Method For Detection Of Mutations Research Articles

A family screening of CD19 gene mutation by PCR-RFLP

Introduction and aim. Mutation(s) in the gene encoding the CD19 molecule affect CD19 protein expression and primary immunodeficiency (PID) occurs. The PCR-RFLP method, which is faster and cheaper than other mutation detection methods, is rarely used in the diagnosis of PID. The study aimed to genetically identify CD19 deficiency, which is a PID, using the PCR-RFLP method. Material and methods. A total of 8 patients and two healthy controls were included in the study and the relevant region genotypes in the CD19 gene were determined by performing PCR-RFLP analysis. Results. The index case, newborn baby and mother were also included in the study. It was determined that the index case (P6) was homozygous mutant, the newborn baby (P7) and mother (P8) had heterozygous genotype. Based on this situation, one child (P1) was found to be homozygous mutant, mother (P2), father (P3) and other children (P4 and P5) had heterozygous genotype in the family, which was determined to be related to the first case. Conclusion. In our study, it has been shown that PCR-RFLP is a method that can be used in the diagnosis of PID by determining genotypes using PCR-RFLP, and especially in terms of rapid genetic testing of family screenings.

Enzyme-Free Colorimetric Method for Fast Detection of Pik3ca Gene Mutation by Praseodymia Nanorods

Enzyme-Free Colorimetric Method for Fast Detection of Pik3ca Gene Mutation by Praseodymia Nanorods

A universal probe system for low-abundance point mutation detection based on endonuclease IV.

Single base mutations are closely related to cancer diagnosis and treatment. The fluorescent probe method is one of the important methods to detect single-base mutations. We constructed a universal probe detection system based on endonuclease IV and the DNA strand displacement reaction. The system uses two toehold strand displacement reactions to relay the mutation information to the universal strand. There is no need to design the probe one-by-one for each mutation point during multi-site detection. It has the advantages of simple operation, rapid detection, and low cost. We used this method to detect common clinical mutation sites (PTEN R130Q/EGFR L858R/PTEN rs1473918395), and the detection limit can reach 0.1%-1%. The detection system can provide a new rapid and economical method for clinical single-base mutation detection, and has broad application prospects in diagnosis and prognostic evaluation.

Nuclease-Assisted, Multiplexed Minor-Allele Enrichment: Application in Liquid Biopsy of Cancer.

The use of next-generation sequencing (NGS) to profile genomic variation of individual cancer species is revolutionizing the practice of clinical oncology. In liquid biopsy of cancer, sequencing of circulating-free DNA (cfDNA) is gradually applied to all stages of cancer diagnosis and treatment, serving as complement or replacement of tissue biopsies. However, analysis of cfDNA obtained from blood draws still faces technical obstacles due in part to an excess of wild-type DNA originating from normal tissues and hematopoietic cells. The resulting low-level mutation abundance often falls below routine NGS detection sensitivity and limits reliable mutation identification that meets clinical sensitivity and specificity standards. Despite sample preparation advances that reduce sequencing error rates via use of unique molecular identifiers (molecular barcodes) and error-suppression algorithms, excessive amounts of sequencing are still required to detect mutations at allelic frequency levels below 1%. This requirement reduces throughput and increases cost.In this chapter, we describe a sensitive multiplex mutation detection method that enriches mutation-containing DNA during sample preparation, prior to sequencing, thereby increasing signal-to-noise ratios and providing low-level mutation detection without excessive sequencing depth. We couple targeted next-generation sequencing with wild-type DNA removal using Nuclease-assisted Minor-allele Enrichment using Probe Overlap, NaME-PrO, a recently developed method to eliminate wild-type sequences from multiple targets simultaneously. A step by step guide to library preparation and data analysis are provided as well as some precautions during the sample handling.

