scholarly journals DETECTION OF SOMATIC MUTATIONS IN THE BRAF GENE BY PYROSEQUENCING

2021 ◽  
Vol 20 (5) ◽  
pp. 75-83
Author(s):  
O. P. Dribnokhodova ◽  
E. A. Dunaeva ◽  
G. V. Leshkina ◽  
E. A. Yarygina ◽  
A. Yu. Bukharina ◽  
...  
Keyword(s):  
Mutant Allele ◽  
Somatic Mutations ◽  
Needle Aspiration ◽  
Clinical Samples ◽  
Braf Gene ◽  
Allele Ratio ◽  
Rare Mutations ◽  
Frequent Mutations ◽  
Sufficient Concentration ◽  
Pyrosequencing Technology

Introduction. Detection of somatic mutations in the BRAF gene can be used in clinical oncology to clarify the diagnosis, select therapy and assess the prognosis of the disease. Pyrosequencing technology makes it possible to identify both already known and new mutations, as well as to determine the mutant allele ratio in the sample.The aim of the study was to develop the pyrosequencing-based method for detecting mutations in 592–601 codons of the BRAF gene.Material and Methods. The nucleotide sequences were obtained using «PyroMark Q24» instrument. The sensitivity and specificity of the method were estimated using dilutions of plasmid DNA samples containing the intact BRAF gene fragment mixed with sequence containing one of the mutations V600E, V600R, V600K, V600M, and K601E. The clinical testing was performed on 200 samples from thyroid nodules.Results. The developed method makes it possible to determine samples containing 2 % of the mutant allele for mutations V600K and V600R, 3 % for V600E and V600M, and 10 % for K601E. The pyrogram signal values for samples without mutations ranged from 0 to 19.5 % for different mutations. An analysis algorithm was developed to confirm the presence and differentiation of mutations in the 600 codon at a low proportion of the mutant allele based on the signals ratio on the pyrogram. The 47 clinical samples with mutations were found, 45 with V600E and 1 with V600_K601>E, for one sample, the type of mutation in the 600 codon could not be determined. The proportion of the mutant allele was 3.5–45 %. The concentration of extracted DNA less than 10 copies per mkl was obtained in 47 samples, of which 8 samples were found to have the mutations.Conclusion. The pyrosequencing-based method was developed for the detection of somatic mutations in 592–601 codons of the BRAF gene. The technique provided sufficient sensitivity to detect frequent mutations in the 600 codon and allowed the detection of rare mutations. Extraction of DNA from clinical samples obtained by fine-needle aspiration biopsy in most cases provided a sufficient concentration of DNA, which made it possible to use the technique in combination with cytological analysis without additional sampling. This approach can be applied to determine somatic mutations in DNA fragments of same length for other oncogenes. 

scholarly journals An algorithm to evaluate the efficacy of detecting somatic mutations

2018 ◽  
Vol 8 (2) ◽  
pp. 25
Author(s):  
Thurai Moorthy
Keyword(s):  
Lower Limit ◽  
Late Stage ◽  
Mutant Allele ◽  
Somatic Mutations ◽  
Limit Of Detection ◽  
Clinical Samples ◽  
Wild Type ◽  
Copy Numbers ◽  
Lower Copy ◽  
Assay Conditions

Detection of somatic mutations from late stage solid tumors is a critical part of cancer treatment. Although tumor content is used as a convenient parameter to measure efficacy of detection, it fails to include two basic factors: the lower limit of detection (LLOD), and the ratio of the mutant and wild type allele frequencies.  Recently, the detection of somatic mutations has expanded to liquid biopsy, early stages of cancer and population screening, which all generally carry lower copy numbers of somatic mutations compared to late stage tumors.  With the growing importance of these mutations for targeted chemotherapy and other clinical applications, there is a need re-evaluate the efficacy of detection of somatic mutations.  Hence, a new algorithm, Detection Index (DI), is proposed to standardize the efficacy of all molecular methods and is applicable to all types of clinical samples. Detection Index (DI) is based on two basic determinants: lower limit of detection of the mutant allele, and the ratio of the copies of the mutant allele to that of the wild-type. The benefits of DI include (a) standardization of methods detecting somatic mutations so that laboratory reports will have a uniform interpretation related to clinical picture, and (b) the flexibility to use appropriate amounts of DNA and assay conditions to achieve desired DI. 

