Detection Of Point Mutations Research Articles

Detection of Colletotrichum spp. Resistant to Benomyl by Using Molecular Techniques.

Colletotrichum species is known as the major causal pathogen of red pepper anthracnose in Korea and various groups of fungicides are registered for the management of the disease. However, the consistent use of fungicides has resulted in the development of resistance in many red pepper-growing areas of Korea. Effective management of the occurrence of fungicide resistance depends on constant monitoring and early detection. Thus, in this study, various methods such as agar dilution method (ADM), gene sequencing, allele-specific polymerase chain reaction (PCR), and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were applied for the detection of benzimidazole resistance among 24 isolates of Colletotrichum acutatum s. lat. and Colletotrichum gloeosporioides s. lat. The result of the ADM showed that C. gloeosporioides s. lat. was classified into sensitive and resistant isolates to benomyl while C. acutatum s. lat. was insensitive at ≥1 µg/ml of benomyl. The sequence analysis of the β-tubulin gene showed the presence of a single nucleotide mutation at the 198th amino acid position of five isolates (16CACY14, 16CAYY19, 15HN5, 15KJ1, and 16CAYY7) of C. gloeosporioides s. lat. Allele-specific PCR and PCR-RFLP were used to detect point mutation at 198th amino acid position and this was done within a day unlike ADM which usually takes more than one week and thus saving time and resources that are essential in the fungicide resistance management in the field. Therefore, the molecular techniques established in this study can warrant early detection of benzimidazole fungicide resistance for the adoption of management strategies that can prevent yield losses among farmers.

Model of Colorectal Cancer for Implementation of the HRMA Method for the Genetic Characterization of Human Pathologies

Context: In developing countries and particularly in Sub-Saharan Africa, access to sequencing techniques is limited. In this context, it is necessary to adopt strategies that will allow researchers to work on molecular genetics and genomics and to allow the greatest number of people to benefit from a precision diagnosis that until now has been outsourced to laboratories in Western countries. The high-resolution curve analysis method (HRMA) for the detection of point mutations in diagnostic choices was evaluated here. Methodology and Results: Using genomic DNA from cell lines, the mutation detection sensitivity of the HRMA method was tested on samples containing different percentages of mutated DNA. The results obtained show that the HRMA method can discriminate wild-type samples from those containing a mutation, even for small amounts of mutated DNA in the sample. Conclusion: For the time being, systematic sequencing of all samples for research and diagnosis is a very expensive strategy in our context. The present evaluation allows to consider molecular genetic and genomic studies as well as molecular diagnosis in two steps: (i) screening of samples by the HRMA method; (ii) sequencing of samples containing a mutation by the Sanger method.

Evaluation of clarithromycin and levofloxacin resistance of Helicobacter pylori strains isolated from patients at the Hospital for Tropical Diseases, Ho Chi Minh City

Clarithromycin (CLA) and levofloxacin (LFX) have been recommended as the most effective antibiotics for treating Helicobacter pylori infection. However, the increase in H. pylori’s resistance to antibiotics is an alarming and growing challenge. The study aimed to determine the antimicrobial resistance profiles of H. pylori strains isolated from patients at the Hospital for Tropical Diseases and to detect point mutations in 23S ribosomal RNA (23S rRNA) and gyraseA (gyrA) genes. Point mutations in 23S rRNA and gyrA were detected using Sanger sequencing. Antibiotic resistance was tested by the microdilution method. Out of the 45 isolates, 44 (97.8%) were found to be resistant to at least one antibiotic, 38 (84.4%) resistant to metronidazole (MTZ), and all were sensitive to amoxicillin (AMX) and tetracycline (TET). Sixty percent of the isolated strains were resistant to 3-6 antibiotics; among them, multiple drug resistance (MDR) strains presented twenty percent resistance to more than two classes of antibiotics. Eleven strains (24.4%) carried two mutations associated with CLA and LFX but only nine of them were dual resistant to CLA and LFX, and twenty-four (53.3%) carried either CLA or LFX mutation. The point mutations A2143G in the 23S rRNA and N87K (Asn87Lys) in the gyrA were detected in the CLA and LFX resistant strains, respectively. The prevalence of MDR, especially CLA, MTZ, and LFX resistance, in the H. pylori isolates suggests that the use of these antibiotics need to be more considerable and cautious.