A simple method for detection of mutations in amino acid 452 of the Spike protein of SARS-CoV-2 using restriction enzyme analysis

Variants of Concern or Interest of SARS-CoV-2 (VOC or VOI), the coronavirus responsible for COVID-19, have emerged in several countries. Mutations in the amino acid 452 of the Spike protein are particularly important and associated with some of these variants: L452R, present in Delta VOC, and L452Q, present in Lambda VOI. These mutations have been associated with both increased infectivity and evasion of protective immune response. A search on GISAID to detect the number of sequences harboring the L452R mutation and the frequency of Delta VOC among them, showed that since August 2021, most of these sequences belong to the Delta VOC. Restriction enzyme analysis is proposed as a rapid method to detect L452R. A small amplicon from the Spike gene was digested with MspI. A 100% concordance was observed between digestion and sequencing results. The mutation L452Q can also be detected by restriction analysis, allowing the identification of putative Lambda VOIs. The proposed methodology, which allows screening of a great number of samples, could provide a faster information on the prevalence of Delta VOC cases.

Investigation of EGFR mutations in non-small cell lung cancer usually undetectable by PCR methods.

Epidermal growth factor receptor (EGFR) mutations are the most significant genomic drivers of non-small cell lung cancer (NSCLC) and determine the efficacy of EGFR tyrosine kinase inhibitor (EGFR-TKI) therapy. PCR methods are used clinically for the detection of EGFR mutations. The Scorpion Amplification Refractory Mutation System (Scorpion-ARMS) and the cobas® EGFR Mutation Test v2 (cobas v2) are widely used PCR methods. However, those PCR methods only selectively detect the common EGFR mutations. The aim of the present study was to reveal the true frequency of EGFR mutations in NSCLC by investigating EGFR mutations usually undetectable by PCR methods by using direct sequencing. A total of 70 Japanese patients who underwent lung resection for NSCLC between September 2016 and March 2019 were included in the present study. Subsequently, PCR methods and direct sequencing were performed. In total, 29 mutations were detected by cobas v2. In total, 41 patients were identified as EGFR wild-type by cobas v2, among whom direct sequencing detected mutations in 3 patients. Subsequent Scorpion-ARMS was performed in the 3 patients in whom direct sequencing detected mutations. In total, one exon 21 L858R + G863D compound mutation was identified as a L858R single mutation, and two other mutations were undetectable. Moreover, 1 patient who was ‘wild-type’ on cobas v2 but ‘EGFR mutation’ on direct sequencing developed recurrence after surgery and responded to EGFR-TKI treatment. In present study, the percentage of undetectable EGFR mutations by cobas v2 was 9.4% in 32 mutations. It was inferred that the cause of the discrepancy in the mutation type (L858R + G863D in exon 21, and L858R in exon 21) between cobas v2 and Scorpion ARMS was due to the different limit of detection between these two PCR methods. In conclusion, the findings of the present study suggested that a selective mutation detection method may decrease the opportunity of patients with NSCLC to receive EGFR-TKI therapy. Thus, the development of a screening test to determine the EGFR status as wild-type or mutant is required for EGFR-TKI therapy.

Highly Sensitive and Rapid Diagnosis of Mutations in Hematological Malignancies By CRISPR Technology

With the rapid development of genomics and molecular diagnosis technologies, the treatment of hematological malignancies has entered the era of precision medicine, where individualized treatment is provided based on the unique genetic profile of each patient. Commonly-used techniques for gene mutation detection include polymerase chain reaction (PCR), sequencing approaches and real-time fluorescence quantitative PCR. However, these methods have drawbacks in sensitivity, specificity, or speed. Here, we established a mutation detection method based on CRISPR technology, which can achieve a highly sensitive, specific, and rapid diagnosis. Through a rapid release of genomic DNA, isothermal amplification, mutation enrichment, and Cas12a fluorescence reaction, this method can complete the whole diagnosis process from drawing blood to obtaining the results within one hour, without any large instruments or expensive reagents. Taking the detection of FLT3 gene D835Y/V/H/F drug-resistant mutations as an example, the sensitivity can reach 0.001%, which means 10 copies of mutation template can be detected from 999,990 copies of wild-type template. Then we used this method to detect 112 patients with acute myeloid leukemia, resulting in 11 FLT3-D835Y/V/H/F positive cases, which were verified by next-generation sequencing while missed by Sanger sequencing. We also applied this method to detect other common mutations in hematological malignancies, including NRAS gene G12D, IDH2 gene R172K, and JAK2 gene V617F. These results fully proved the high sensitivity, specificity, efficiency and universality of this method. Thus it has the potential to become a novel molecular diagnostic technology, and help point-of-care, early screening, prognosis determination and medication guidance in hematological malignancies. DisclosuresNo relevant conflicts of interest to declare.

