High-resolution Melting Method Research Articles

Response of Some Pepper Genotypes to Cucumber Mosaic Virus (CMV) and Discrimination of Kilis Isolates Using High-Resolution Melting (HRM) Method

The escalating global population, diminishing agricultural lands, and the overarching global climate crisis are significantly impacting pepper cultivation. These challenges exacerbate the vulnerability of pepper plants to various biotic and abiotic factors, particularly viral diseases, resulting in diminished yield and quality. Cucumber mosaic virus (CMV), a notable concern for peppers in the Solanaceae family, is causing substantial quality and yield losses, with no effective chemical control methods currently available. This study focuses on exploring the genetic structure of CMV isolates obtained from pepper production areas in Kilis province and comparing these regions through the High-Resolution Melting (HRM) method. CMV isolates, cultivated in tobacco plants, have their partial coat protein sequences compared with those of other CMV isolates registered in the gene bank. The nucleotide sequences of identified CMV isolates are phylogenetically grouped and compared using an HRM graph. The HRM graph effectively distinguishes Kilis 3 and Kilis 4 isolates, clustered similarly to sequence patterns, from other isolates. The study highlights the utility of HRM analyses in identifying differences between isolates before determining sequence patterns. In the gene bank comparison, Kilis CMV isolates distinguished from others. Similarities were observed with isolates from Iran's Balsam (Impatiens balsamina - LC066478), Türkiye's Radish (Raphanus sativus - LC0665051), and Wild Turnip (Rapistrum rugosum - LC066514, LC066511, LC066517). The study found that Kilis 7 CMV isolate, transferred mechanically to 24 different pepper genotypes (C. annuum) from the local population, revealed susceptibility to CMV in the 24 lines developed from the Elbeyli population in Kilis province.

Effect of DNMT3A R882H Hot Spot Mutations on DDX43 Promoter Methylation in Acute Myeloid Leukemia.

Epigenetic alterations have been observed in many hematological malignancies, including acute myeloid leukemia (AML). Many of these alterations result from mutations in DNA methyl transferase (DNMT) enzymes, disabling them to methylate target genes in a proper way. In this case-control study, we investigated the association between R882H mutation in DNMT3A gene and DDX43 gene methylation in patients with AML. 47 AML patients and 6 controls were included in this study. After DNA extraction, amplification refractory mutation system (ARMS)-PCR was used to evaluate R882H mutations in DNMT3A gene. The high-resolution melting (HRM) method was used to determine the methylation changes of the DDX43 gene promoter. R882H mutation was only found in 10.6% (5 out of 47) of AML patients. The frequency of DDX43 gene methylation was significantly higher in patients without R882H mutations compared to patients with R882H mutations (P < 0.05). The DNMT3A R882H mutation is typically present in a minority of AML patients. Nevertheless, this mutation is associated with a reduced frequency of methylation in the DDX43 promoter region.

High-resolution melt curve analysis: An approach for variant detection in the TPO gene of congenital hypothyroid patients in Bangladesh.

TPO (Thyroid Peroxidase) is known to be one of the major genes involved in congenital hypothyroid patients with thyroid dyshormonogenesis. The present study aims to validate high-resolution melting (HRM) curve analysis as a substitute method for Sanger sequencing, focusing on the frequently observed non-synonymous mutations c.1117G>T, c.1193G>C, and c.2173A>C in the TPO gene in patients from Bangladesh. We enrolled 36 confirmed cases of congenital hypothyroid patients with dyshormonogenesis to establish the HRM method. Blood specimens were collected, and DNA was extracted followed by PCR and Sanger sequencing. Among the 36 specimens, 20 were pre-sequenced, and variants were characterized through Sanger sequencing. Following pre-sequencing, the 20 pre-sequenced specimens underwent real-time PCR-HRM curve analysis to determine the proper HRM condition for separating the three variations from the wild-type state into heterozygous and homozygous states. Furthermore, 16 unknown specimens were subjected to HRM analysis to validate the method. This method demonstrated a sensitivity and specificity of 100 percent in accurately discerning wild-type alleles from both homozygous and heterozygous states of c.1117G>T (23/36; 63.8%), c.1193G>C (30/36; 83.3%), and c.2173A>C (23/36; 63.8%) variants frequently encountered among 36 Bangladeshi patients. The HRM data was found to be similar to the sequencing result, thus confirming the validity of the HRM approach for TPO gene variant detection. In conclusion, HRM-based molecular technique targeting variants c.1117G>T, c.1193G>C, and c.2173A>C could be used as a high throughput, rapid, reliable, and cost-effective screening approach for the detection of all common mutations in TPO gene in Bangladeshi patients with dyshormonogenesis.

