Targeted Next-Generation Sequencing (NGS) Enables for the First Time the Detection of Point Mutations, Molecular Insertions and Deletions, as Well as Leukemia-Specific Fusion Genes in AML in a Single Procedure.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 706-706
Author(s):  
Alexander Kohlmann ◽  
Vera Grossmann ◽  
Hans-Ulrich Klein ◽  
Sonja Schindela ◽  
Martin Dugas ◽  
...  
Keyword(s):  
Genetic Characterization ◽  
Snp Array ◽  
Point Mutations ◽  
Fusion Genes ◽  
Targeted Next Generation Sequencing ◽  
One Step ◽  
Equity Ownership ◽  
Next Generation Sequencing Ngs ◽  
First Time ◽  
Generation Sequencing

Abstract Abstract 706 Today, the genetic characterization necessary for optimal treatment of acute myeloid leukemia (AML) requires a combination of different labor-intensive methods such as chromosome banding analysis, sequencing for the detection of molecular mutations, and RT-PCR for the confirmation of characteristic fusion genes. DNA sequence enrichment from complex genomic samples using microarrays has recently been proposed to enable a targeted Next-Generation Sequencing (NGS) approach. Here, we combined 454 PicoTiterPlate (PTP) pyrosequencing with long-oligonucleotide sequence capture arrays to evaluate whether this technique allows a comprehensive genetic characterization in a one-step procedure (Roche Diagnostics Corporation, Branford, CT). 6 AML cases were analyzed with either known chromosomal aberrations–inversions and translocations–leading to fusion genes (CBFB-MYH11, RUNX1-RUNX1T1, MLL-MLLT3, MLL-unidentified fusion partner) or molecular mutations (KIT, FLT3-ITD, FLT3-TKD, and KRAS). A custom 1.91 Mb microarray was designed to contain capture probes for all coding regions of 92 target genes with relevance in leukemia, including e.g. KIT, NF1, KRAS, CEBPA, NPM1, FLT3, IKZF1, or TP53 (1559 exons). In addition, the complete genomic regions were targeted for the genes CBFB, RUNX1, and MLL (NimbleGen 385K format; Madison, WI). Starting with 20 μg of genomic DNA, this array design allowed a median 207-fold DNA enrichment of the targeted genomic loci. For sequencing, 454 Titanium chemistry was applied and in median 56.1 Mb of sequence data were generated per patient (median number of reads: 178.146). In median, 66.0% of reads were mapped to the original sequence capture array design, resulting in 18.7-fold median coverage per patient. The applied NGS data analysis pipeline used algorithms to map the obtained reads both exactly against the human genome, but also searched for chimeric sequences mapping to different regions in the genome. By this approach all corresponding fusion genes were detected as RUNX1-RUNX1T1 as well as the reciprocal RUNX1T1-RUNX1; CBFB-MYH11 and MYH11-CBFB; and MLL-MLLT3 and MLLT3-MLL, respectively. Interestingly, in one case a translocation t(11;19)(q23;p13) had been observed in chromosome banding analysis and the involvement of the MLL gene had been proven by FISH. However, using RT-PCR neither MLL-MLLT1 nor MLL-ELL fusion transcripts could be amplified. In contrast, the NGS approach identified chimeric reads containing both MLL and ELL sequences and, in addition, chimeric reads which were composed of SFRS14 (splicing factor, arginine/serine-rich 14; also located on 19p13 centromeric of ELL) and MLL. This suggested that a deletion had occurred in the breakpoint area and thus prevented the formation of a reciprocal ELL-MLL fusion gene. To confirm this assumption we performed a SNP array analysis (Affymetrix genome-wide human SNP array 6.0) and data from the SNP microarrays demonstrated a 615 kb deletion on 19p13, flanked by ELL and SFRS14, spanning from chr19: 18,346,048 - 18,961,490. Furthermore, with NGS it was possible to detect all molecular mutations identified by conventional methods including point mutations (KRAS G12C, FLT3-TKD D835Y), deletions (KIT D419X), and insertions (FLT3-ITD: 63 base pair length mutation). In conclusion, we demonstrated for the first time that fusion genes, point mutations, as well as deletions and insertions can be detected in a one-step methodological approach using the combination of a targeted DNA sequence enrichment assay followed by NGS technology. Furthermore, the genomic representation of only one of the partner genes of a chimeric fusion on this capture platform is sufficient to identify also any potentially unknown partner gene. As such, this novel assay has a strong potential to become an important method for a comprehensive genetic characterization of leukemias and other malignancies. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment. Grossmann:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Next-Generation Sequencing (NGS) in CMML, MDS and AML Detects Molecular Mutations in Oncogenes and Allows the Identification of Balanced Chromosomal Abnormalities with Extraordinary Sensitivity and Specificity.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 144-144
Author(s):  
Vera Grossmann ◽  
Alexander Kohlmann ◽  
Claudia Haferlach ◽  
Hans-Ulrich Klein ◽  
