Rapid Detection Of Mutations Research Articles

Developing Ultrasensitive Library-Aliquot-Based Droplet Digital PCR for Detecting T790M in Plasma-Circulating Tumor DNA of Non-small-Cell-Lung-Cancer Patients.

A T790M secondary mutation in epidermal-growth-factor receptor (EGFR) is the most well-established EGFR-tyrosine-kinase-inhibitor (TKI) resistance marker in non-small-cell lung cancer (NSCLC). The current methods to rapidly and accurately detect T790M in clinical practice are not satisfactory because of several obstacles, including the unavailability of tumor-tissue rebiopsies and the low DNA copy number of T790M in circulating tumor DNA (ctDNA). Here, we develop library-aliquot-based droplet digital PCR (LAB-ddPCR) to increase detection sensitivity without affecting accuracy. This new LAB-ddPCR method is performed using aliquots of the ctDNA precapture next-generation-sequencing (NGS) library, in which the isolated ctDNA was amplified and enriched. We show that the LAB-ddPCR can precisely distinguish between T790M wild-type and mutation alleles without introducing extra false-positive signals. In a cohort of 70 post-TKI NSCLC patients, the LAB-ddPCR identified 41 T790M-positive cases (sensitivity 58.57%), but ddPCR only detected T790M in 27 cases (sensitivity 38.57%). Taking the ARMS-PCR result from matched tumor rebiopsies into consideration, the LAB-ddPCR method is better than ddPCR. In conclusion, the LAB-ddPCR ctDNA test offers a feasible and flexible option for the rapid and accurate detection of the T790M secondary mutation, which is helpful in dynamically monitoring drug response and disease progression throughout the therapeutic regimen.

Abstract 3253: Droplet digital PCR is a sensitive method for detecting refractory acute myeloid leukemia (AML) clones in peripheral blood and saliva

Abstract Droplet digital PCR (ddPCR) is a highly sensitive and rapid method for detecting mutant allele frequency (MAF). In preliminary work, our lower limit of detection for common myeloid gene mutations was 0.001% in peripheral blood and bone marrow compared to 0.1% with flow cytometry and 0.01% with real-time quantitative PCR, and turnaround time is 1 day. Furthermore, we detected leukemic mutant alleles in peripheral blood (PB), introducing the possibility of sparing painful bone marrow biopsy procedures to determine treatment response. Thus, we hypothesized that ddPCR is a feasible and accurate method for monitoring leukemic disease burden in PB for the prospective care of patients (pts) with AML. Eighteen patients (pts) with de novo, relapse/refractory, and secondary AML were recruited to an IRB-approved study (NCT02435550) and bone marrow (BM), peripheral blood (PB), and saliva were collected at standard clinical visits. Gene mutations were identified by whole-exome sequencing (WES) of BM specimens at study entry. For ddPCR interrogation, genomic DNA was isolated (Qiagen), and select primers and probes (Bio-Rad/IDT) were developed based on variants identified in WES data. Case-specific primers and probes were validated on archived specimens obtained at study entry. 12/18 pt mutanomes met criteria for primer/probe design. 8 pts are in the primer/probe design and validation stage and 4 have completed validation and serial analyses. WES identified, and ddPCR confirmed, at least 1 mutation per patient at the study entry timepoint. The mutations included NRAS G13R, NRAS G12A, CSF3R T618I, and IDH2 R172K. In 2 cases, we observed a reduction in both PB and saliva MAF that were consistent with the reduction in both BM and PB blasts after treatment, resulting in complete remissions. Although PB blasts were reduced in a third pt receiving ruxolitinib, the persistence of their CSF3R MAF in PB indicated a resistant AML clone. WES revealed the presence of NRAS G13R variant in a secondary AML pt; however, WES did not detect this NRAS G13R variant in a cryopreserved BM specimen obtained at the pts MDS diagnosis. Interestingly, ddPCR was able to detect NRAS G13R variant at 0.1% MAF in a PB sample obtained at MDS diagnosis, demonstrating the ultrasensitive detection of rare variants within a sample, and highlighting the subclonal evolution of this pt's malignancy. Rapid detection of myeloid-related somatic mutations in a variety of tissue sources (i.e., saliva, PB) will allow for noninvasive monitoring of AML tumor burden. ddPCR may be used to observe molecular response to treatment and to detect molecular residual disease and relapse prior to clinically indicated BM biopsies. Citation Format: Kimberly E. Hawkins, Cesia Salan, Madeleine Turcotte, Lauren T. Vaughn, Mei Zhang, Yanping Zhang, Barry Sawicki, Glenda G. Anderson, Nosha Farhadfar, Hemant S. Murthy, Biljana N. Horn, Helen L. Leather, Paul Castillo, Maxim Norkin, John W. Hiemenz, Randy A. Brown, William Slayton, Jack W. Hsu, John R. Wingard, Christopher R. Cogle, Leylah M. Drusbosky. Droplet digital PCR is a sensitive method for detecting refractory acute myeloid leukemia (AML) clones in peripheral blood and saliva [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3253.