Abstract 7326: TP53 p.Pro47Ser polymorphism in breast cancer susceptibility in Mali

Abstract Background: The aim of this study was to assess the potential association of p.Pro47Ser polymorphisms of TP53 with the risk of developing breast cancer in the Malian population. Methods: We analyzed DNA samples from 72 breast cancer patients and 121 unrelated healthy, age-matched individuals to examine the presence of the polymorphisms. The genotyping of TP53 polymorphism was performed using PCR-RFLP and HRM (high-resolution melting) methods. Results: The genotypic frequencies of PP, PS, and SS were found to be 63.9% PP, 30.6% PS, and 5.6% SS in breast cancer patients, compared to 70.2% PP, 22.3% PS, and 7.4% SS in the control group. The frequencies of P and S alleles of TP53 p.P47S polymorphism were 79.2% vs 81.4%, 20.8% vs 18.6%, and 5% vs 4.5% in patients and controls, respectively. In this study, no significant correlation was observed between TP53 p.Pro47Ser polymorphism and breast cancer in the Malian population (Pro/Ser vs Pro/Pro: OR = 1.55, 95% CI = 0.80-3.05, P = 0.19; Ser/Ser vs Pro/Pro: OR = 1.27, 95% CI = 0.34-4.74, P = 0.71; and Ser vs Pro allele: OR = 1.39, 95% CI = 0.80-2.32, P = 0.24). Conclusion: Despite the absence of a genetic association between TP53 p.Pro47Ser and the risk of breast cancer in Malian patients, our findings suggest that individuals carrying at least one minor Ser allele are not protected against the disease. Citation Format: Brehima Diakite, Yaya Kassogue, Mamoudou Maiga, Oumar Kassogue, Kadidiatou Cisse, Fousseyni Diarra, Cheick Traore B. Traore, Bakarou Kamate, Jane L. Holl, Lifang Hou. TP53 p.Pro47Ser polymorphism in breast cancer susceptibility in Mali [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7326.

DNA barcoding combined with high-resolution melting analysis to discriminate rhubarb species and its traditional Chinese patent medicines.

Introduction: Rhubarb is a frequently used and beneficial traditional Chinese medicine. Wild resources of these plants are constantly being depleted, meaning that rhubarb products have been subjected to an unparalleled level of adulteration. Consequentially, reliable technology is urgently required to verify the authenticity of rhubarb raw materials and commercial botanical drugs. Methods: In this study, the barcode-DNA high-resolution melting (Bar-HRM) method was applied to characterize 63 rhubarb samples (five Polygonaceae species: Rheum tanguticum, Rh. palmatum, Rh. officinale, Rumex japonicus and Ru. sp.) and distinguish the rhubarb contents of 24 traditional Chinese patent medicine (TCPM) samples. Three markers, namely ITS2, rbcL and psbA-trnH, were tested to assess the candidate DNA barcodes for their effectiveness in distinguishing rhubarb from its adulterants. A segment from ITS2 was selected as the most suitable mini-barcode to identify the botanical drug rhubarb in TCPMs. Then, rhubarbs and TCPM samples were subjected to HRM analysis based on the ITS2 barcode. Results: Among the tested barcoding loci, ITS2 displayed abundant sites of variation and was effective in identifying Polygonaceae species and their botanical origins. HRM analysis based on the ITS2 mini-barcode region successfully distinguished the authenticity of five Polygonaceae species and eight batches of TCPMs. Of the 18 TCPM samples, 66.7 % (12 samples) were identified as containing Rh. tanguticum or Rh. officinale. However, 33.3 % were shown to consist of adulterants. Conclusions: These results demonstrated that DNA barcoding combined with HRM is a specific, suitable and powerful approach for identifying rhubarb species and TCPMs, which is crucial to guaranteeing the security of medicinal plants being traded internationally.