Martin Dugas ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Next Generation Sequencing ◽  
Candidate Genes ◽  
Point Mutations ◽  
Diagnostic Methods ◽  
Next Generation ◽  
Coding Regions ◽  
Equity Ownership ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Abstract Abstract 144 PicoTiterPlate (PTP) pyrosequencing allows the detection of low-abundance oncogene aberrations in complex samples even with low tumor content. Here, we compared deep sequencing data of two Next-Generation Sequencing (NGS) assays to detect molecular mutations using a PCR-based strategy and, in addition, to uncover inversions, translocations, and insertions in a targeted sequence enrichment workflow (454 Life Sciences, Roche Diagnostics Corporation, Branford, CT). First, we studied 95 patients (CMML, n=81; AML, n=6; MDS, n=3; MPS, n=3; ET, n=2) using the amplicon approach and investigated seven candidate genes with relevance in oncogenesis of myeloid malignancies: TET2, RUNX1, JAK2, MPL, KRAS, NRAS, and CBL. 43 primer pairs were designed to cover the complete coding regions of TET2, RUNX1 (beta isoform), and hotspot regions of the latter genes. In total, 4128 individual PCR reactions were performed with DNA isolated from bone marrow mononuclear cells, followed by product purification, fluorometric quantitation, and equimolar pooling of the corresponding 43 amplicon products to generate one single sequence library per patient. For sequencing, a 454 8-lane PTP was used applying standard FLX chemistry and representing one patient per lane. The median number of base pairs sequenced per patient was 9.23 Mb. For each amplicon a median of 840 reads was generated (coverage range: 485–1929 reads). As initial proof-of-concept analysis 27 of the 95 patients with known mutations (n=32) as detected by conventional sequencing or melting curve analyses were investigated (range of cells carrying the respective mutation: 1.1% for JAK2 V617F to 98.14% for TET2 C1464X). In all cases, 454 NGS confirmed results from routine diagnostic methods (GS Amplicon Variant Analyzer software version 2.0.01). We then investigated the remaining 69 CMML patients: In median, 2 variances (range 1–8 variances), i.e. differences in comparison to the reference sequence, per patient were detected. These variances included both point mutations in all candidate genes and large deletions (12-19 bp) in CBL, RUNX1, and TET2. Only 20/81 patients of the CMML-cohort (24.69%) were without any detectable mutation. Secondly, in a cohort of six AML bone marrow specimens a custom NimbleGen array (385K format; Madison, WI) was used to perform a targeted DNA sequence enrichment procedure. In total, capture probes spanning 1.91 Mb were designed to represent all coding regions of 92 target genes (1559 exons) with relevance in hematological malignancies (e.g. KIT, NF1, TP53, BCR, ABL1, NPM1, or FLT3). In addition, the complete genomic regions were targeted for RUNX1, CBFB, and MLL. For sequencing, 454 Titanium chemistry was applied, loading three patients per lane on a 2-lane PTP including three molecular identifiers (MIDs) each. Data analysis was performed using the GS Reference Mapper software version 2.0.01. For the enrichment assay, the median enrichment of the targeted genomic loci was 207-fold, as assessed by ligation-mediated LM-PCR. Overall, 1,098,132 reads were generated in the two lanes, yielding a total sequence length of 386,097,740 bases. In median, 96.52% of the sequenced bases mapped against the human genome, and 66.0% were derived from the customized NimbleGen array capture probes, resulting in a median coverage of 18.7-fold . With this method it was possible to detect and confirm point mutations (KIT, FLT3-TKD, and KRAS) and insertions (FLT3-ITD). Moreover, by capturing chimeric DNA fragments and generating reads mapping to both fusion partners this approach detected balanced aberrations, i.e. inv(16)(p13q22) and the translocations t(8;21)(q22;q22) or t(9;11)(p22;q23). In conclusion, both assays to specifically sequence targeted regions with oncogenic relevance on a NGS platform demonstrated promising results and are feasible. The amplicon approach is more suitable for detection of mutations in a routine setting and is ideally suited for large genes such as TET2, ATM, and NF1, which are labor-intensive to sequence conventionally. The array-based capturing assay is characterized by a complex and time-consuming workflow with low-throughput. However, the ability to detect balanced genomic aberrations which are detectable thus far only by cytogenetics and FISH has the potential to become an important diagnostic assay, especially in tumors in which cytogenetics can not be applied successfully. Disclosures: Grossmann: MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Dicker:MLL Munich Leukemia Laboratory: Employment. Kazak:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

scholarly journals Yield of Rare Variants Detected by Targeted Next-Generation Sequencing in a Cohort of Romanian Index Patients with Hypertrophic Cardiomyopathy