scholarly journals Mutation Profile of Aggressive Pheochromocytoma and Paraganglioma with Comparison of TCGA Data

Cancers ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 2389
Author(s):  
Yun Mi Choi ◽  
Jinyeong Lim ◽  
Min Ji Jeon ◽  
Yu-Mi Lee ◽  
Tae-Yon Sung ◽  
...  
Keyword(s):  
Somatic Mutations ◽  
The Cancer Genome Atlas ◽  
Sequencing Analysis ◽  
Poor Overall Survival ◽  
Sdhb Mutation ◽  
Targeted Next Generation Sequencing ◽  
Mutation Profile ◽  
Next Generation Sequencing Analysis ◽  
Number Variation ◽  
Frequent Mutations

In pheochromocytoma and paraganglioma (PPGL), germline or somatic mutations in one of the known susceptibility genes are identified in up to 60% patients. However, the peculiar genetic events that drive the aggressive behavior including metastasis in PPGL are poorly understood. We performed targeted next-generation sequencing analysis to characterize the mutation profile in fifteen aggressive PPGL patients and compared accessible data of aggressive PPGLs from The Cancer Genome Atlas (TCGA) with findings of our cohort. A total of 115 germline and 34 somatic variants were identified with a median 0.58 per megabase tumor mutation burden in our cohort. The most frequent mutation was SDHB germline mutation (27%) and the second frequent mutations were somatic mutations for SETD2, NF1, and HRAS (13%, respectively). Patients were subtyped into three categories based on the kind of mutated genes: pseudohypoxia (n = 5), kinase (n = 5), and unknown (n = 5) group. In copy number variation analysis, deletion of chromosome arm 1p harboring SDHB gene was the most frequently observed. In our cohort, SDHB mutation and pseudohypoxia subtype were significantly associated with poor overall survival. In conclusion, subtyping of mutation profile can be helpful in aggressive PPGL patients with heterogeneous prognosis to make relevant follow-up plan and achieve proper treatment.

scholarly journals Using the Minor Variant Finder software to identify and quantify the allelic burden level of somatic mutations in oncohematologic diseases

2020 ◽  
Vol 15 (2) ◽  
pp. 85-91
Author(s):  
T. N. Subbotina ◽  
I. E. Maslyukova ◽  
A. A. Faleeva ◽  
P. A. Nikolaeva ◽  
A. S. Khazieva ◽  
...  
Keyword(s):  
Mutant Allele ◽  
Sanger Sequencing ◽  
Quantitative Assessment ◽  
Somatic Mutations ◽  
Myeloproliferative Neoplasms ◽  
Mutant Gene ◽  
Positive Sample ◽  
Allele Burden ◽  
Minor Variant ◽  
Allelic Burden

Background. There are problems related to both quantitative assessment of an allele burden level of a mutant gene and interpretation of results in DNA samples with the burden level of the mutant allele less than 15–20 %, when using Sanger sequencing for analyzing somatic mutations. Applied Biosystems (USA) has developed new software Minor Variant Finder, which allows determining mutations with the allele burden level from 5 %.The objective: to determine the allele burden level and identification of minor variants of somatic mutations in the ASXL1, JAK2 genes and BCR-ABL oncogene using Minor Variant Finder software in patients with myeloproliferative neoplasms.Materials and methods. The level of mutant allele burden for 15 patients with myeloproliferative neoplasms was determined by the identified mutations using the Minor Variant Finder software, after analysis of point somatic mutations in the ASXL1, JAK2 genes and BCR-ABL oncogene by Sanger sequencing.Results. The allele burden level in all 5 ASXL1-positive samples and BCR-ABL-positive sample was determined as higher than 20 % using the Minor Variant Finder software. The allele burden level in 2 cases was higher than 20 % and in 7 cases lower than 20 %, when we analyzed 9 JAK2-positive samples.Conclusion. Minor Variant Finder software can be used to estimate the allele burden level and to identify minor variants of somatic mutations in the ASXL, JAK2 and BCR-ABL genes.

scholarly journals First isolation of Mycobacterium ulcerans from Swabs and Fine-Needle-Aspiration Specimens in Togo.

