tissues that tested MSH2 methylation positive (1% and 5%) which confirmed that our established MS-HRM can be widely applied to various types of samples. Conclusion MS-HRM standard curve can be used for the detection of the methylation level of MSH2 in EMPD tumor samples and other cancerous tissues potentially, which presents a promising candidate as a quantitative assay to analyze MSH2 promoter methylation in routine pathological procedure.

Rifampicin and isoniazid drug resistance among patients diagnosed with pulmonary tuberculosis in southwestern Uganda.

Multidrug-resistant tuberculosis (MDR-TB) has become a major threat to the control of tuberculosis globally. Uganda is among the countries with a relatively high prevalence of tuberculosis despite significant control efforts. In this study, the drug resistance of Mycobacterium tuberculosis to rifampicin (RIF) and isoniazid (INH) was investigated among patients diagnosed with pulmonary tuberculosis in Southwestern Uganda. A total of 283 sputum samples (266 from newly diagnosed and 17 from previously treated patients), collected between May 2018 and April 2019 at four different TB diagnostic centres, were assessed for RIF and INH resistance using high-resolution melt curve analysis. The overall prevalence of monoresistance to INH and RIF was 8.5% and 11% respectively, while the prevalence of MDR-TB was 6.7%. Bivariate analysis showed that patients aged 25 to 44 years were at a higher risk of developing MDR-TB (cOR 0.253). Furthermore, among the newly diagnosed patients, the prevalence of monoresistance to INH, RIF and MDR-TB was 8.6%, 10.2% and 6.4% respectively; while among the previously treated cases, these prevalence rates were 5.9%, 23.5% and 11.8%. These rates are higher than those reported previously indicating a rise in MTB drug resistance and may call for measures used to prevent a further rise in drug resistance. There is also a need to conduct frequent drug resistance surveys, to monitor and curtail the development and spread of drug-resistant TB.

CRISPR-Cas9 gene editing and rapid detection of gene-edited mutants using high-resolution melting in the apple scab fungus, Venturia inaequalis

Apple scab, caused by the fungal pathogen Venturia inaequalis, is the most economically important disease of apple (Malus x domestica) worldwide. To develop durable control strategies against this disease, a better understanding of the genetic mechanisms underlying the growth, reproduction, virulence and pathogenicity of V. inaequalis is required. A major bottleneck for the genetic characterization of V. inaequalis is the inability to easily delete or disrupt genes of interest using homologous recombination. Indeed, no gene deletions or disruptions in V. inaequalis have yet been published. Using the melanin biosynthesis pathway gene trihydroxynaphthalene reductase (THN) as a target for inactivation, which has previously been shown to result in a light-brown colony phenotype when transcriptionally silenced using RNA interference, we show, for the first time, that the CRISPR-Cas9 gene editing system can be successfully applied to the apple scab fungus. More specifically, using a CRISPR-Cas9 single guide RNA (sgRNA) targeted to the THN gene, delivered by a single autonomously replicating Golden Gate-compatible plasmid, we were able to identify six of 36 stable transformants with a light-brown phenotype, indicating an ∼16.7% gene inactivation efficiency. Notably, of the six THN mutants, five had an independent mutation. As part of our pipeline, we also report a high-resolution melting (HRM) curve protocol for the rapid detection of CRISPR-Cas9 gene-edited mutants of V. inaequalis. This protocol identified a single base pair deletion mutation in a sample containing only 5% mutant genomic DNA, indicating high sensitivity for mutant screening. In establishing CRISPR-Cas9 as a tool for gene editing in V. inaequalis, we have provided a strong starting point for studies aiming to decipher gene function in this fungus. The associated HRM curve protocol will enable CRISPR-Cas9 transformants to be screened for gene inactivation in a high-throughput and low-cost manner, which will be particularly powerful in cases where the CRISPR-Cas9-mediated gene inactivation efficiency is low.

A hybrid CNN-LSTM model for high resolution melting curve classification

High resolution melting (HRM) curve analysis is an efficient, correct, and rapid technique for analyzing real-time polymerase chain reaction (PCR) results. HRM curves are formed based on increasing temperature and decreasing amount of fluorescent dye in real-time PCR process. The shapes of them are unique for each species due to the sequence, length, and GC content of species' DNA. In the literature, the classification of HRM curves is usually conducted through visual inspection and a limited number of data mining methods have been used to classify these curves. However, it becomes challenging as the number of species and their samples and the number of closely related species increase. In this study, a hybrid classification model, which is based on convolutional neural network (CNN) and long short-term memory (LSTM) models, is proposed to classify HRM curves, efficiently. In the proposed CNN-LSTM model, CNN model was used for feature extraction, and LSTM model was used for classification. It takes both the HRM curves and derivative curves as inputs and gives the predicted species of HRM curves as outputs. The performance of the proposed CNN-LSTM model was compared with that of CNN and support vector machines (SVM) approaches. The results show that the proposed CNN-LSTM model outperforms other models. The accuracy, macro-average of F1, specificity, precision, and recall values of the proposed model were 0.96±0.02,0.95±0.02,1±0,0.96±0.02, and 0.96±0.02, respectively.