Diagnostics ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 1061
Author(s):  
Miruna Mihaela Micheu ◽  
Nicoleta-Monica Popa-Fotea ◽  
Nicoleta Oprescu ◽  
Stefan Bogdan ◽  
Monica Dan ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Hypertrophic Cardiomyopathy ◽  
Rare Variants ◽  
Causal Variant ◽  
Next Generation ◽  
Targeted Next Generation Sequencing ◽  
Novel Variants ◽  
Next Generation Sequencing Ngs ◽  
First Time ◽  
Generation Sequencing

Background: The aim of this study was to explore the rare variants in a cohort of Romanian index cases with hypertrophic cardiomyopathy (HCM). Methods: Forty-five unrelated probands with HCM were screened by targeted next generation sequencing (NGS) of 47 core and emerging genes connected with HCM. Results: We identified 95 variants with allele frequency < 0.1% in population databases. MYBPC3 and TTN had the largest number of rare variants (17 variants each). A definite genetic etiology was found in 6 probands (13.3%), while inconclusive results due to either known or novel variants were established in 31 cases (68.9%). All disease-causing variants were detected in sarcomeric genes (MYBPC3 and MYH7 with two cases each, and one case in TNNI3 and TPM1 respectively). Multiple variants were detected in 27 subjects (60%), but no proband carried more than one causal variant. Of note, almost half of the rare variants were novel. Conclusions: Herein we reported for the first time the rare variants identified in core and putative genes associated with HCM in a cohort of Romanian unrelated adult patients. The clinical significance of most detected variants is yet to be established, additional studies based on segregation analysis being required for definite classification.

scholarly journals The ICR96 exon CNV validation series: a resource for orthogonal assessment of exon CNV calling in NGS data

2017 ◽  
Vol 2 ◽  
pp. 35 ◽  
Author(s):  
Shazia Mahamdallie ◽  
Elise Ruark ◽  
Shawn Yost ◽  
Emma Ramsay ◽  
Imran Uddin ◽  
...  
Keyword(s):  
Sequencing Data ◽  
Targeted Next Generation Sequencing ◽  
Negative Results ◽  
Targeted Ngs ◽  
Predisposition Genes ◽  
Next Generation Sequencing Ngs ◽  
Ngs Data ◽  
Validation Series ◽  
Generation Sequencing ◽  
Dependent Probe

Detection of deletions and duplications of whole exons (exon CNVs) is a key requirement of genetic testing. Accurate detection of this variant type has proved very challenging in targeted next-generation sequencing (NGS) data, particularly if only a single exon is involved. Many different NGS exon CNV calling methods have been developed over the last five years. Such methods are usually evaluated using simulated and/or in-house data due to a lack of publicly-available datasets with orthogonally generated results. This hinders tool comparisons, transparency and reproducibility. To provide a community resource for assessment of exon CNV calling methods in targeted NGS data, we here present the ICR96 exon CNV validation series. The dataset includes high-quality sequencing data from a targeted NGS assay (the TruSight Cancer Panel) together with Multiplex Ligation-dependent Probe Amplification (MLPA) results for 96 independent samples. 66 samples contain at least one validated exon CNV and 30 samples have validated negative results for exon CNVs in 26 genes. The dataset includes 46 exon CNVs in BRCA1, BRCA2, TP53, MLH1, MSH2, MSH6, PMS2, EPCAM or PTEN, giving excellent representation of the cancer predisposition genes most frequently tested in clinical practice. Moreover, the validated exon CNVs include 25 single exon CNVs, the most difficult type of exon CNV to detect. The FASTQ files for the ICR96 exon CNV validation series can be accessed through the European-Genome phenome Archive (EGA) under the accession number EGAS00001002428.

scholarly journals Targeted Next Generation Sequencing (NGS) to Diagnose Hereditary Hemolytic Anemias