The sensitive detection of IVSII-1(G˃A) mutation in beta globin gene using a Nano-based ligation genotyping system

Beta-thalassemia (β-thalassemia) is a globally genetic diseases, and is most prevalent in the Middle East, particularly in Iran. Carrier detection and prenatal diagnosis are the best ways to managing it, and to prevent new community cases from emerging. We report on a simple method for rapid detection of the worst β-thalassemia point mutation in Iran (IVS-II-1 G>A), using a nano-based ligation assay, this was performed using probes with labeled magnetic nanoparticles and quantum dots. After optimizing the technique, 50 DNA samples were genotyped with this method. We found a frequency of 72% for IVSII-1 (G˃A) mutation (42% heterozygote, and 30% mutant homozygote) with a highly sensitive nano-based ligation genotyping system, offering excellent sensitivity and specificity for point mutation detection; it has been demonstrated to be inaccurate, sensitive, cost-effective, and rapid technique for single nucleotide polymorphism (SNP) genotyping.

Validation of Ion TorrentTM Inherited Disease Panel with the PGMTM Sequencing Platform for Rapid and Comprehensive Mutation Detection.

Quick and accurate molecular testing is necessary for the better management of many inherited diseases. Recent technological advances in various next generation sequencing (NGS) platforms, such as target panel-based sequencing, has enabled comprehensive, quick, and precise interrogation of many genetic variations. As a result, these technologies have become a valuable tool for gene discovery and for clinical diagnostics. The AmpliSeq Inherited Disease Panel (IDP) consists of 328 genes underlying more than 700 inherited diseases. Here, we aimed to assess the performance of the IDP as a sensitive and rapid comprehensive gene panel testing. A total of 88 patients with inherited diseases and causal mutations that were previously identified by Sanger sequencing were randomly selected for assessing the performance of the IDP. The IDP successfully detected 93.1% of the mutations in our validation cohort, achieving high overall gene coverage (98%). The sensitivity for detecting single nucleotide variants (SNVs) and short Indels was 97.3% and 69.2%, respectively. IDP, when coupled with Ion Torrent Personal Genome Machine (PGM), delivers comprehensive and rapid sequencing for genes that are responsible for various inherited diseases. Our validation results suggest the suitability of this panel for use as a first-line screening test after applying the necessary clinical validation.

Rapid detection of mutation in isocitrate dehydrogenase 1 and 2 genes using mass spectrometry.

The 2016 World Health Organization classification of tumors of the central nervous system was recently revised. Mutations in the isocitrate dehydrogenase 1 (IDH1) and IDH2 genes and chromosome 1p/19q codeletion are especially important for both the integrated diagnosis and the determination of surgical strategy. To establish a method for intraoperative molecular diagnosis, a simple, rapid method was developed for the measurement of 2-hydroxyglutarate (2-HG), a specific oncometabolite formed in the presence of IDH gene mutation, using liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS). This method requires only 10min to measure the level of 2-HG from tissue preparation to completion of examination. Using this method, the level of 2-HG was analyzed in 105 patients with diffuse infiltrating glioma, and showed that IDH mutated glioma had significantly higher level of 2-HG compared to IDH wild-type glioma. Receiver operating characteristic curve analysis showed the area under the curve, sensitivity, and specificity were 0.9815, 97.5, and 100%, respectively. In contrast, tumor grade and presence of chromosome 1p/19q codeletion in the IDH mutated glioma could not be predicted from the level of 2-HG. Measurement of 2-HG level using LC/ESI-MS/MS can provide rapid and accurate information of mutation status in the IDH gene.