Association of Promoter Methylation Patterns with Expression of MAPK14 in Tissue of Papillary Thyroid Cancer Patients.

Thyroid cancer is one of the most prevalent malignancies worldwide. Genetic and epigenetic alterations are one of the main causes of thyroid tumor that is responsible to the activation of oncogenes as well as the inactivation of tumor suppressor genes. This research aimed to investigate the relationship of promoter methylation patterns with the expression of P38α in Iranian patients with thyroid cancer. We collected 40 thyroid tumor samples and 40 adjacent normal thyroid samples from 40 Iranian patients with papillary thyroid cancer. The promoter methylation pattern of P38α gene was investigated by methylation-sensitive high-resolution melting (MS-HRM) method. Moreover, mRNA expression of P38α was investigated by Real-Time PCR method. Further validation of the obtained results was performed by the Cancer Genome Atlas (TCGA) dataset. The obtained results indicated that the expression of the P38α (MAPK-14) gene in the thyroid cancer sample was considerably higher than tumor margin sample. Also, P38α gene promoter methylation was higher in thyroid margin tissue as compared to tumor tissue. These results were additionally confirmed by TCGA analysis. The receiver operating characteristic (ROC) curve analysis showed a high accuracy of P38α gene expression as a diagnostic biomarker for thyroid malignancy. Our study demonstrated that the P38α expression level gene was associated with thyroid cancer pathogenesis among the Iranian population. We suggested that this gene expression might be used as a biomarker for diagnosis of thyroid tumor.

The Effect of DNA Concentration on the HRM Performance in Detecting Jak2 p.V617F Variant in Patients with Myeloproliferative Neoplasms

Introduction: Janus kinase 2 (JAK2) is an intracellular signaling protein. JAK2 p. V617F is a common variant in normal karyotype myeloproliferative neoplasms (MPN). Highresolution melting (HRM) analysis is one of the essential methods for detecting the JAK2 p.V617F variant. In this study, we have investigated the effect of DNA concentration on detecting the JAK2 p.V617F variant using the HRM method. Methods: Genomic DNA was extracted from human blood and diluted ten times in distilled water from 1 to 0.03; afterward, HRM was conducted for each dilution (triplicate). Using SPSS v.20.0 software, the mean Tm of each dilution was calculated and compared. Results: The HRM results revealed the JAK2 wild type and variant to have Tms of 81/64°C and 80/76°C, respectively. At the endpoint of the pre-amplification, the dilutions had different emissions. The statistical analysis revealed no statistically significant differences in Tm between samples with varying DNA concentrations (P value &gt; 0.05). Conclusion: There have been no significant differences obtained in the analysis of JAK2 p.V617F point variant in different DNA dilutions, implying that the HRM analysis has no relation to DNA concentration.

Rapid Detection of Predominant SARS-CoV-2 Variants Using Multiplex High-Resolution Melting Analysis.