A Rapid Genotyping Assay for CSF Accelerates Diagnosis and Treatment Initiation for CNS Malignancies

The overlapping clinical and radiographic features of CNS neoplasms, combined with low yields of conventional CSF studies, complicate diagnostic workflows and delay treatment initiation. Neurosurgical biopsies of high-risk lesions are frequently required, and decisions regarding extent of surgical resection are challenging. In order to address these challenges, we previously developed the Targeted Rapid Sequencing (TetRS) assay to detect highly recurrent point mutations characteristic of CNS neoplasms from CSF, within 80 minutes of sample collection. This qPCR-based panel offers parallel detection of MYD88, TERT promoter, IDH1/2, BRAF and H3F3A point mutations. In this study, we describe the clinical performance and real-time utilization of this assay in a large, single-center prospective patient sample. We asked whether rapid point mutation detection from CSF liquid biopsies can (1) accelerate times to diagnosis and (2) provide a basis for safely initiating disease-directed therapies in the absence of solid tissue sampling. We collected CSF from 98 patients with new CNS lesions suspicious for neoplasms over an 18-month period. Patient enrollment, selection of specific assay variants, and utilization of results in clinical decision-making were at the discretion of providing physicians. IRB approval was obtained. In this diverse cohort, 40 of 98 patients ultimately established a diagnosis of a CNS neoplasm. A point mutation was detected by TetRS in 15 out of 40 patients with a neoplasm (37.5%); this included cases of primary and secondary CNS lymphoma (PCNSL), glioblastoma, IDH-mutant brainstem glioma and H3K27M-mutant diffuse midline glioma. In time-to-event analyses, we found that detection of a mutation by TetRS accelerated the return of diagnostic information from CSF sampling when compared to contemporaneous and historical control cases in which a mutation was not detected (median 0.5 vs 10.5 days, P<0.01). We found multiple cases in which mutation detection by TetRS helped to avoid neurosurgical biopsy. In one representative case, detection of the MYD88 L265P mutation established the diagnosis of PCNSL; methotrexate and rituximab were initiated within 3 days of CSF sampling. In this patient, TetRS results over serial CSF samplings correlated with treatment response and detecting disease recurrence. In another representative case, a lesion initially thought to be an H3K27M-mutant diffuse midline glioma on clinical and radiographic grounds was instead diagnosed as an IDH-mutant brainstem glioma based on TetRS detection of the IDH1 R132G mutation in CSF. This enabled rapid initiation of fractionated radiation, as well as targeted IDH inhibitor therapy; the patient experienced clinical and radiographic response over 10 months of follow-up. TetRS thus represents a novel, targeted point mutation panel that can rapidly and specifically diagnose a broad array of CNS neoplasms. We demonstrate the potential of this novel tool to accelerate diagnosis, follow treatment response and detect recurrence in selected patients without solid tissue sampling. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

TMIC-71. SPATIAL ANALYSIS OF GENETIC HETEROGENEITY AND TUMOR MICROENVIRONMENT INTERACTIONS IN GLIOBLASTOMA

Abstract Genetic variation can be considered a driver of somatic evolution in glioblastoma (GBM); however, the tumor microenvironment (TME) is a key contributor to the selection process and evolutionary potential of cancer cells. Although diverse ecosystems exist in GBM which impact tumor cell fitness and characterize an aggressive pathophysiology, little is known about how genetically distinct subpopulations of GBM cells interact with the surrounding TME. Therefore, we sought to investigate how the varying clonal genotypes of GBM cells are influenced by different types of selective pressures within the TME. Our imaging-based strategy is centered around Specific-to-Allele PCR Florescence In Situ Hybridization (STAR-FISH), an in situ PCR based probe hybridization method for detection of point mutations and copy number variations at the single-cell level. Using STAR-FISH to illuminate the spatial distribution of clones with mutations in the TERT promoter, we integrated multicolor immunofluorescent staining, DNA-FISH, reflectance imaging, and computational cellular analysis methods to achieve highly-multiplexed single cell spatial profiling of intact GBM specimens. With this strategy we are able to simultaneously evaluate tumor tissues for the presence of hypoxic, angiogenic, and inflammatory niches, extracellular matrix organization, nuclear morphology, mutational status, and several oncogenic amplifications on the same slide and location. We analyzed 20 matched primary and recurrent GBM samples, imaging several distinct regions of the same tumor. Our initial results have revealed interesting and potentially novel distributions of subclones residing within specific microenvironments. These results include TERT mutation positive clones enriched in areas possessing more structured extracellular matrix, as well as unique arrangements of infiltrating immune cells in the angiogenic tumor niche. This research underscores the importance of the TME in selecting for certain phenotypes that characterize somatic evolution, highlighting novel genetic and spatial dependencies which sustain heterogeneity in GBM.