High Resolution Melting Analysis for Rapid Detection of PIK3CA Gene Mutations in Bladder Cancer: A Mutated Target for Cancer Therapy.

PIK3CA gene mutations have clinical importance and their presence is associated with therapy response. They are also considered as a molecule for targeted therapy. As regards to their importance, genetic variation within a population as well as among different populations, this study was conducted to detect common mutations of exons 9 and 20 and other probable mutations in PIK3CA gene as well as their frequencies in Iranian bladder cancer patients. Paired tumor and adjacent normal tissues samples were obtained from 50 bladder cancer patients. Mutations of PIK3CA gene were detected using High Resolution Melting (HRM) analysis which is ahighly sensitive, repeatable, rapid, and cost-effective technique. To determine the precision of the HRM analysis, Sanger sequencing analysis was used. The result showed that mutations were present in 10% (5/50) of the subjects. The majority of these cases (4/5) had the mutation(s) in exon 9, spanning over five different mutations, among which three of them were actually novel mutations. Further analysis showed that 2 cases had simultaneous mutations for exon 9. In addition to novel mutations, the PIK3CA mutation rate observed in Iranian bladder patients was not as frequent as previous reports and COSMIC. HRM can be used as a rapid and sensitive method for mutation screening. Dysregulation of PIK3CA gene in bladder cancer reveals its potentials as a mechanistic link for cancer development, which in turn suggests its special use in interventional studies for targeted therapy.

Performance of probe polymerization-conjunction-agarose gel electrophoresis in the rapid detection of KRAS gene mutation.

This study aimed to develop a simple and rapid method to detect KRAS gene mutations for conventional clinical applications under laboratory conditions. The genotype of mutation sites was determined based on the occurrence of target bands in the corresponding lanes of the reaction tubes through polymerization-conjunction of the probes, probe purification and amplification, and agarose gel electrophoresis. Circulating DNA samples were obtained from the plasma of 72 patients with lung cancer, which were identified based on six mutation sites (G12S, G12R, G12C, G12D, G12A, and G12V) of codon 12 of the KRAS gene. The detection results were compared with direct sequencing data. The proposed detection method is characterized by simple operation, high specificity, and high sensitivity (2%). This method can detect the mutations of three samples at G12S, G12R, and G12A. In the direct sequencing spectra of these samples, the genotype could not be determined due to the lack of evident sequencing peaks that correspond to the basic group of mutations. In conclusion, a simple and rapid method was established based on probe polymerization-conjunction-agarose gel electrophoresis for detecting KRAS gene mutations. This method can be applied to the conventional mutation detection of inhomogeneous samples.

A Fluorescence Quenching Assay Based on Molecular Beacon Formation through a Ligase Detection Reaction for Facile and Rapid Detection of Point Mutations.

A fluorescence quenching assay based on a ligase detection reaction was developed for facile and rapid detection of point mutations present in a mixed population of non-variant DNA. If the test DNA carried a targeted mutation, then the two allele-specific primers were ligated to form a molecular beacon resulting in the expected fluorescence quenching signatures. Using this method, we successfully detected as low as 5% mutant DNA in a mixture of wild-type DNA (t test at 99% confidence level).