Clinical characteristics, disease evolution and survival in patients with chronic myeloid leukemia, BCR-ABL1 (+) and T315I mutation.

The T315I mutation in patients with chronic myeloid leukemia (CML) has been associated with therapeutic resistance and an unfavourable prognosis. To study the frequency of T315I mutation in patients with CML, BCR-ABL(+), their clinical characteristics, disease evolution, and median survival. We studied 75 patients with CML and BCR-ABL1(+). T315I mutation was detected by digital droplet PCR and BCR-ABL1 was analyzed by RT-PCR. A comparative analysis was performed by sex, age, disease phase, risk group, treatment, molecular response (MR), and median survival in T315I(+) and T315I(-) patients. T315I mutation was detected in 11 patients (14.7%). No significant difference was found in the phase, risk group, and first-line therapy. A significantly higher proportion of T315I (+) did not achieve MR >3.5 log: 8 (72.7%) vs. 22 (34.4%) (p=0.023). The lowest mean BCR-ABL1 levels were significantly higher in the CML T315I(+) group compared to the CML T315I(-) group: 12.1±6.0 vs. 3.77±1.28 (p=0.009). The median survival of T315I (+) patients was significantly shorter: 73 months vs. 175 months (p<0.0001, CI 95%). Our data confirm the world experience on the frequency of T315I mutation, including the unfavourable evolution, resistance to TKI treatment and short survival. ddPCR is a highly sensitive method for early detection of genetic mutations which gives the chance for effective treatment.

Deep Learning and Pathomics Analyses Reveal Cell Nuclei as Important Features for Mutation Prediction of BRAF-Mutated Melanomas

Image-based analysis as a method for mutation detection can be advantageous in settings when tumor tissue is limited or unavailable for direct testing. In this study, we utilize two distinct and complementary machine-learning methods of analyzing whole-slide images for predicting mutated BRAF. In the first method, whole-slide images of melanomas from 256 patients were used to train a deep convolutional neural network to develop a fully automated model that first selects for tumor-rich areas (area under the curve= 0.96) and then predicts for mutated BRAF (area under the curve= 0.71). Saliency mapping was performed and revealed that pixels corresponding to nuclei were the most relevant to network learning. In the second method, whole-slide images were analyzed using a pathomics pipeline that first annotates nuclei and then quantifies nuclear features, showing that mutated BRAF nuclei were significantly larger and rounder than BRAF‒wild-type nuclei. Finally, we developed a model that combines clinical information, deep learning, and pathomics that improves the predictive performance for mutated BRAF to an area under the curve of 0.89. Not only does this provide additional insights on how BRAF mutations affect tumor structural characteristics, but machine learning‒based analysis of whole-slide images also has the potential to be integrated into higher-order models for understanding tumor biology.

The BRAF V600E Mutation Detection by quasa Sensitive Real-Time PCR Assay in Northeast Romania Melanoma Patients

Background: The prevalence of melanoma in Romanian patients is underestimated. There is a need to identify the BRAF V600E mutation to accurately treat patients with the newest approved BRAF inhibitor therapy. This is a pilot study in which we first aimed to choose the optimal DNA purification method from formalin fixation and paraffin embedding (FFPE) malignant melanoma skin samples to assess the BRAF mutation prevalence and correlate it with clinical pathological parameters. Methods: 30 FFPE samples were purified in parallel with two DNA extraction kits, a manual and a semi-automated kit. The extracted DNA in pure and optimum quantity was tested for the BRAF V600E mutation using the quantitative allele-specific amplification (quasa) method. quasa is a method for the sensitive detection of mutations that may be present in clinical samples at low levels. Results: The BRAF V600E mutation was detected in 60% (18/30) samples in patients with primary cutaneous melanoma of the skin. BRAFV600E mutation was equally distributed by gender and was associated with age &gt;60, nodular melanoma, and trunk localization. Conclusions: The high prevalence of BRAF V600E mutations in our study group raises awareness for improvements to the national reporting system and initiation of the target therapy for patients with malignant melanoma of the skin.