2019 ◽  
Author(s):  
Tchalare Kondi Makagni ◽  
Maman Issaka ◽  
Piten Ebekalisai ◽  
Disse Kodjo ◽  
Essossimna A. Kadanga ◽  
...  
Keyword(s):  
Fine Needle Aspiration ◽  
Slow Growth ◽  
Buruli Ulcer ◽  
Needle Aspiration ◽  
Mycobacterium Ulcerans ◽  
Clinical Samples ◽  
Direct Examination ◽  
Fine Needle ◽  
Pcr Targeting

Abstract Background Buruli ulcer is a skin disease caused by a mycobacterium called Mycobacterium ulcerans . It is prevalent in more than 33 countries on several continents but West Africa is the most affected. The isolation in culture of the bacteria is difficult because of its slow growth and the facilities required. In Togo, studies have been done on the risk factors for Mycobacterium ulcerans infection and the detection of cases by the Ziehl-Neelsen and PCR technique on clinical and environmental samples, but to date no data of isolates from clinical samples are available. The purpose of this study was to perform an in vitro culture of M. ulcerans from swab and fine needle aspiration samples through the confirmation stages of direct examination and IS2404 -PCR. Method A total of 70 clinical samples from Togo and 10 clinical isolates from Benin are analyzed by the three techniques indicated in the diagnosis, in particular the direct examination of acid-fast bacilli (AFB) using the Ziehl-Neelsen staining, the PCR targeting the IS2404 sequence, and the culture after transport of the samples in a transport medium made of Middlebrook 7H9 medium supplemented with a mixture of PANTA and OADC and decontamination by the modified Petroff method. Results The application of the three techniques of diagnosis for clinical samples yielded 44.28% of positivity rates on direct examination of AFB, 35.71% on culture and 77.14% on qPCR IS2404 with a significantly higher rate for qPCR (0.0001). All samples positive for Ziehl-Neelsen staining and culture were also positive for qPCR. Conclusion : Our results show that the culture, despite it difficulty and the slow growth of the bacteria, can be carried out with recommended tools of the mycobacteria culture and a good method of decontamination of the samples can improve the positivity rates. Its realization will allow the assessment of the in vitro sensitivity to the antibiotics used in the treatment and the discovery of new strains of Mycobacterium ulcerans .

scholarly journals Closed-Tube PCR with Nested Serial Invasion Probe Visualization Using Gold Nanoparticles

2017 ◽  
Vol 63 (4) ◽  
pp. 852-860 ◽  
Author(s):  
Jianping Wang ◽  
Bingjie Zou ◽  
Yinjiao Ma ◽  
Xueping Ma ◽  
Nan Sheng ◽  
...  
Keyword(s):  
Personalized Medicine ◽  
Gold Nanoparticle ◽  
Somatic Mutations ◽  
Dna Analysis ◽  
Circulating Tumor Dna ◽  
Clinical Samples ◽  
Nucleotide Polymorphisms ◽  
Biomarker Analysis ◽  
Closed Tube ◽  
Signal Readout