Coronavirus disease 2019, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses a considerable threat to global public health. This study developed and evaluated a rapid, low-cost, expandable, and sequencing-free high-resolution melting (HRM) assay for the direct detection of SARS-CoV-2 variants. A panel of 64 common bacterial and viral pathogens that can cause respiratory tract infections was employed to evaluate our method's specificity. Serial dilutions of viral isolates determined the sensitivity of the method. Finally, the assay's clinical performance was assessed using 324 clinical samples with potential SARS-CoV-2 infection. Multiplex HRM analysis accurately identified SARS-CoV-2 (as confirmed with parallel reverse transcription-quantitative PCR [qRT-PCR] tests), differentiating between mutations at each marker site within approximately 2 h. For each target, the limit of detection (LOD) was lower than 10 copies/reaction (the LOD of N, G142D, R158G, Y505H, V213G, G446S, S413R, F486V, and S704L was 7.38, 9.72, 9.96, 9.96, 9.50, 7.80, 9.33, 8.25, and 8.25 copies/reaction, respectively). No cross-reactivity occurred with organisms of the specificity testing panel. In terms of variant detection, our results had a 97.9% (47/48) rate of agreement with standard Sanger sequencing. The multiplex HRM assay therefore offers a rapid and simple procedure for detecting SARS-CoV-2 variants. IMPORTANCE In the face of the current severe situation of increasing SARS-CoV-2 variants, we developed an upgraded multiplex HRM method for the predominant SARS-CoV-2 variants based on our original research. This method not only could identify the variants but also could be utilized in subsequent detection of novel variants since the assay has great performance in terms of flexibility. In summary, the upgraded multiplex HRM assay is a rapid, reliable, and economical detection method, which could better screen prevalent virus strains, monitor the epidemic situation, and help to develop measures for the prevention and control of SARS-CoV-2.

PTEN promoter methylation and expression in endometrial cancer tissues.

Introduction: Some gene expression regulation in cancers can be controlled by epigenetic change like methylation. PTENpromoter methylation and expression were evaluatedin endometrial cancer. Methods: The study was run on 39 tumor tissues of endometrial cancer patients and 41 normal endometrial tissues. After total RNA extraction, cDNAsynthesis was done by reverse transcription of the total (real-time PCR) using SYBER Green master mix. DNA extraction and bisulfite treatment were conducted and methylation was semiquantified by the methylation-sensitive high-resolution melting method. Finally, promoter methylation quantification of the total number of 25 tumors and 22 non-neoplastic tissues was done. Results: PTEN gene expression showed a significant decrease in endometrial cancer tissues. Promoter methylation was significantly lower in the non-neoplastic group (7.2; p<0.001). In addition, PTEN promoter methylation was observed in 52.0% of tumor tissues compared with 13.6% in thenon-neoplastic group(p=0.06). There were no significant correlations between PTEN expression and methylation andclinicopathological features in endometrial cancer patients (p>0.05). Conclusion: PTEN gene expression in endometrial cancer tissues decreased because of its promoter hypermethylation.

Joint Effects Between CDKN2B/P15 Methylation and Environmental Factors on the Susceptibility to Gastric Cancer.

The incidence of gastric cancer has long been at a high level in China, seriously affecting the health of Chinese people. This case‒control study was performed to identify gene methylation biomarkers of gastric cancer susceptibility. A total of 393 gastric cancer cases and 397 controls were included in this study. Gene methylation in peripheral blood leukocytes was detected by a methylation-sensitive high-resolution melting method, and the Helicobacter pylori antibody presence was semi-quantified in serum by ELISA. Individuals with total methylation of CDKN2B/P15 had a 1.883-fold (95%CI: 1.166-3.040, P = 0.010) risk of gastric cancer compared with unmethylated individuals. Individuals with both CDKN2B/P15 and NEUROG1 methylation had a higher risk of gastric cancer (OR = 2.147, 95% CI: 1.137-4.073, P = 0.019). The interaction between CDKN2B/P15 and NEUROG1 total methylation on gastric cancer risk was affected by the pattern of adjustment. In addition, the joint effects between CDKN2B/P15 total methylation and environmental factors, such as freshwater fish intake (OR = 6.403, 95% CI = 2.970-13.802, P < 0.001), irregular diet (OR = 5.186, 95% CI = 2.559-10.510, P < 0.001), unsanitary water intake (OR = 2.238, 95% CI = 1.144-4.378, P = 0.019), smoking (OR = 2.421, 95% CI = 1.456-4.026, P = 0.001), alcohol consumption(OR = 2.163, 95% CI = 1.309-3.576, P = 0.003), and garlic intake(OR = 0.373, 95% CI = 0.196-0.709, P = 0.003) on GC risk were observed, respectively. However, CDKN2B/P15 and NEUROG1 total methylation were not associated with gastric cancer prognosis. CDKN2B/P15 methylation in peripheral blood may be a potential biomarker for evaluating susceptibility to gastric cancer. The joint effects between CDKN2B/P15 methylation and environmental factors may also contribute to gastric cancer susceptibility.