The Spatio-Temporal Pattern and Transition Mode of Recessive Cultivated Land Use Morphology in the Huaibei Region of the Jiangsu Province

Examining land use transition is a new way of building on the comprehensive research on Land Use/Cover Change (LUCC). Research on transition law and characteristics is important for improving the theory of land use transition and the practice of land resource management, and for being able to provide a basis and reference for promoting socio-economic transformation. Based on the relevant statistical data concerning cultivated land use in the Huaibei area of the Jiangsu Province from 1995 to 2020, and by understanding the county as a unit to be measured, this paper constructed a multi-dimensional (economic–social–ecological) functional index system of recessive morphology, analyzed the spatio-temporal pattern of the transition of cultivated land use, identified transition point mutations, and established the transition mode by adopting multi-dimensional time series point mutation detection and the piecewise linear regression method. The findings suggest that the index of recessive cultivated land use morphology in the Huaibei region of the Jiangsu Province presents a trend of “slow decline to significant growth to stable growth”. Moreover, the index presented evolutionary characteristics such as “high in the middle and east while low in the west”, as well as “the relatively balanced distribution between counties”, thus indicating that the degree of transition deepened, it showed a homogeneous development trend, and the transition process presented obvious “ladder” stage characteristics; therefore, the authors suggest making scientific use of cultivated land resources, in accordance with local conditions, in order to make the land use transition of cultivated land efficient, green, and sustainable.

Excerno: Using Mutational Signatures in Sequencing Data to Filter False Variants Caused by Clinical Archival.

The accurate detection of point mutations from pathology slides using sequencing data is of great importance in cancer genomics and precision oncology. Formalin-fixation paraffin-embedding (FFPE) is a widely used technique to preserve pathology tissues. The FFPE process introduces artificial C > T mutations in next-generation sequencing, so we set out to develop excerno, a method to score and filter such spurious variants. FFPE mutational artifacts follow a mutational signature. By using the FFPE signature and Bayes' formula, we can calculate the probability of a mutation resulting from the FFPE process and use this probability to filter FFPE variants. We implement this method as the excerno R package. We tested excerno by simulating mutations across all 60-baseline mutational signatures from the Catalog of Somatic Mutations in Cancer (COSMIC) and combining them with mutations following the FFPE mutational signature. The sensitivity and specificity of excerno are adversely affected by the cosine similarity between the baseline and FFPE signatures (). Higher percentages of FFPE mutations () result in increased sensitivity and reduced specificity. The specificity and sensitivity of excerno can be predicted as linear model with an interaction term using and , with an and 0.79, respectively. Finally, we tested excerno using six RNA sequencing cancer samples and observed concordant trends of specificity and sensitivity with respect to our simulated data. The excerno R package can be used to annotate and filter FFPE-induced mutations in cancer genomics. Our method is adversely affected by and .