A Sensitive PCR-Based Method for Somatic Mutations Enrichment and Screening.

BackgroundEGFR and KRAS are the most frequently mutated genes in lung cancers, occurring in about 60% of all cases. Mutation genes assay has emerged as a promising blood-based biomarker for monitoring cancer dynamics noninvasively. However, detection can be challenging in patients where plasma often contains low levels of tumor-derived DNA fragments.MethodsWe have developed a nuclease-based enrichment assay for detecting mutant alleles. The procedure is based on Surveyor endonuclease cleaves mismatched DNA molecules, and these DNA fragments were enriched for mutation screening. We screened lung cancer specimens for mutations in exons 18 and 21 of EGFR, and the majority of activating mutations in lung cancer occur in codons 12 (G12X) and 13 (G13X) of exon 2 of the KRAS gene. The method screened all mutant genes with the same pair primers and three relevant TaqMan probes.ResultsThe method can effectively remove wild-type sequences and enrich mutation DNA, and the sensitivity detectable mutant allele frequencies (MAF) achieved 0.001%. The method increases the sensitivity and efficiency of mutation DNA for cancers screening. This highlights the importance of complex DNA variation like mutations in exon 21 of EGFR and exon 2 of the KRAS gene detected by the same probe.ConclusionWe developed a simple and sensitive methodology for mutation gene screening. The method is a cost-effective and sensitive method for mutation DNA enrichment and detection.

Analytical Performance of NGS-Based Molecular Genetic Tests Used in the Diagnostic Workflow of Pheochromocytoma/Paraganglioma.

Simple SummaryThe escalating use of Next Generation Sequencing in the routine clinical setting greatly facilitates the genetic diagnosis of hereditary cancer syndromes. However, these novel methods pose new and unique challenges. In our study we sought to demonstrate the evolution of these techniques, especially whole exome sequencing and targeted panel sequencing. This study highlights the multi-layered workflow and how each step affects the diagnostic outcome and demonstrates the effectiveness of an in-house developed targeted panel sequencing for hereditary endocrine tumor syndromes.Next Generation Sequencing (NGS)-based methods are high-throughput and cost-effective molecular genetic diagnostic tools. Targeted gene panel and whole exome sequencing (WES) are applied in clinical practice for assessing mutations of pheochromocytoma/paraganglioma (PPGL) associated genes, but the best strategy is debated. Germline mutations of at the least 18 PPGL genes are present in approximately 20–40% of patients, thus molecular genetic testing is recommended in all cases. We aimed to evaluate the analytical and clinical performances of NGS methods for mutation detection of PPGL-associated genes. WES (three different library preparation and bioinformatics workflows) and an in-house, hybridization based gene panel (endocrine-onco-gene-panel- ENDOGENE) was evaluated on 37 (20 WES and 17 ENDOGENE) samples with known variants. After optimization of the bioinformatic workflow, 61 additional samples were tested prospectively. All clinically relevant variants were validated with Sanger sequencing. Target capture of PPGL genes differed markedly between WES platforms and genes tested. All known variants were correctly identified by all methods, but methods of library preparations, sequencing platforms and bioinformatical settings significantly affected the diagnostic accuracy. The ENDOGENE panel identified several pathogenic mutations and unusual genotype–phenotype associations suggesting that the whole panel should be used for identification of genetic susceptibility of PPGL.

Integrated Genome and Transcriptome Sequencing to Solve a Neuromuscular Puzzle: Miyoshi Muscular Dystrophy and Early Onset Primary Dystonia in Siblings of the Same Family.