Abstract BACKGROUND Detecting DNA biomarkers related to personalized medicine could improve the outcome of drug therapy. However, personalized medicine in a resource-restrained hospital is very difficult because DNA biomarker detection should be performed by well-trained staff and requires expensive laboratory facilities. METHODS We developed a gold nanoparticle–based “Tube-Lab” to enable DNA analysis in a closed tube. Gold nanoparticle–modified probes (GNPs) were used to construct an inexpensive and simple DNA sensor for signal readout. The method consists of 3 steps (template amplification, sequence identification, and GNP-based signal readout), bridged by an invasive reaction. With temperature control at each step, the 3 reactions proceed sequentially and automatically in a closed tube without any liquid transfer. We used Tube-Lab to detect different biomarkers in blood, tissue, and plasma, including US Food and Drug Administration–approved pharmacogenomic biomarkers (single nucleotide polymorphisms, somatic mutations). RESULTS The combination of PCR-based template replication and invader-based signal amplification allowed detection of approximately 6 copies of input DNA and the selective pick up 0.1% mutants from large amounts of background DNA. This method highly discriminated polymorphisms and somatic mutations from clinical samples and allowed a “liquid biopsy” assay with the naked eye. CONCLUSIONS Tube-Lab provides a promising and cost-effective approach for DNA biomarker analysis, including polymorphisms and somatic mutations from blood DNA, tissue DNA, or circulating tumor DNA in plasma, which are critical for personalized medicine.

scholarly journals Coamplification at Lower Denaturation Temperature-PCR Increases Mutation-Detection Selectivity of TaqMan-Based Real-Time PCR

2009 ◽  
Vol 55 (4) ◽  
pp. 748-756 ◽  
Author(s):  
Jin Li ◽  
Lilin Wang ◽  
Pasi A Jänne ◽  
G Mike Makrigiorgos
Keyword(s):  
Somatic Mutations ◽  
Kinase Inhibitors ◽  
Hot Spot ◽  
Resistance Mutation ◽  
Clinical Samples ◽  
Nucleotide Polymorphisms ◽  
Wild Type ◽  
Denaturation Temperature ◽  
Low Level ◽  
Single Temperature

Abstract Background: DNA genotyping with mutation-specific TaqMan® probes (Applied Biosystems) is broadly used in detection of single-nucleotide polymorphisms but is less so for somatic mutations because of its limited selectivity for low-level mutations. We recently described coamplification at lower denaturation temperature-PCR (COLD-PCR), a method that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences during the PCR. We demonstrate that combining COLD-PCR with TaqMan technology provides TaqMan genotyping with the selectivity needed to detect low-level somatic mutations. Methods: Minor-groove binder-based or common TaqMan probes were designed to contain a nucleotide that matches the desired mutation approximately in the middle of the probe. The critical denaturation temperature (Tc) of each amplicon was then experimentally determined. COLD-PCR/TaqMan genotyping was performed in 2 steps: denaturation at the Tc, followed by annealing and extension at a single temperature (fast COLD-PCR). The threshold cycle was used to identify mutations on the basis of serial dilutions of mutant DNA into wild-type DNA and to identify TP53 (tumor protein p53) and EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] mutations in tumors. Results: COLD-PCR/TaqMan genotyping identified G>A mutations within TP53 exon 8 (codon 273 mutation hot spot) and C>T mutations within the EGFR gene (drug-resistance mutation T790M) with a selectivity improvement of 15- to 30-fold over regular PCR/TaqMan genotyping. A second round of COLD-PCR/TaqMan genotyping improved the selectivity by another 15- to 30-fold and enabled detection of 1 mutant in 2000 wild-type alleles. Use of COLD-PCR/TaqMan genotyping allowed quantitative identification of low-level TP53 and T790 mutations in colon tumor samples and in non-small-cell lung cancer cell lines treated with kinase inhibitors. Conclusions: The major improvement in selectivity provided by COLD-PCR enables the popular TaqMan genotyping method to become a powerful tool for detecting low-level mutations in clinical samples.

scholarly journals BRAF Gene Mutation (V600E) in Aspiration Cytology of Patients With Suspected Papillary Thyroid Carcinoma

2021 ◽  
Author(s):  
Mohammadhossein Dadgarnia ◽  
Mohsen Abouii ◽  
Mohammadhossein Baradaranfar ◽  
Sedighe Vaziribozorg ◽  
Vahid Zand
Keyword(s):  
Papillary Thyroid Carcinoma ◽  
Thyroid Carcinoma ◽  
Gene Mutation ◽  
Significant Relationship ◽  
Needle Aspiration ◽  
Primary Treatment ◽  
Papillary Thyroid ◽  
Braf Gene ◽  
Aspiration Cytology ◽  
Cross Sectional