Rapid detection of mutations in erm(41) and rrl associated with clarithromycin resistance in Mycobacterium abscessus complex by denaturing gradient gel electrophoresis

Clarithromycin resistance is increasing dramatically among Mycobacterium abscessus complex. The main resistance mechanisms are mutations in the erm(41) and rrl genes. Here we report PCR-based denaturing gradient gel electrophoresis (DGGE) as an alternative method for rapidly detection of mutations in erm(41) and rrl among M. abscessus isolates. Four primer sets targeting the full-length erm(41) gene and a 354bp fragment of the rrl gene were designed. A combination of 16 different DGGE patterns were observed for erm(41) gene, including 16 in M. abscessus subsp. abscessus and 1 in M. abscessus subsp. massiliense. Six DGGE patterns were obtained for rrl gene. Mutations in the erm(41) and rrl detected by DGGE were 100% identical to mutations detected by DNA sequencing. This is the first report to identify PCR-based DGGE as a practical, relatively inexpensive technique for rapidly detecting mutations in the erm(41) and rrl genes associated with clarithromycin resistance in M. abscessus complex.

KRAS mutation testing in borderline ovarian tumors and low-grade ovarian carcinomas with a rapid, fully integrated molecular diagnostic system.

Epithelial ovarian neoplasms are a heterogeneous group of tumors, including various malignancies with distinct clinicopathologic and molecular features. Mutations in BRAF and KRAS genes are the most frequent genetic aberrations found in low-grade serous ovarian carcinomas and serous and mucinous borderline tumors. Implementation of targeted therapeutic strategies requires access to highly specific and highly sensitive diagnostic tests for rapid determination of mutation status. One candidate for such test is fully integrated, real-time polymerase chain reaction-based Idylla™ system for quick and simple detection of KRAS mutations in formaldehyde fixed-paraffin embedded tumor samples. The primary aim of this study was to verify whether fully integrated real-time polymerase chain reaction-based Idylla system may be useful in determination of KRAS mutation status in patients with borderline ovarian tumors and low-grade ovarian carcinomas. The study included tissue specimens from 37 patients with histopathologically verified ovarian masses, operated on at the Department of Obstetrics and Gynecology, Nicolaus Copernicus University Collegium Medicum in Bydgoszcz (Poland) between January 2009 and June 2012. Based on histopathological examination of surgical specimens, 30 lesions were classified as low-grade ovarian carcinomas and 7 as borderline ovarian tumors. Seven patients examined with Idylla KRAS Mutation Test tested positive for KRAS mutation. No statistically significant association was found between the incidence of KRAS mutations and histopathological type of ovarian tumors. Mean survival of the study subjects was 48.51 months (range 3-60 months). Presence of KRAS mutation did not exert a significant effect on the duration of survival in our series. Our findings suggest that Idylla KRAS Mutation Test may be a useful tool for rapid detection of KRAS mutations in ovarian tumor tissue.

Clinical utility of circulating DNA analysis for rapid detection of actionable mutations to select metastatic colorectal patients for anti-EGFR treatment

While tumor-tissue remains the 'gold standard' for genetic analysis in cancer patients, it is challenged with the advent of circulating cell-free tumor DNA (ctDNA) analysis from blood samples. Here, we broaden our previous study on the clinical validation of plasma DNA in metastatic colorectal cancer patients, by evaluating its clinical utility under standard management care. Concordance and data turnaround-time of ctDNA when compared with tumor-tissue analysis were studied in a real-time blinded prospective multicenter clinical study (n = 140 metastatic colorectal patients). Results are presented according to STARD criteria and were discussed in regard with clinical outcomes of patients. Much more mutations were found by ctDNA analysis: 59%, 11.8% and 14.4% of the patients were found KRAS, NRAS and BRAF mutant by ctDNA analysis instead of 44%, 8.8% and 7.2% by tumor-tissue analysis. Median tumor-tissue data turnaround-time was 16 days while 2 days for ctDNA analysis. Discordant samples analysis revealed that use of biopsy, long delay between tumor-tissue and blood collection and resection of the tumor at time of blood draw, tumor site, or type of tissue analyzed seem to affect concordance. Altogether, the clinical data with respect to the anti-epidermal growth factor receptor response (RAS status) and the prognosis (BRAF status) of those discordant patients do not appear contradictory to the mutational status as determined by plasma analysis. Lastly, we present the first distribution profile of the RAS and BRAF hotspot mutations as determined by ctDNA analysis (n = 119), revealing a high proportion of patients with multiple mutations (45% of the population and up to 5 mutations) and only 24% of WT scored patients for both genes. Mutation profile as determined from ctDNA analysis with using various detection thresholds highlights the importance of the test sensitivity. Our study showed that ctDNA could replace tumor-tissue analysis, and also clinical utility of ctDNA analysis by considerably reducing data turnaround time.