Molecular Detection of gyrA Mutation in Clinical Strains of Klebsiella pneumoniae

Multi-drug resistant (MDR) Klebsiella pneumoniae strains cause the majority of community acquired and life-threatening infections. We aimed to detect the gyrA mutations in the clinical strains of nalidixic acid and ciprofloxacin resistant K. pneumoniae isolated from the patients with urinary tract infections. Bacterial strains were isolated from the patients with urinary tract infections admitted to a major hospital in Tehran, Iran (2017-2018). Bacterial identification was done according to standard microbiological tests. Antimicrobial susceptibility testing for quinolones and fluoroquinolone antibiotics was done using both disc diffusion and minimal inhibitory concentrations (MICs) methods. PCR-RFLP was used to detect the probable mutation in the gyrA gene in nalidixic acid and ciprofloxacin resistant strains. Finally sequencing was performed to detect point mutations in isolated K. pneumoniae strains. One hundred K. pneumonia isolates were recovered from the urine samples of the clinical cases. Antibiotic resistance testing showed that among all K. pneumoniae isolates, 26% and 19% of the strains were resistant to nalidixic acid and ciprofloxacin respectively. MIC value was ≥4 μg/ml for ciprofloxacin resistant isolates. The results of RFLP on gyrA PCR amplicons using HinfI restriction enzyme showed point mutation in this gene in 46% of nalidixic acid and ciprofloxacin resistant K. pneumonia. The data obtained from the sequencing confirmed the RFLP results and indicated the presence of point mutations in codons 83 and 87 in the gyrA gene which leads to the substitution of different amino acids in gyrA protein. Our findings indicated a relative increased rate of resistance against quinolones and fluoroquinolone antibiotics that raised a concern about extensive dissemination of clinical strains of nalidixic acid and ciprofloxacin resistant K. pneumonia. Point mutation of gyrA gene was responsible for the resistance in our strains however to gain more insight into the molecular characterization of quinolone-resistant isolates, other possible mechanisms of the resistance should also be investigated.

Gold island‐enhanced multiplex quantum dots fluorescent system for biomedical analysis of circulating tumor nucleic acids

AbstractCirculating tumor nucleic acids (CTNAs) have been employed as the potential markers for tumor diagnosis and management, which are highly correlated with the tumorigenesis, progression, and metastasis processes. Therefore, it is important to develop a highly‐sensitive and reliable method for the detection of CTNAs, especially the multiplex point mutation detection of blood‐derived CTNAs. Herein, a versatile gold island‐enhanced multiplex quantum dots (GIEM‐QDs) fluorescent platform was constructed for the highly‐sensitive detection of CTNAs in serum. The GIEM fluorescent strategy was designed as the highly‐efficient signal giving‐out mode which could amplify the fluorescence intensity of QDs, thus realizing a homogeneous platform for the enrichment, multiplex detection, and point mutations monitoring of CTNAs with the principle of base‐stacking. A high sensitivity of 10 amol and desirable specificity was achieved, and the performance index for analysis of clinical tumor patients’ samples indicated that the GIEM‐QDs fluorescent strategy could realize multiplex point mutation detection of CTNAs in complex blood samples. Hence, this platform could achieve a high detection rate in clinical samples that suitably met the clinical requirements for multiplex detection and point mutations monitoring of CTNAs, and thus has the potential to serve as the tumor liquid biopsy strategy based on CTNAs.

Second-line Therapy for Helicobacter Pylori Eradication: State of the Art.

Helicobacter pylori (H. pylori) is a Gram negative spiraliform bacterium that colonizes the human stomach. It is the most common cause of chronic gastritis, peptic ulcer, and gastric carcinoma. The eradication therapy is based on the combination of a proton pump inhibitor and several antibiotics such as amoxicillin, metronidazole, clarithromycin, levofloxacin or tetracycline. The most commonly used regimens for eradication in the first line are triple, sequential and concomitant therapy, despite the last European Guidelines suggesting a quadruple therapy already at the first attempt in areas with high resistance rates. However, the rise in antibiotic resistance is the main reason for a marked increase in first-line therapy failure. Clarithromycin resistancea, is especially acknowledged as the most important event resulting in failure. Up to 20% of patients are intended not to eradicate, therefore they will need a second line therapy. Currently, the most used rescue regimens are levofloxacin-based triple therapy and bismuth-containing quadruple therapy, despite guidelines suggesting to use a combination of antibiotics that have not been included in previous treatments. Nitazoxanide is a novel antibiotic with promising results. Additionally, an interesting field worth of investigation is the antibiotic susceptibility based approach, which could help choose antibiotics with confirmed effectiveness in vitro. Analysis of antibiotic resistance may be performed by both bacterial culture and molecular biology techniques, able to detect point mutations conferring resistance. This is a particularly interesting approach, since it may personalize the therapy, thus optimizing the regimen and maximizing the probability of success.