This study was attempted to investigate the prevalence of BRAF gene mutation (V600E) in aspiration cytology of patients with suspected papillary thyroid carcinoma (PTC). Seventy-six patients suspected of having PTC who were referred for fine-needle aspiration (FNA) biopsy were included in this cross-sectional study. Ultrasound-guided FNA was taken from the thyroid masses, and samples were sent for cytologic evaluation. Simultaneously, the samples were sent to a genetic laboratory to check the status of BRAFV600E mutation. Patients with FNA positive for PTC were assigned in one group, and those with FNA negative for PTC were assigned to another group. Cytological and molecular results were compared with those of histopathology and sonography. The results showed that the prevalence of the BRAF gene (V600E) mutation in our study was 21.1% (16 out of 76 patients). In addition, the results showed a significant relationship between gene mutation and pathologic findings so that the highest gene mutation was significantly detected in patients with FNA positive for PTC (P=0.001). Also, our results showed a significant relationship between gene mutation and some sonographic findings (calcification, P=0.004) and no significant relation in the other sonographic findings (hypoechoic changes, P=1.12 and regular changes, P=0.194). According to the results of the present study, BRAF mutation (V600E) can be an effective indicator for definitive diagnosis and primary treatment of PTC in suspected cases.

Somatic Mutation of SF3B1, a Gene Encoding a Core Component of RNA Splicing Machinery, in Myelodysplasia with Ring Sideroblasts

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3-3 ◽  
Author(s):  
Luca Malcovati ◽  
Elli Papaemmanuil ◽  
Eva Hellström-Lindberg ◽  
Jacqueline Boultwood ◽  
David Bowen ◽  
...  
Keyword(s):  
Somatic Mutation ◽  
Rna Splicing ◽  
Mutant Allele ◽  
Somatic Mutations ◽  
Point Mutations ◽  
Refractory Anemia ◽  
Core Component ◽  
Ring Sideroblasts ◽  
Risk Of Progression ◽  
Splicing Machinery