Rapid detection of hot spot mutations of FGFR3 gene with PCR-high resolution melting assay

To identify the causative mutations in five individuals affected with dyschondroplasia and develop an efficient procedure for detecting hot spot mutations of the FGFR3 gene. Genomic DNA was extracted from peripheral blood samples with a standard phenol/chloroform method. PCR-Sanger sequencing was used to analyze the causative mutations in the five probands. PCR-high resolution melting (HRM) was developed to detect the identified mutations. A c.1138G>A mutation in exon 8 was found in 4 probands, while a c.1620C>G mutation was found in exon 11 of proband 5 whom had a mild phenotype. All patients were successfully distinguished from healthy controls with the PCR-HRM method. The results of HRM analysis were highly consistent with that of Sanger sequencing. The Gly380Arg and Asn540Lys are hot spot mutations of the FGFR3 gene among patients with ACH/HCH. PCR-HRM analysis is more efficient for detecting hot spot mutations of the FGFR3 gene.

A refined method to study gene dosage changes in vitro using CRISPR/Cas9

AimsGene dosage can have a major impact on cell biology, although, hitherto, it has been difficult to study using in vitro models. We sought to refine and accelerate the development...

Rapid and accurate detection of KRAS mutations in colorectal cancers using the isothermal-based optical sensor for companion diagnostics.

Although KRAS mutational status testing is becoming a companion diagnostic tool for managing patients with colorectal cancer (CRC), there are still several difficulties when analyzing KRAS mutations using the existing assays, particularly with regard to low sensitivity, its time-consuming, and the need for large instruments. We developed a rapid, sensitive, and specific mutation detection assay based on the bio-photonic sensor termed ISAD (isothermal solid-phase amplification/detection), and used it to analyze KRAS gene mutations in human clinical samples. To validate the ISAD-KRAS assay for use in clinical diagnostics, we examined for hotspot KRAS mutations (codon 12 and codon 13) in 70 CRC specimens using PCR and direct sequencing methods. In a serial dilution study, ISAD-KRAS could detect mutations in a sample containing only 1% of the mutant allele in a mixture of wild-type DNA, whereas both PCR and direct sequencing methods could detect mutations in a sample containing approximately 30% of mutant cells. The results of the ISAD-KRAS assay from 70 clinical samples matched those from PCR and direct sequencing, except in 5 cases, wherein ISAD-KRAS could detect mutations that were not detected by PCR and direct sequencing. We also found that the sensitivity and specificity of ISAD-KRAS were 100% within 30 min. The ISAD-KRAS assay provides a rapid, highly sensitive, and label-free method for KRAS mutation testing, and can serve as a robust and near patient testing approach for the rapid detection of patients most likely to respond to anti-EGFR drugs.

Abstract 2715: Precision-designed, rapid and low-cost single molecule detection of mutations from circulating tumor DNA