DEVELOPMENT OF GENETIC DIVERSITY OF DIFFERENT BRINJAL GERMPLASM BY USING DNA MARKERS

Brinjal is an important Solanaceous vegetable in many countries of Asia and Africa. It is a good source of minerals and vitamins in tropical diets hence analysis of Genetic diversity of eggplant germplasm is important from agronomic and breeding point of view. The present study was about genetic diversity of different eggplant germplasm by using PCR based DNA markers. PCR is a technique used to amplify a specific DNA sequence. It is a widely used method for detection of particular DNA sequence in a pool of unknown DNA sequence. 4 eggplant germplasm were collected for the identification of genetically diverse line among them. Total 10 ISSR, 15 RAPD, and 5 SSR primers were used. Among them 2 ISSR primer showed the specific polymorphic band. UBC 820 for AS 33 and AS 63 genotypes and UBC 844 for AS 888 and AS 555 genotypes. These genetically distinct genotypes identified using ISSR markers could be potential sources of germplasm for eggplant improvement by detection of point mutation and detection of diseases if any present in those germplasm, henceforth increasing the crop.

Detection of A2143G, A2142C, and A2142G Point Mutations with Real-Time PCR in Stool Specimens from Children Infected with Helicobacter pylori.

Reports have indicated an increasing prevalence of clarithromycin resistance in children relative to adults. Thus, it is important to investigate primary clarithromycin resistance before therapy to avoid treatment failure. A2142G, A2143G, and A2142C point mutations in the peptidyltransferase region of the 23S ribosomal RNA (rRNA) of Helicobacter pylori (H. pylori) strains isolated from children with gastrointestinal symptoms and asymptomatic children were evaluated via real-time polymerase chain reaction (RT-PCR) using fecal DNA samples. The presence of H. pylori was determined using a fecal H. pylori antigen enzyme-linked immunosorbent assay (ELISA) kit from the stools of children (n = 543). A2143G, A2142C, and A2142G point mutations were detected via RT-PCR and confirmed by sequencing the 23S rDNA. Fecal H. pylori antigen testing was positive in 101 symptomatic (49) and asymptomatic (52) children. A significant difference was found between the 0–5- and 5–18-year-old groups in terms of the A2143G and A2142G point mutations (p = 0.001). The A2142C mutation was not detected. There was a significant difference in the A2143G mutation between the symptomatic and asymptomatic 5–18-year-old children (p = 0.019). Macrolides are frequently used to treat upper respiratory tract infections in children due to their selective pressure effect. We suggest that H. pylori strains carrying mutations in the 23S RNA subunit conferring clarithromycin resistance may lead to an intense inflammatory response in the gastric epithelial cells, allowing them to proliferate more rapidly and causing possible diarrhea, halitosis, or abdominal pain in children.

Hypertriglyceridemia as a main feature associated with 17q12 deletion syndrome-related hepatocyte nuclear factor 1β-maturity-onset diabetes of the young.

SummaryHepatocyte nuclear factor 1β (HNF1B) gene is located on chromosome 17q12. It is a transcription factor implicated in the early embryonic development of multiple organs. HNF1B-associated disease is a multi-system disorder with variable clinical phenotypes. There are increasing reports suggesting that the 17q12 deletion syndrome should be suspected in patients with maturity-onset diabetes of the young type 5 (MODY5) due to the deletion of HNF1B gene. In contrast to classical 17q12 syndrome in childhood with neurological disorders and autism, patients with HNF1B-MODY deletion rarely had neuropsychological disorders or learning disabilities. The diagnosis of 17q12 deletion syndrome highlighted the phenotypic heterogeneity of HNF1B-MODY patients. In this study, we report the clinical course of a Thai woman with young-onset diabetes mellitus and hypertriglyceridemia as a predominant feature due to HNF1B deletion as part of the 17q12 deletion syndrome. Our findings and others suggest that hypertriglyceridemia should be considered a syndromic feature of HNF1B-MODY. Our case also highlights the need to use sequencing with dosage analyses to detect point mutations and copy number variations to avoid missing a whole deletion of HNF1B.Learning pointsMaturity-onset diabetes of the young type 5 (MODY5) may be caused by heterozygous point mutations or whole gene deletion of HNF1B. Recent studies revealed that complete deletion of the HNF1B gene may be part of the 17q12 deletion syndrome with multi-system involvement.The length of the deletion can contribute to the phenotypic variability in patients with HNF1B-MODY due to whole gene deletion.Using next-generation sequencing alone to diagnose MODY could miss a whole gene deletion or copy number variations. Specialized detection methods such as microarray analysis or low-pass whole genome sequencing are required to accurately diagnose HNF1B-MODY as a component of the 17q12 deletion syndrome.Molecular diagnosis is necessary to distinguish other acquired cystic kidney diseases in patients with type 2 diabetes which could phenocopy HNF1B-MODY.Hypertriglyceridemia is a possible metabolic feature in patients with HNF1B-MODY due to 17q12 deletion syndrome.