Abstract Abstract 3 Myelodysplastic syndromes (MDS) are myeloid neoplasms characterized by dysplasia in one or more cell lines, ineffective hematopoiesis, and variable risk of progression to acute myeloid leukemia (AML). As any other neoplasm, MDS is expected to be driven by mutation, and its clonal evolution is likely a multistep process in which several genetic events occur. Somatic mutations of TET2 have been found in about 25% of MDS patients, while additional mutant genes (including ASXL1, ETV6, EZH2, IDH1, IDH2, RUNX1, and TP53) have been detected in smaller proportions of patients, particularly in those with poor prognosis. Refractory anemia with ring sideroblasts (RARS) is a phenotypically well-defined subtype of MDS, characterized by 15% or more ring sideroblasts (RS, erythroblasts with perinuclear iron-loaded mitochondria) in the bone marrow. We reasoned that the identification of recurrently mutated genes in RARS could provide novel insights into molecular pathogenesis of MDS, and used massively parallel sequencing technology to identify somatically acquired point mutations across all protein-coding exons in the genome in 8 patients with RARS. We identified 62 point mutations across the 8 patients, and the mutation spectrum showed a predominance of transitions, especially C>T/G>A mutations. Within 5/8 patients studied, the observed proportion of reads reporting a mutant allele showed significantly greater variability than expected by chance, indicating that the population of malignant cells was genetically heterogeneous. In 6/8 RARS patients, we identified recurrent somatic mutations (found in granulocytes but not in T-lymphocytes) in a gene that encodes a core component of the RNA splicing machinery, SF3B1. Based on the proportion of reads reporting the mutant allele, the mutations all appeared to be heterozygous and present in the dominant clone of cells. To characterize the spectrum and frequency of SF3B1 mutations in greater detail, both in myeloid malignancies and other cancers, we undertook targeted resequencing of the gene. In particular, we studied patients with MDS, myelodysplastic/myeloproliferative neoplasm (MDS/MPN) or AML evolving from MDS. Somatic mutations of SF3B1 were found in 150/533 (28.1%) patients with MDS, 16/83 (19.3%) patients with MDS/MPN, and 2/38 (5.3%) patients with AML. The gene was also mutated in 1–5% of diverse other tumor types. All mutations appeared to be heterozygous substitutions, and we observed no frameshift indels, splice site mutations or nonsense substitutions. The mutations clustered in exons 12–15 of the gene, and K700E accounted for 97/168 (57.7%) of the variants observed. SF3B1 mutations were less deleterious than expected by chance, implying that the mutated protein retains structural integrity with altered function. Gene expression profiling revealed SF3B1 mutations are associated with down-regulation of key gene networks, including core mitochondrial pathways. Close relationships were found between mutant SF3B1 and presence of RS (P<.001), and between mutant allele burden and percentage of RS (P=.002). Overall, 83/105 (79%) of patients with RARS, 30/54 (57.7%) of those with refractory cytopenia with multilineage dysplasia and RS, and 12/18 (66.7%) of those with RARS associated with marked thrombocytosis (RARS-T) carried a somatic mutation of SF3B1. On the other hand, 97% of patients carrying a mutant SF3B1 had RS, and the mutant gene had a positive predictive value for RS of 97.7% (95% CI, 93.5–99.5%). We then studied the prognostic significance of the genetic lesion. In multivariable analysis including established risk factors, SF3B1 mutations were independently associated with better overall survival (HR=0.18, P=.028) and lower risk of progression to AML (HR=0.32, P=.048). In conclusion, mutations in SF3B1 implicate abnormalities of mRNA splicing, a pathway not previously known as a target for mutation, in the pathogenesis of MDS. The close relationship between this molecular lesion and RS is consistent with a causal relationship, and makes SF3B1 the first gene to be strongly associated with a specific morphological feature in MDS. Finally, SF3B1 mutations are independent predictors of favorable clinical outcome, and their detection may improve risk assessment in MDS. The first two authors equally contributed to this paper, which is on behalf of the International Cancer Genome Consortium Chronic Myeloid Disorders Working Group. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Bisulfite-converted duplexes for the strand-specific detection and quantification of rare mutations

2017 ◽  
Vol 114 (18) ◽  
pp. 4733-4738 ◽  
Author(s):  
Austin K. Mattox ◽  
Yuxuan Wang ◽  
Simeon Springer ◽  
Joshua D. Cohen ◽  
Srinivasan Yegnasubramanian ◽  
...  
Keyword(s):  
Massively Parallel Sequencing ◽  
Massively Parallel ◽  
Clinical Samples ◽  
Low Frequencies ◽  
Parallel Sequencing ◽  
Molecular Barcoding ◽  
Bisulfite Treatment ◽  
Rare Mutations ◽  
Template Dna ◽  
Generation Sequencing

The identification of mutations that are present at low frequencies in clinical samples is an essential component of precision medicine. The development of molecular barcoding for next-generation sequencing has greatly enhanced the sensitivity of detecting such mutations by massively parallel sequencing. However, further improvements in specificity would be useful for a variety of applications. We herein describe a technology (BiSeqS) that can increase the specificity of sequencing by at least two orders of magnitude over and above that achieved with molecular barcoding and can be applied to any massively parallel sequencing instrument. BiSeqS employs bisulfite treatment to distinguish the two strands of molecularly barcoded DNA; its specificity arises from the requirement for the same mutation to be identified in both strands. Because no library preparation is required, the technology permits very efficient use of the template DNA as well as sequence reads, which are nearly all confined to the amplicons of interest. Such efficiency is critical for clinical samples, such as plasma, in which only tiny amounts of DNA are often available. We show here that BiSeqS can be applied to evaluate transversions, as well as small insertions or deletions, and can reliably detect one mutation among >10,000 wild-type molecules.

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