2020 ◽  
Author(s):  
Rishab Bharadwaj ◽  
Thulasi Raman ◽  
Ravikumar Thangadorai ◽  
Deenadayalan Munirathnam
Keyword(s):  
Next Generation Sequencing ◽  
Gene Sequencing ◽  
Accurate Diagnosis ◽  
Next Generation ◽  
Diagnostic Challenge ◽  
Targeted Next Generation Sequencing ◽  
Appropriate Treatment ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Hereditary hemolytic anemias present a unique diagnostic challenge due to their wide phenotypic and genotypic spectrum. Accurate diagnosis is essential to ensure appropriate treatment. We report two cases, which presented as hemolytic anemias, but initial workup was inconclusive and they were finally diagnosed with the help of Next Generation Sequencing (Dehydrated Hereditary Stomatocytosis and Kӧln Hemoglobinopathy). The introduction of gene sequencing to aid diagnosis of these disorders is a revolutionary step forward and should be incorporated earlier in the workup of such patients.

scholarly journals Molecular Genetics and Interferon Signature in the Italian Aicardi Goutières Syndrome Cohort: Report of 12 New Cases and Literature Review

2019 ◽  
Vol 8 (5) ◽  
pp. 750 ◽  
Author(s):  
Jessica Garau ◽  
Vanessa Cavallera ◽  
Marialuisa Valente ◽  
Davide Tonduti ◽  
Daisy Sproviero ◽  
...  
Keyword(s):  
Interferon Signaling ◽  
Cerebral Calcification ◽  
Interferon Signature ◽  
Interferon Stimulated Genes ◽  
Functional Investigation ◽  
Genetically Determined ◽  
Next Generation Sequencing Ngs ◽  
First Time ◽  
Generation Sequencing ◽  
Italian Cohort

Aicardi-Goutières syndrome (AGS) is a genetically determined early onset encephalopathy characterized by cerebral calcification, leukodystrophy, and increased expression of interferon-stimulated genes (ISGs). Up to now, seven genes (TREX1, RNASEH2B, RNASEH2C, RNASEH2A, ADAR1, SAMHD1, IFIH1) have been associated with an AGS phenotype. Next Generation Sequencing (NGS) analysis was performed on 51 AGS patients and interferon signature (IS) was investigated in 18 AGS patients and 31 healthy controls. NGS identified mutations in 48 of 51 subjects, with three patients demonstrating a typical AGS phenotype but not carrying mutations in known AGS-related genes. Five mutations, in RNASEH2B, SAMHD1 and IFIH1 gene, were not previously reported. Eleven patients were positive and seven negatives for the upregulation of interferon signaling (IS > 2.216). This work presents, for the first time, the genetic data of an Italian cohort of AGS patients, with a higher percentage of mutations in RNASEH2B and a lower frequency of mutations in TREX1 than those seen in international series. RNASEH2B mutated patients showed a prevalence of negative IS consistent with data reported in the literature. We also identified five novel pathogenic mutations that warrant further functional investigation. Exome/genome sequencing will be performed in future studies in patients without a mutation in AGS-related genes.

scholarly journals Genetic Variants Detected Using Cell-Free DNA from Blood and Tumor Samples in Patients with Inflammatory Breast Cancer

2020 ◽  
Vol 21 (4) ◽  
pp. 1290
Author(s):  
Jennifer S. Winn ◽  
Zachary Hasse ◽  
Michael Slifker ◽  
Jianming Pei ◽  
Sebastian M. Arisi-Fernandez ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Inflammatory Breast Cancer ◽  
Triple Negative ◽  
Genetic Profile ◽  
Cell Free Dna ◽  
Targeted Next Generation Sequencing ◽  
Pathogenic Variants ◽  
Free Dna ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

We studied genomic alterations in 19 inflammatory breast cancer (IBC) patients with advanced disease using samples of tissue and paired blood serum or plasma (cell-free DNA, cfDNA) by targeted next generation sequencing (NGS). At diagnosis, the disease was triple negative (TN) in eleven patients (57.8%), ER+ Her2- IBC in six patients (31.6%), ER+ Her2+ IBC in one patient (5.3%), and ER- Her2+ IBC in one other patient (5.3%). Pathogenic or likely pathogenic variants were frequently detected in TP53 (47.3%), PMS2 (26.3%), MRE11 (26.3%), RB1 (10.5%), BRCA1 (10.5%), PTEN (10.5%) and AR (10.5%); other affected genes included PMS1, KMT2C, BRCA2, PALB2, MUTYH, MEN1, MSH2, CHEK2, NCOR1, PIK3CA, ESR1 and MAP2K4. In 15 of the 19 patients in which tissue and paired blood were collected at the same time point, 80% of the variants detected in tissue were also detected in the paired cfDNA. Higher concordance between tissue and cfDNA was found for variants with higher allele fraction in tissue (AFtissue ≥ 5%). Furthermore, 86% of the variants detected in cfDNA were also detected in paired tissue. Our study suggests that the genetic profile measured in blood cfDNA is complementary to that of tumor tissue in IBC patients.

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