The importance of SMA screening tests in newborns – review

Spinal Muscular Atrophy (SMA) is an autosomal recessive neuromuscular disease caused by the loss of alfa motor neurons occuring with a frequency of 1 of 11000 births. It leads to muscular weakness and is cause of high rate of mortality in children. 95% cases of SMA are caused by homozygous deletion in SMN1 which is identified as the SMA disease-determining gene. The number of copies of SMN2 determines the phenotype of the disease. Screening tests for SMA can detect 95-98% of the mutations in SMN1, but cannot detect point mutations. The SMA genetic tests are characterized by high sensitivity and specificity, they are inexpensive and therefore can be used on a large scale. Generally DNA is isolated from the dry blood spot and then subjected to PCR analysis. The SMN2 copy number is determined using the MLPA technique. Screening tests allow the diagnosis of SMA even before the onset of symptoms. Numerous scientific studies show that early treatment in most patients with detected SMA allows for better neuromotor development in children. The most beneficial effects of the SMA therapy are visible in patients treated pre-symptomatically - and this is only possible thanks to the use of screening tests. In this review we present the importance of SMA screening tests in newborns according to the latest scientific reports.

Amplification refractory mutation system based real-time PCR (ARMS-qPCR) for rapid resistance characterization of Tribolium castaneum to phosphine

Resistance of Tribolium castaneum to phosphine is related to point mutations in DNA code corresponding to amino acid changes associated with a core metabolic enzyme dihydrolipoamide dehydrogenase (DLD), but the mutation patterns vary among different resistant populations. Thus, there is a great need to develop a cost-effective method to detect core mutations in T. castaneum, which would be the key factor to understand the molecular basis of phosphine resistance. Amplification refractory mutation system-based quantitative Real-Time PCR (ARMS-qPCR) is an ideal method that can rapidly detect point mutations. Here, the P45S and G131D mutations existed in the DLD of T. castaneum selected from strong Chinese resistance phenotypes, and the DLD P45S mutation, which represents a strong phosphine resistance allele, was confirmed as the most abundant mutation to determine strong resistance genotypes. Our study found that 85 out of 120 beetles carried the P45S resistance allele, including 51 homozygous and 34 heterozygous individuals. Moreover, there was a strong linear relationship (R2 = 0.917) between the resistance ratio and the resistance allele frequency among the strongly resistant populations. Our data showed that the ARMS-qPCR method that we developed could rapidly determine strong resistance phenotypes of T. castaneum to phosphine by detecting the DLD P45S mutation. These results not only provide a detailed example for developing an ARMS-qPCR-based method to characterize pesticide resistance, but also support further elucidation of the molecular basis of phosphine resistance.