Re-Evaluation of Genotyping Methodologies in Cattle: The Proficiency of Imputation

In dairy cattle, identifying polymorphisms that contribute to complex economical traits such as residual feed intake (RFI) is challenging and demands accurate genotyping. In this study, we compared imputed genotypes (n = 192 cows) to those obtained using the TaqMan and high-resolution melting (HRM) methods (n = 114 cows), for mutations in the FABP4 gene that had been suggested to have a large effect on RFI. Combining the whole genome sequence (n = 19 bulls) and the cows' BovineHD BeadChip allowed imputing genotypes for these mutations that were verified by Sanger sequencing, whereas, an error rate of 11.6% and 10.7% were encountered for HRM and TaqMan, respectively. We show that this error rate seriously affected the linkage-disequilibrium analysis that supported this gene candidacy over other BTA14 gene candidates. Thus, imputation produced superior genotypes and should also be regarded as a method of choice to validate the reliability of the genotypes obtained by other methodologies that are prone to genotyping errors due to technical conditions. These results support the view that RFI is a complex trait and that searching for the causative sequence variation underlying cattle RFI should await the development of statistical methods suitable to handle additive and epistatic interactions.

Importance of STAT3 Polymorphisms on the Risk and Clinical Characteristics of Rheumatoid Arthritis.

Signal transducer and activator of transcription 3 (STAT3) has been introduced as one of the critical genetic factors in the pathogenesis of rheumatoid arthritis (RA). Single nucleotide polymorphisms (SNPs) in microRNA binding sites, known as miRSNPs, are a class of common variants in the 3' untranslated regions of genes targeted by miRNAs. miRSNPs unbalance gene expression by disrupting the binding regions of microRNAs. In this study, we intended to evaluate the association of two miRSNPs with the risk of RA development and its clinical features. We studied 120 Iranian patients with RA and 125 non-RA subjects as controls. The genotypes and alleles of rs1053005 and rs1053023 in each individual were assessed by the high-resolution melting method. The distribution of STAT3 variants did not differ markedly in RA patients compared to healthy controls. Stratification analysis revealed that rs1053005 was linked with a higher concentration of C-reactive protein and an increased erythrocyte sedimentation rate, two indicators of inflammation and disease activity in RA patients. The rs1053023 variant was correlated with higher levels of creatinine as an indicator of renal involvement. Our data demonstrate an association between STAT3 variants and clinical characteristics of RA, such as disease activity and probably kidney impairment. However, we did not observe a significant relationship between the two targeted variants and a predisposition to RA.

Distinguishing venomous jellyfish species via high resolution melting analysis

Accurate species identification is critical for highly venomous jellyfish studies particularly for evaluation of their distribution as it is important not only for understanding population dynamics but also predicting risk to locals and tourists. Hazardous jellyfish species have been determined using methods such as comparative morphology and chemistry, which are less useful and practical in some cases. A few molecular methods have been developed in aiding hazardous jellyfish species identification. However, no studies have focused on designing and developing high resolution melting (HRM) assays to distinguish jellyfish species from each other. This is despite several studies showing the potential of HRM in effectively distinguishing plants and animals. In this study, we developed an HRM method to differentiate jellyfish species in five box jellyfish families (Carukiidae, Chirodropidae, Chiropsalmidae, Chiropsellidae, and Tripedaliidae) and Physaliidae found in Thai waters. All jellyfish samples (10 species) were able to be distinguished by the HRM with 16S rRNA primers. Even closely related species in the same genus could be clearly discriminated by their melting profiles. The melting curves of the 10 species were then used as references. By comparing the normalized melting curves of twenty-two jellyfish specimens with the reference curves, the tested specimens were assigned into species. In addition, HRM results of all jellyfish specimens tested in this study were similar to those of morphological based identification by experts. This is the first attempt of using HRM analysis to determine species of jellyfish collected from Thai waters.