Abstract Detecting and quantifying mutations from circulating tumor DNA (ctDNA) is a new approach for longitudinally monitoring cancer patients throughout the course of treatment. However, general limitations to the clinical utility of ctDNA include sample concentration, overall assay cost, and result turnaround time. We have developed a novel molecular assay that utilize single color digital droplet PCR (ddPCR) to both genotype and quantify the number of tumor derived DNA molecules in a given sample. Our assay routinely detects as few as three mutant DNA molecules per a reaction volume, can be tested efficiently for fewer than ten dollars per reaction, and generates useable mutation information within four hours. Additionally, the assay can be rapidly configured to detect different mutations specifically identified in any cancer patient. To demonstrate the single molecule sensitivity and specificity for clinically relevant hotspot mutations, we have validated the assay using multiple input sources including high quality cell line DNA, formalin-fixed paraffin embedded tissue (FFPET) DNA, and ctDNA. Our ddPCR assay utilizes a novel primer design that is not rely on fluorescent probes. The overall simplicity of assay design enables one to detect nearly any coding mutation; practically, this means that any cancer or DNA sample can be tested efficiently. We have created customized precision mutation assays for two individual cancer patients. After extracting ctDNA from 500µL of plasma we prepare a controlled mixed wild type and mutant standard curves which contain between 150-3 detectable mutant molecules of interest per reaction. We then assay the patient ctDNA sample in parallel to the controlled standard curve and generate clustering confidence intervals. We have designed precision mutation assays targeting KRAS A146V, AKT1 E17K, and TP53 R175H for Patient A as well as KRAS G12V and PIK3CA H1047R for Patient B. The results of our single-color precision mutation quantification assays support the original mutation findings from the primary tumors. In one case, from 1µL of un-amplified ctDNA equivalent to less than 0.5ng of DNA, we identified 4 mutant molecules per ddPCR reaction volume. We have verified the presence of hotspot mutations from un-amplified patient derived ctDNA and quantified the number of mutant molecules present in each ctDNA sample. This assay has many features that make it amenable to automation. We are currently developing a massively high throughput process to design assays for tens of thousands of mutations in a matter of weeks. Overall this is an extremely low cost, highly sensitive and scalable diagnostic technology. We anticipate that this technology will be a valuable tool for rapidly monitoring ctDNA longitudinally in cancer patients. Citation Format: Christina M. Wood-Bouwens, Christine Handy, Billy Lau, Hanlee Ji. Precision-designed, rapid and low-cost single molecule detection of mutations from circulating tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2715. doi:10.1158/1538-7445.AM2017-2715

Targeted next generation sequencing in patients with infantile bilateral striatal necrosis

Background: Infantile bilateral striatal necrosis (IBSN) is a heterogeneous histo- pathologically condition defined by the involvement of the neostriata that can be observed in a very large number of neurological conditions clinically dominated by the presence of movement disorders. Methods: In a prospective study over the course of 1 year, we applied targeted next generation sequencing (NGS) of 78 genes linked to IBSN, using Sure Design Tool from Agilent Technologies. In addition, we studied biomarkers of known causes of IBSN, including blood interferon signature and free-thiamine and pterins in CSF. Results: We included 21 patients (16 male, age 1 month to 8 years; media ± SD 2.1 ± 1.6). Onset of symptoms was acute in almost all patients. Interferon signature was altered in two patients with Aicardi-Goutierès syndrome. Low free thiamine and elevated pterins in CSF were detected in 2 and 3 patients, respectively. T2-hyperintensities in other regions besides the neostriata were observed in 3/21 patients. We established firm molecular diagnosis in about 38% (8/21) of patients, with mutations been identified in the following genes: RNASEH2B, IFIH1, PDH1A, ADAR, SLC25A19 and NDUFAF6 (3 patients), including two novel mutations in SLC25A19 (c.580T>C) and ADAR (c.2187_2198delTGGCCTTTTGGAinsGT). We presented here the second reported pedigree of mutation in SLC25A19. The mutation is different as that found in the only other described family with SLC25A19 deficiency. Discussion: Targeted NGS leads to rapid and cost-effective detection of causative mutations in IBSN. We have reached a diagnostic rate similar to that described in the literature. Our study expands the very few descriptions of patients with SLC25A19 mutation.

Amplification Refractory Mutation System – Polymerase Chain Reaction for Rapid Detection of rpoB Gene Mutations in Mycobacterium tuberculosis