NGS4THAL, a One-Stop Molecular Diagnosis and Carrier Screening Tool for Thalassemia and Other Hemoglobinopathies by Next-Generation Sequencing

Thalassemia is one of the most common genetic diseases and a major health threat worldwide. Accurate, efficient, and scalable analysis of next-generation sequencing (NGS) data is much needed for its molecular diagnosis and carrier screening. We developed NGS4THAL, a bioinformatics analysis pipeline analyzing NGS data to detect pathogenic variants for thalassemia and other hemoglobinopathies. NGS4THAL realigns ambiguously mapped NGS reads derived from the homologous Hb gene clusters for accurate detection of point mutations and small insertions/deletions. It uses a combination of complementary structural variant (SV) detection tools and an in-house database of control data containing specific SVs to achieve accurate detection of the complex SV types. Detected variants are matched with those in HbVar (A Database of Human Hemoglobin Variants and Thalassemia Mutations), allowing recognition of known pathogenic variants, including disease modifiers. Tested on simulation data, NGS4THAL achieved high sensitivity and specificity. For targeted NGS sequencing data from samples with laboratory-confirmed pathogenic Hb variants, it achieved 100% detection accuracy. Application of NGS4THAL on whole genome sequencing data from unrelated studies revealed thalassemia mutation carrier rates for Hong Kong Chinese and Northern Vietnamese that were consistent with previous reports. NGS4THAL is a highly accurate and efficient molecular diagnosis tool for thalassemia and other hemoglobinopathies based on tailored analysis of NGS data and may be scaled for population carrier screening.

Application of Single-Stranded DNA and PNA Probes for Electrochemical Detection of miRNA 141

miRNA are known as short RNA molecules that usually consist of 18 – 25 nucleotides and are considered as posttranscriptional regulators of gene expression occurring in plants and animals [1]. Over 60% of all genes which encode proteins are controlled by miRNA [2]. miRNA is engaged in various processes including differentiation, proliferation and apoptosis as well as inflammation. Hence, they might be considered as potential biomarkers of diseases such as cancer, neurodegenerative and cardiovascular [3]. A huge challenge is the actual detection of miRNA because of their short length, low concentration and matrix interferences. The miRNA analysis can be performed with the following techniques such as real-time PCR, northern blotting and microarray [4-6]. However, the above mentioned techniques are rather sophisticated and are costly. An alternative could be the application of electrochemical techniques which are characterized with low detection limits, possibility of miniaturization and high sensitivity. In such a case miRNA could be detected using biosensors containing receptor layers formed of single-stranded DNA or PNA.Herein, we present the studies on the development of ssDNA/PNA – based receptor layers for the electrochemical analysis of miRNA 141, which is a biomarker of pancreatic cancer. Various receptor layers were tested including unlabelled ssDNA, methylene blue-labelled DNA as well as PNA-based. The formation of ssDNA/PNA receptor layer along with its binding to miRNA was confirmed using QCM and SPR techniques. The electrochemical studies revealed that it was not possible to obtain low detection limits for miRNA when MB-labelled ssDNA or PNA molecules were applied. On the contrary, detection limit of 10-4 nM was reached with the application of unlabelled ssDNA molecules. The further analysis showed the possibility of detection of point mutation in ssDNA or miRNA sequence as well as miRNA detection in spiked serum sample.Bibliography:[1] D. P. Bartel, Cell 116 (2004) 281–297[2] A. Krek, D. Grun, M. N. Poy, R. Wolf, L. Rosenberg, E. J. Epstein, P. MacMenamin, I. da Piedade, K. C. Gunsalus, M. Stoffel, N. Rajewsky, N. Nat. Genet. 37 (2005) 495−500.[3] H. Hwang, J. T. Mendell, Br. J. Cancer 94 (2006) 776–780.[4] C. Chen, D. A. Ridzon, A. J. Broomer, Z. Zhou, D. H. Lee, J. T. Nguyen, M. Barbisin, N. L. Xu, V. R. Mahuvakar, M. R. Andersen, K. Q. Lao, K. J. Livak, K. J.Guegler, Nucleic Acids Res. 33 (2005) e179[5] A. Valoczi, C. Hornyik, N. Varga, J. Burgyan, S. Kauppinen, Z. Havelda, Nucleic Acids Res. 32 (2004) e175[6] A. M. Krichevsky, K. S. King, C. P. Donahue, K. Khrapko, K. S.Kosik, RNA 9 (2003) 1274−1281.

Exome Sequencing as a Tool for Detecting Point Mutations and Deletions in Patients With Hypogonadotropic Hypogonadism.

Exome Sequencing as a Tool for Detecting Point Mutations and Deletions in Patients With Hypogonadotropic Hypogonadism.