Association between EPHA5 methylation status in peripheral blood leukocytes and the risk and prognosis of gastric cancer.

PurposeAltered DNA methylation, genetic alterations, and environmental factors are involved in tumorigenesis. As a tumor suppressor gene, abnormal EPHA5 methylation was found in gastric cancer (GC) tissues and was linked to the initiation, progression and prognosis of GC. In this study, the EPHA5 methylation level in peripheral blood leukocytes (PBLs) was detected to explore its relationship with GC risk and prognosis.MethodsA total of 366 GC cases and 374 controls were selected as the subjects of this study to collect their environmental factors, and the EPHA5 methylation status was detected through the methylation-sensitive high-resolution melting method. Logistic regression analysis was utilized to evaluate the associations among EPHA5 methylation, environmental factors and GC risk. Meanwhile, the propensity score (PS) was used to adjust the imbalance of some independent variables.ResultsAfter PS adjustment, EPHA5 Pm (positive methylation) was more likely to increase the GC risk than EPHA5 Nm (negative methylation) (ORb = 1.827, 95% CI [1.202–2.777], P = 0.005). EPHA5 Pm had a more significant association with GC risk in the elderly (ORa = 2.785, 95% CI [1.563–4.961], P = 0.001) and H. pylori-negative groups (ORa = 2.758, 95% CI [1.369–5.555], P = 0.005). Moreover, the combined effects of EPHA5 Pm and H. pylori infection (ORca = 3.543, 95% CI [2.233–5.621], P < 0.001), consumption of alcohol (ORca = 2.893, 95% CI [1.844–4.539], P < 0.001), and salty food intake (ORca = 4.018, 95% CI [2.538–6.362], P < 0.001) on increasing the GC risk were observed. In addition, no convincing association was found between EPHA5 Pm and the GC prognosis.ConclusionsEPHA5 methylation in PBLs and its combined effects with environmental risk factors are related to the GC risk.

P02.08.A The relations of focal and total DNA methylation in gliomas

Abstract Background The role of epigenetic events in gliomagenesis is undoubtful. However, the role of specific pathological events is not so clear. It was shown that loss in total DNA methylation correlates with higher tumor malignancy and oxidative DNA damage. But promoter methylation of many genes was reported to be significant for gliomas’ malignancy and predictive for the treatment outcome. In carcinogenesis in general global DNA hypomethylation and focal hypermethylation coexist. The aim of our project was to evaluate the correlation between total DNA methylation and promoter methylation of selected genes. Material and Methods We analysed glioma tissues from 60 patients. For total DNA methylation analysis we used the radiolabelling method with TLC separation of nucleotides and content estimation with phosphoimager. For promoter methylation analysis we have chosen: MGMT (O-6-Methylguanine-DNA Methyltransferase), MPG (DNA-3-methyladenine glycosylase), GJA1 (Gap junction alpha-1 protein / connexin 43). The promoter methylation level was evaluated with the methylation-sensitive high-resolution melting (MS-HRM) method. Results Total DNA methylation was reversely correlated with brain tumor grade, confirming that 5-methylcytosine loss is important step in gliomagenesis. From 3 genes only MPG promoter methylation showed clear low reverse correlation with tumor grade. Promoter methylation in GJA1 show low correlation with both, MGMT and MPG, but there was no link between MGMT and MPG. IDHwt presence was significantly correlated with higher tumor grade. Promoter methylations in MPG and GJA1 were better correlated with IDH status than in MGMT. Conclusion There is a clear correlation between total DNA methylation and tumor malignancy. Gene promoter methylation is not highly correlated with total DNA methylation and shows low significance in selected cases. Promoter methylation showed clear correlation with tumor grade only in MPG case. That suggests diverse mechanisms steering DNA methylation in general and local changes. It also shows that total DNA methylation is best predictor of tumor grade.