Multidrug-resistant tuberculosis (MDR-TB) has become a serious worldwide threat including in Nepal. MDR-TB refers to the pathological condition whereby Mycobacterium tuberculosis becomes resistant to the first line of drug treatment i.e. rifampin and isoniazid. Resistance to rifampin (RIF) is mainly caused by the mutations in the rpoB gene which codes for the β-subunit of RNA polymerase. In this study, Amplification Refractory Mutation System – Polymerase Chain Reaction (ARMS – PCR) technique has been used to detect mutations in the rpoB gene of Mycobacterium tuberculosis. Total DNA samples of 34 phenotypic MDR-TB were subjected to ARMS – PCR using three different codon specific primers (516, 526 and 531). These three codons occupy large portion of total mutation responsible for rifampin resistance. Out of the total DNA samples, all were bearing mutation in at least one of the three codons mentioned. Of those bearing mutation, the highest number had mutation in codon 531 (97.05 %) followed by codon 516 (17.64 %) and finally in codon 526 (11.76%) respectively. Hence, ARMS – PCR may be used as an alternative diagnostic technique for detection of rifampin resistance in Mycobacterium tuberculosis strains, especially for a developing country like Nepal.Int. J. Appl. Sci. Biotechnol. Vol 5(1): 81-85

Evaluation of the Ion Torrent PGM sequencing workflow for the routine rapid detection of BRCA1 and BRCA2 germline mutations

PurposeConventional methods used to identify BRCA1/2 germline mutations in hereditary cancers are time-consuming and expensive, due to the large size of the genes. The recent introduction of next generation sequencing (NGS) benchtop platforms is a great promise, which is rapidly revolutionizing genetic screening in diagnostic and clinical applications. We recently transferred our methodology for routine BRCA1/2 mutation screening (denaturing High Performance Liquid Chromatography plus Sanger sequencing) to the Ion Torrent PGM platform with the Ion Ampliseq BRCA1 and BRCA2 panel and tested the performance of the system. MethodsWe first validated the NGS approach in a cohort of 33 patients who had previously undergone genetic diagnosis in our laboratory by conventional methods. Then, we tested 29 newly diagnosed and uncharacterized patients by NGS, and Sanger sequencing was used to confirm results from the NGS platform. ResultsIn the validation cohort, all previously identified single nucleotide variants, insertions and deletions (also composed of multiple bases and within complex homopolymeric stretches) were identified by NGS in their correct zygosity status except for variants in a complex multinucleotide region within intron 7 of BRCA1 gene. NGS approach was further able to identify previously undetected variants. In the prospective cohort, almost all (99.3%) called variants were confirmed by Sanger. In both cohorts, in addition to the false positive (31) and false negative (110) results in the intron 7 of BRCA1 gene, the NGS method detected 10 false positives, that were solved by Sanger. ConclusionsThe Ion Torrent PGM NGS approach in BRCA1/2 germline mutation identification is highly sensitive, easy to use, faster and cheaper than traditional approaches. Therefore, according to other recently published works, we highly recommend this system for routine diagnostic testing on BRCA1/2 genes, along with Sanger confirmation of the called variants, and support the usefulness of the approach also in other routine genetic analysis.

Rapid and Reliable Detection of Nonsyndromic Hearing Loss Mutations by Multicolor Melting Curve Analysis

Hearing loss is a common birth defect worldwide. The GJB2, SLC26A4, MT-RNR1 and MT-TS1 genes have been reported as major pathogenic genes in nonsyndromic hearing loss. Early genetic screening is recommended to minimize the incidence of hearing loss. We hereby described a multicolor melting curve analysis (MMCA)-based assay for simultaneous detection of 12 prevalent nonsyndromic hearing loss-related mutations. The three-reaction assay could process 30 samples within 2.5 h in a single run on a 96-well thermocycler. Allelic types of each mutation could be reproducibly obtained from 10 pg ~100 ng genomic DNA per reaction. For the mitochondrial mutations, 10% ~ 20% heteroplasmic mutations could be detected. A comparison study using 501 clinical samples showed that the MMCA assay had 100% concordance with both SNaPshot minisequencing and Sanger sequencing. We concluded that the MMCA assay is a rapid, convenient and cost-effective method for detecting the common mutations, and can be expectedly a reliable tool in preliminary screening of nonsyndromic hearing loss in the Chinese Han population.

QMC-PCRx: a novel method for rapid mutation detection

AimsWe previously described the quick multiplex consensus PCR (QMC-PCR) as a method for rapid mutation screening in low-quality template. QMC-PCR has two-stages: a prediagnostic multiplex (PDM) reaction followed by a...