Detection of the presence of polymorphisms in the DPYD gene in patients with colorectal and non-colorectal digestive tumors.

e15530 Background: Recently the European Medicines Agency (EMA) and the Spanish Medicines Agency (AEMPS) recommend performing a phenotypic and/or genotypic test before starting treatment with fluoropyrimidines The objective of this work has been to analyze the detection of polymorphisms in the DPYD gene in order to predict toxicity to fluoropyrimidines in our study population. Methods: Prospective study of 6 months duration in which patients diagnosed with CRC and non-CRC digestive tumors with an age range of 18-99 years have been included. The technique is performed on a sample of DNA extracted from lymphocytes in peripheral blood in an EDTA tube, performing a specific fluorescent allele amplification study (AMRS), which detects point mutations, insertions or deletions of DNA. Results: During the study period, 260 patients have been included, of whom 16 mutations have been detected, all in heterozygosity. Conclusions: Various variants of DPYD have been reported to cause the absence or reduction of dihydropyrimidine dehydrogenase (DPP) activity. Through this technique we avoid the risk of serious toxicity with the administration of these drugs.[Table: see text]

The prevalence and prognostic impact of mutations promoting chromatin remodelling dysregulation in non-resectable or recurrent/metastatic adenoid cystic carcinoma.

6087 Background: Few effective drug therapies exist for patients with recurrent/metastatic (R/M) adenoid cystic carcinoma (ACC). Mutations in genes encoding chromatin remodelling proteins are described in almost 50% of ACC patients (pts). Novel drug therapies are being developed to target these pathways, and may have clinical utility in this sub-group. We sought to classify mutations in chromatin remodelling genes in ACC based on predicted pathogenicity and to determine the impact of chromatin remodelling dysregulation (CRD) on clinical outcomes. Methods: Matched clinical-genomic data from 269 pts with non-resectable or R/M ACC were included in this study. 130 pts were prospectively recruited to an ethically approved study. For these pts, DNA extracted from FFPE tissue was sequenced on a commercially available platform to detect point mutations, indels and copy number variation in 324 genes. In addition, clinical-genomic data from 139 ACC pts were collected from cBioPortal (MetTropism, Cell 2021) for analysis. Mutations were classified as pathogenic using COSMIC, ClinVar and OncoKB. Univariate survival analysis was performed to determine the impact of one or more mutations in chromatin remodelling genes on survival from first recurrence or metastasis. p values determined using Kaplan-Meier and log-rank. Results: In 269 pts with non-resectable or R/M ACC, mutations promoting CRD were identified in ARID1A (11%), CREBBP (5%), EP300 (5%), KMT2C (1%), KMT2D (5%), KDM6A (13%), and SETD2 (3%). CRD mutations were identified in 94/269 (35%) pts, with 27/269 (10%) having 2 or more CRD mutations. For patients in whom CRD mutations were present survival from recurrence was decreased (median OS 4.6 v 8.2 years, HR 1.65 (95% confidence interval (CI) 1.13-2.40), p = 0.01). Analysis of each individual CRD gene identified that association with decreased survival from recurrence was significant for mutations in KDM6A (n = 35; median OS 4.2 vs 6.5 years; HR 2.01 (95% CI 1.25-3.22); p = 0.004), CREBBP (n = 11; median OS 4.2 vs 5.9 years; HR 2.97 (95% CI 1.20-7.38); p = 0.019) and SETD2 (n = 7; median OS 3.7 vs 5.9; HR 2.47 (95% CI 1.002-6.09); p = 0.042). Previous studies have identified NOTCH pathway activation and TP53 loss-of-function as prognostic. In a secondary analysis of pts without NOTCH activation and/or TP53 mutations (n = 202), OS from recurrence was decreased in those with CRD mutations (n = 58 median OS 5.7 vs 9.2 years, HR 1.5 (CI 95% 0.95-2.55), p = 0.07). Conclusions: We have identified a novel prognostic group of ACC pts characterised by mutations promoting CRD, which may have potential therapeutic options. Alterations in EP300/CREBBP/ARID1A may provide a rationale for treatment with CREBBP/EP300 inhibitors currently in clinical development. Significant co-occurrence with NOTCH gain of function may provide a rationale for future combination studies.