BackgroundNeuromuscular disorders (NMD), many of which are hereditary, affect muscular function. Due to advances in high-throughput sequencing technologies, the diagnosis of hereditary NMDs has dramatically improved in recent years.Methods and ResultsIn this study, we report an family with two siblings exhibiting two different NMD, Miyoshi muscular dystrophy (MMD) and early onset primary dystonia (EOPD). Whole exome sequencing (WES) identified a novel monoallelic frameshift deletion mutation (dysferlin: c.4404delC/p.I1469Sfs∗17) in the Dysferlin gene in the index patient who suffered from MMD. This deletion was inherited from his unaffected father and was carried by his younger sister with EOPD. However, immunostaining staining revealed an absence of dysferlin expression in the proband’s muscle tissue and thus suggested the presence of the second underlying mutant allele in dysferlin. Using integrated RNA sequencing (RNA-seq) and whole genome sequencing (WGS) of muscle tissue, a novel deep intronic mutation in dysferlin (dysferlin: c.5341-415A > G) was discovered in the index patient. This mutation caused aberrant mRNA splicing and inclusion of an additional pseudoexon (PE) which we termed PE48.1. This PE was inherited from his unaffected mother. PE48.1 inclusion altered the Dysferlin sequence, causing premature termination of translation.ConclusionUsing integrated genome and transcriptome sequencing, we discovered hereditary MMD and EOPD affecting two siblings of same family. Our results added further weight to the combined use of RNA-seq and WGS as an important method for detection of deep intronic gene mutations, and suggest that integrated sequencing assays are an effective strategy for the diagnosis of hereditary NMDs.

Polymerase chain reaction-restriction fragment length polymorphism method for detection of Calreticulin type-1 and type-2 mutations in myeloproliferative neoplasm

Calreticulin (CALR) mutations are included in the WHO diagnostic criteria of Myeloproliferative Neoplasm (MPN). Presence of CALR mutation, aids in their diagnosis by excluding reactive causes. There are several molecular techniques to detect CALR mutations; however, these require advanced equipment and technical expertise. An in-house “polymerase chain reaction-restriction fragment length polymorphism (PCR- RFLP)” method, using restriction enzymes MseI and MluCI for detection of CALR type-1 and type-2 mutations, was developed. PCR-RFLP method was validated by comparing its results with real-time PCR (RQ-PCR) using CE-IVD marked Ipsogen® CALR RGQ PCR (Qiagen, Germany) kit. All PCR-RFLP positive CALR type-1 and type-2 mutations samples of MPN (n=26) were confirmed positive by RQ-PCR, and all negative samples (n=21) were confirmed to be negative, showing 100% diagnostic sensitivity and specificity for CALR mutation detection by PCR-RFLP. The developed PCR-RFLP method can be used for detection of CALR type-1 and type-2 mutations with only a PCR machine and gel electrophoresis system. It is an inexpensive and easily applicable alternative method which can be adopted even by a small molecular laboratory for detection of CALR mutations.

Digital PCR detection of EGFR somatic mutations in non-small-cell lung cancer formalin fixed paraffin embedded samples

BackgroundDigital PCR (dPCR) is proposed to replace real time PCR and Sanger sequencing for detection and quantification of rare mutations, frequently unnoticed in the mass of tumoral cells. Screening of endothelial growth factor receptor (EGFR) mutations is mandatory before treatment with EGFR-targeted therapy with small-molecule tyrosine kinase inhibitors, which has been approved for the treatment of advanced non-small-cell lung cancer (NSCLC). ObjectiveIn order to establish a cost-effective method for detection of mutations, we optimized dPCR identification of EGFR mutations in exons 18–21, and determined dPCR sensitivity, limits of detection (LoD) and quantification (LoQ). MethodsFor clinical validation, we compared the performance of dPCR and castPCR in 57 NSCL formalin fixed paraffin embedded samples and 10 lung cancer-free formalin fixed paraffin embedded samples. ResultsEGFR mutations DEL19, p.L858R, p.G719X, p.L861Q and p.T790 M were detected by dPCR in 27 samples versus 11 detected by castPCR (p = 0.014). LoD was determined as 100 molecules of DNA/uL and LoQ as 1%. Most of the samples (87%) identified by competitive Allele-Specific TaqMan (castPCR) as wild-type and by dPCR as mutated, presented less than 10% mutated DNA molecules (mean 4.57%). Accuracy of dPCR was 94.44%, as measured with the assay recommended by the College of American Pathologists. ConclusionThese results indicated higher sensibility and specificity of dPCR for screening EGFR mutations in NSCLC biopsies, compared to castPCR.