Single Nucleotide Polymorphism rs6445975 in the PXK Gene Is Correlated with Susceptibility and Clinical Characteristics of Systemic Lupus Erythematosus.

Background:Recently, genome-wide association studies (GWAS) have discovered several single nucleotide polymorphisms (SNPs) and loci associated with the risk of systemic lupus erythematosus (SLE). rs6445975 (T>G; intronic variant) polymorphism in the PXK gene is one of these loci. However, there was an inconsistency between the results of replicative studies on European and Asia ancestry. This study aimed to assess the possible association between rs6445975 polymorphism with SLE risk in the Iranian population.Methods:Genotype and allele distribution of rs6445975 polymorphism were investigated in 110 patients with SLE and 115 healthy controls in Isfahan University of Medical Sciences, Isfahan, Iran in 2019 via real-time PCR high resolution melting method (HRM).Results:GG and TG genotypes, but not TT genotype, were associated with increased risk of SLE (GG vs TT; OR= 7.538; 95%CI [3.47, 17.066] and TG vs TT; OR=2.21; 95%CI [1.06, 4.72]). Inheritance analysis revealed that TG + GG was correlated with the increased risk of SLE disease in the dominant model (OR=3.928; 95%CI [2.056, 7.74]). Moreover, subjects with the G allele were more frequently affected with SLE than individuals with the T allele (OR= 3.55; 95%CI [2.37, 5.36]). The G allele in patients was correlated with serum concentration of CRP, ESR, anti-dsDNA antibody, C3, and C4 and presentation of some clinical manifestations such as kidney involvements and skin lesions (P<0.05).Conclusion:Our findings suggest a substantial association between rs6445975 polymorphism in the PXK gene with susceptibility and clinical characteristics of SLE in the Iranian population.

MiRNA-binding site polymorphismin IL-15RA gene in rheumatoid arthritis and systemic lupus erythematosus: correlation with disease risk and clinical characteristics.

MiRSNPs may interfere with mRNA stability through effects on microRNAs (miRNAs)-mRNA interactions via direct changes in miRNA binding site or effect on the secondary structure of this region and changes in accessibility of this region to miRNAs. Studies have confirmed that an elevated level of interleukin-15 receptor alpha (IL-15RA) has an important role in the pathogenesis of systemic lupus erythematosus(SLE) andrheumatoid arthritis(RA). In the present study, for the first time, we aimed to evaluate the possible correlation between a miRSNP, rs2296135, in IL-15RA gene with the risk of SLE and RA. In this case-control study, 100 SLE patients, 100 RA patients, and 110 healthy participants were enrolled to assess rs2296135 genotypes with real-time PCR high-resolution melting method. According to our findings, AA genotype and A allele of rs2296135 were considerably associated with enhanced risk of RA (for AA genotype, OR = 2.29; 95% CI [1.06-5.02]; for A allele, OR = 1.65; 95% CI [1.10-2.48]). However, this common variant was not significantly correlated with SLE risk in population under study. Stratification analysis in the RA group verified that patients with the A allele had considerably higher serum concentrations of C-reactive protein (CRP) (P < 0.001). In SLE subjects, the frequency of arthritis (P: 0.021) and renal involvement (P: 0.025) in patients with A allele was significantly higher than in other SLE individuals. The current study proposes a substantial association between rs2296135 polymorphism in IL-15RA gene with augmented risk of RA and some clinical characteristics in RA and SLE patients.

Development of Ethyl Methanesulfonate Mutant Edamame Soybean (Glycine max (L.) Merr.) Populations and Forward and Reverse Genetic Screening for Early-Flowering Mutants.