Role of RNF213 polymorphism in defining quasi-moyamoya disease and definitive moyamoya disease.

Quasi-moyamoya disease (QMMD) is moyamoya disease (MMD) associated with additional underlying diseases. Although the ring finger protein 213 (RNF213) c.14576G>A mutation is highly correlated with MMD in the Asian population, its relationship to QMMD is unclear. Therefore, in this study the authors sought to investigate the RNF213 c.14576G>A mutation in the genetic diagnosis and classification of QMMD. This case-control study was conducted among four core hospitals. A screening system for the RNF213 c.14576G>A mutation based on high-resolution melting curve analysis was designed. The prevalence of RNF213 c.14576G>A was investigated in 76 patients with MMD and 10 patients with QMMD. There were no significant differences in age, sex, family history, and mode of onset between the two groups. Underlying diseases presenting in patients with QMMD were hyperthyroidism (n = 6), neurofibromatosis type 1 (n = 2), Sjögren's syndrome (n = 1), and meningitis (n =1). The RNF213 c.14576G>A mutation was found in 64 patients (84.2%) with MMD and 8 patients (80%) with QMMD; no significant difference in mutation frequency was observed between cohorts. There are two forms of QMMD, one in which the vascular abnormality is associated with an underlying disease, and the other in which MMD is coincidentally complicated by an unrelated underlying disease. It has been suggested that the presence or absence of the RNF213 c.14576G>A mutation may be useful in distinguishing between these disease types.

HFE variants in colorectal cancer and their clinicopathological correlations

The study aimed to screen mutation of human homeostatic iron regulator (HFE) in colorectal carcinoma (CRC) and detect their associations with clinicopathological parameters. Expression of HFE was determined by quantitative polymerase chain reaction in matched CRC and non neoplastic colorectal mucosal tissue of 76 patients. Genomic DNA extracted were subjected to high high-resolution melt curve analysis and Sanger sequencing to detect mutations in HFE. The associations of the identified mutations with a variety of clinical features were determined. Approximately 60% of CRC showed low HFE expression. Of the ten 10 mutations identified in exons 2 and 4, c.187C>G (H63D), c845G>A (C282Y), c.193A>T (S65C), g.3828T>C, g.5795T>C, and g.5728G>A were known mutations. Four novel mutations were discovered; : c.184G>A, c.220T>G, c.322A>C, and c.324T>C. Heterozygous H63D and C282Y mutations were seen in 71% and 49% of cancer tissue, respectively. Tumour site (p=0.048) and gender (p=0.039) were significantly associated with H63D and C282Y mutation status, respectively. Local spread of cancer was significantly associated with C282Y mutations in CRC cancer and adjacent non-neoplastic tissue (p=0.029 & and p=0.004, respectively). There was a statistically significant association between H63D and C282Y negativity in matched non-neoplastic colorectal mucosa tissue and pathological staging of cancer (p=0.047 & and p=0.001, respectively). Patients with H63D and C282Y mutations in cancer tissue tend to have higher survival rates. Hence HFE mutations are common in CRC and are associated with clinicopathological parameters, implying the potential clinical significance of HFE mutations in colorectal carcinogenesis.

Abstracts

Abstracts

Rapid detection of Mycoplasma mycoides subsp. capri and Mycoplasma capricolum subsp. capripneumoniae using high-resolution melting curve analysis

Mycoplasma capricolumsubsp.subsp. capripneumonia (Mccp) and Mycoplasma mycoidessubsp.sbusp. capri (Mmc) cause caprine pleuropneumonia (CCPP) and mycoplasmal pneumonia in goats and sheep (MPGS), respectively. These diseases cannot be identified on clinical symptoms alone and it is laborious to distinguish them using biochemical methods. It is therefore important to establish a simple, rapid identification method for Mccp and Mmc. Here, we report a high-resolution melting (HRM) curve analysis using specific primers based on the Mmc 95010 strain MLC_0560 and Mccp F38 strain MCCPF38_00984 gene sequences. The method was highly specific with intra- and inter-batch coefficients of variation < 1%. The lower limit of detection for Mccp and Mmc was 55 copies/μL and 58 copies/μL, respectively. HRM and fluorescence qPCR results were compared using 106 nasal swabs and 47 lung tissue samples from goats (HRM-qPCR coincidence rate 94.8%; 145/153). Mycoplasma isolation and identification was performed on 30 lung tissue samples and 16 nasal swabs (HRM-culturing coincidence rate 87.0%; 40/46). HRM analysis was more sensitive than fluorescence qPCR and Mycoplasma isolation, indicating the practicality of HRM for accurate and rapid identification of Mccp and Mmc, and diagnosis and epidemiology of CCPP and MPGS.

Cover Image, Volume 93, Number 9, September 2021

Front Cover Caption: The cover image is based on the Research Article High-resolution melting curve FRET-PCR rapidly identifies SARS-CoV-2 mutations by Subarna Barua et al., https://doi.org/10.1002/jmv.27139.