Benchmarking and optimization of a high-throughput sequencing based method for transgene sequence variant analysis in biotherapeutic cell line development.

In recent years, High-Throughput Sequencing (HTS) based methods to detect mutations in biotherapeutic transgene products have become a key quality step deployed during the development of manufacturing cell line clones. Previously we reported on a higher throughput, rapid mutation detection method based on amplicon sequencing (targeting transgene RNA) and detailed its implementation to facilitate cell line clone selection. By gaining experience with our assay in a diverse set of cell line development programs, we improved the computational analysis as well as experimental protocols. Here we report on these improvements as well as on a comprehensive benchmarking of our assay. We evaluated assay performance by mixing amplicon samples of a verified mutated antibody clone with a non-mutated antibody clone to generate spike-in mutations from ∼60% down to ∼0.3% frequencies. We subsequently tested the effect of 16 different sample and HTS library preparation protocols on the assay's ability to quantify mutations and on the occurrence of false-positive background error mutations (artifacts). Our evaluation confirmed assay robustness, established a high confidence limit of detection of ∼0.6%, and identified protocols that reduce error levels thereby significantly reducing a source of false positives that bottlenecked the identification of low-level true mutations.

Super-ARMS: A new method for plasma ESR1 mutation detection

BackgroundESR1 mutation is an important mechanism of drug resistance and recurrence in hormone receptor-positive breast cancer patients during AI treatment. Patient could still benefit from treatment with fulvestrant after ESR1 mutated. ObjectiveAt present, there is still no suitable method to detect ESR1 mutation in plasma as clinical promotion method. We aim to improve from ARMS-PCR to get a method with higher sensitivity but no additional cost is incurred. MethodsWe designed new primers for ESR1. Then positive and negative standard sample was used for sensitivity and specificity tests. Lastly, we collected patient peripheral blood sample and analyzed the performance of Super-ARMS in plasma ctDNA samples. ResultsA total of 207 patients were enrolled in this study, including 142 prime breast cancer (PBC) patients and 65 metastasis breast cancer(MBC) patients. The mutation rate was as high as 27.9%(12/43) in MBC patients with AI treatment. But only 2.97%(3/101) in PBC patients with AI and 0% in both MBC or PBC patient without AI. There was no significant difference in Super-ARMS results compared with DDPCR method. ConclusionSuper-ARMS is a method that has sensitivity close to DDPCR and has the convenience and low price of ARMS-PCR for plasma ctDNA ESR1 mutation detection. It has obvious advantages compared with other method such NGS and DDPCR as clinical promotion method.

The Clinical Impact of Proteomics in Amyloid Typing

The Clinical Impact of Proteomics in Amyloid Typing

Methodological Research on Rapid Detection and Genotyping of NPM1 Gene Mutations in Leukemia

To establish a method for rapid detection and typing of NPM1 mutations in acute myeloid leukemia (AML) by fluorescence melting curve analysis technology. A pair of primers and a fluorescent single-stranded probe (molecule beacon) were designed for the mutant genes mutA, mutB, mutD in exon 12 of nucleopsin (NPM1) and wild type. With a real-time qPCR, the A, B, and D gene mutations of NPM1 were detected and typed by different-melting curve peak value of the probe through RT-PCR. This method could detected the mutations of A, B, and D in NPM1 effectively with a sensitivity of 1%. Furthermore, 62 AML clinical samples were evaluated by the method. In the results, the detection rate and typing of NPM1 mutations were consistent with the sequencing results of clinical samples. There are three features in the method of fluorescence melting curve analysis: stable PCR system, easy to operate, and the easily distinguishable results. The method might meet the demand for rapid typing of NPM1 gene mutation in early diagnosis or concomitant diagnosis of AML.

P02.06 A Novel Loop-Mediated Isothermal Amplification Method for Robust Detection of EGFR Mutations

P02.06 A Novel Loop-Mediated Isothermal Amplification Method for Robust Detection of EGFR Mutations