Induced mutation is a viable breeding strategy that is widely utilized in the development of elite plant varieties. We aimed to improve a variety of edamame by constructing novel mutant populations using the ethyl methanesulfonate in soybeans (Glycine max (L.) Merr.). In the M2 population, the flowering stage showed a considerable standard deviation compared to the wild type, confirming that the mutant populations had the expected DNA mutations. To identify the DNA mutations in the mutant populations, we used the targeting induced local lesions in genomes (TILLING) method, which is a reverse genetic method, to search for soybean flowering-related gene mutants. A total of 30 mutants from E1, E3, E4, and PhyA1 genes, which are known to be highly effective genes, or their homologous gene for flowering and maturation found in soybean quantitative trait locus analyses were isolated from our TILLING screening. Among these mutants, there were eleven nonsynonymous substitution mutants, one nonsense mutant, and two single nucleotide deletion mutants that could be expected to reduce or eliminate gene function. The e1, e3, and e4 mutants obtained in this study flowered considerably earlier than the wild type. In particular, the e1 mutant with a nonsynonymous substitution flowered approximately 1 month after sowing regardless of the sowing date, and its harvest date was approximately 1 month earlier than that of the wild type. Mutations identified using the TILLING method could not only be used as gel-based DNA markers with the same manipulation method, but the mutations could also be detected as DNA markers by the high-resolution melting method. These results indicate that mutations achieved without chromosome modification by crossbreeding are effective for early and practical improvement of superior varieties and that efficient selection of mutants by reverse genetics is an effective method for the identification of genetic modifications. The edamame mutant populations developed in this study are believed to possess various useful alleles which may be applicable in the search for mutations that lead to improved edamame yield and eating quality beyond the flowering stage.

Assessment of the association between TNIP1 polymorphism with clinical features and risk of systemic lupus erythematosus.

Over the past decades, TNIP1 has been identified as a strong risk locus in multiple genome-wide association studies (GWAS), spanning multiple populations and various autoimmune diseases. TNIP1 is a polyubiquitin-binding protein that works as a physiological inhibitor of NF-κB and maintains immune homeostasis. Some studies have confirmed that TNIP1 is downregulated in autoimmune diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). In the current study, for the first time, we evaluated the possible association between rs6889239 polymorphism in the TNIP1 gene with the risk and clinical characteristics of RA and SLE in the Iranian population. In this case-control study, 115 patients with RA, 115 patients with SLE, and 115 unrelated healthy subjects were enrolled to estimate rs6889239 genotypes with real-time PCR high resolution melting (HRM) method. Our results demonstrated considerable associations between CC genotype and C allele of rs6889239 with augmented risk of SLE (OR for CC genotype= 2.23; 95%CI [1.175-4.307], OR for C allele= 1.84; 95%CI [1.254-2.720]). However, there was an insignificant association between genotypes and allele frequencies of rs6889239 with the occurrence risk of RA in the population under study (p > 0.05). Additionally, stratification analysis specified that the C allele in rs6889239 was linked with the incidence of renal involvement in SLE patients and lower age of onset in the RA group (p < 0.05). These findings propose a significant association between TNIP1 polymorphism and higher risk of SLE and some clinical characteristics of RA and SLE.

Molecular Diagnostics of Lung Cancer in Serous Effusion Samples

For molecular diagnostics of lung cancer samples, often only a small amount of material is available. The ever-increasing number of biomarker testing is in contrast to the amount of material obtained. In that case, cytological specimens, such as serous effusion samples, are one possible option. Effusion samples were prepared as sediment smears or cytospins or as a cell block if needed. Suitable tumor cells areas were marked by a cytopathologist and used for molecular diagnostics, including fast track analysis, parallel sequencing, and/or fluorescence in situ hybridization. In 62 cases of malignant effusion with cells of pulmonary adenocarcinoma, molecular diagnostics were carried out. A fast-track result with the high-resolution melting method for hotspot mutation of KRAS Exon 2 and EGFR exon 21 and fragment length analysis of EGFR exon 19 was available for 43 out of 47 samples (92%). Parallel sequencing was successful for 56 out of 60 samples (93.3%). In the same period, 108 FISH analyses were performed for MET amplification, followed by ROS1, RET, and ALK translocation analysis. If only a limited amount of tissue/biopsy is available, a malignant effusion is advisable to perform on the molecular diagnostics with a high success rate.