scholarly journals Syndrome of progressive bone marrow failure and pancreatic insufficiency remains cryptic despite whole exome sequencing: variant of Shwachman-Diamond syndrome or new condition?

2017 ◽  
Vol 5 (6) ◽  
pp. 748-752 ◽  
Author(s):  
Matthew C. Fadus ◽  
Eric T. Rush ◽  
Christina K. Lettieri
Keyword(s):  
Bone Marrow ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Bone Marrow Failure ◽  
Pancreatic Insufficiency ◽  
Whole Exome ◽  
Shwachman Diamond Syndrome

Diagnostic Efficacy of Whole-Exome Sequencing in 250 Patients with Congenital Bone Marrow Failure

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4385-4385
Author(s):  
Hideki Muramatsu ◽  
Yusuke Okuno ◽  
Kenichi Yoshida ◽  
Sayoko Doisaki ◽  
Asahito Hama ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Clinical Practice ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Sanger Sequencing ◽  
Bone Marrow Failure ◽  
Exome Capture ◽  
Diagnostic Efficacy ◽  
Hematopoietic Stem ◽  
Whole Exome

Abstract Introduction: Congenital bone marrow failure syndromes (CBMFSs) are a heterogeneous class of diseases with overlapping phenotypes. Therefore, a precise and comprehensive genetic diagnostic system is strongly warranted to arrive at appropriate clinical decisions to avoid ineffective therapies and/or lethal complications of allogeneic hematopoietic stem cell transplantation. However, a large panel of newly identified causative genes of CBMFSs have been identified in recent years; therefore, it is virtually impossible to establish a routine genetic diagnostic test using conventional Sanger sequencing. Whole-exome sequencing (WES) is a promising solution for the diagnosis of inherited diseases because it tests virtually all genes simultaneously. For the introduction of WES into clinical practice, it is necessary to clarify whether this technique has superior diagnostic efficacy to conventional clinical genetic tests. Methods: We performed WES in 250 patients with CBMFSs lacking genetic diagnoses. Exome capture was performed using the SureSelect® Human All Exon V3–5 kit (Agilent Technologies, Santa Clara, CA, USA), which covers all known coding exons, followed by massively parallel sequencing using the HiSeq 2000 Sequencing System (Illumina, San Diego, CA, USA). Our established pipeline for WES (genomon: http://genomon.hgc.jp/exome/) detected >20,000 candidate variants per patient. Diagnoses were based on variants of 130 genes with pathogenicities confirmed by published studies. Results: Genetic diagnoses were possible in 68 patients (27%). The best efficacy was achieved in patients with Fanconi anemia [35/73, 48%; FANCG (n = 17), FANCA (n = 14), FANCB (n = 1), FANCF (n = 1), SLX4 (n = 1), and BRCA2 (n = 1)], although Sanger sequencing was not applied because of the large sizes of its causative genes. Encouraging results were obtained in patients with Diamond–Blackfan anemia [11/ 61, 18%; RPS26 (n = 3), RPS7 (n = 2), RPS19 (n = 2), RPL5 (n = 2), RPL35A (n = 1), and RPL11 (n = 1)] and dyskeratosis congenita [7/29, 24%; TERT (n = 3), TINF2 (n = 2), and DKC1 (n = 2)]. Five genetic diagnoses (7%) were inconsistent with clinical diagnoses, possibly because of overlapping disease phenotypes. Conclusion: Relative to conventional genetic testing, WES was found to be effective for the diagnoses of CBMFSs. Furthermore, the efficacy of WES will increase as our knowledge of gene mutations expands. In conclusion, the use of WES in clinical practice is warranted. Disclosures No relevant conflicts of interest to declare.

Whole Exome Sequencing Shows a Paucity Of Somatic Gene Mutations In Pediatric Idiopathic Bone Marrow Failure Syndrome

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3708-3708
Author(s):  
Atsushi Narita ◽  
Hideki Muramatsu ◽  
Kenichi Yoshida ◽  
Yusuke Okuno ◽  
Asahito Hama ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Somatic Mutations ◽  
Bone Marrow Failure ◽  
Gene Mutations ◽  
Bone Marrow Failure Syndrome ◽  
Cell Lineages ◽  
Bone Marrow Failure Syndromes ◽  
Whole Exome

Abstract Introduction Pancytopenia accompanied by a severe decrease in bone marrow (BM) cellularity in children can be due to a broad variety of underlying disorders. Appropriate classification of bone marrow failure syndrome in children is challenging, particularly in relation to the morphological distinction between aplastic anemia (AA), refractory cytopenia of childhood (RCC), and refractory cytopenia with multilineage dysplasia (RCMD). The goal of this study was to characterize the molecular pathogenesis of these conditions by identifying the full spectrum of gene mutations in 29 patients with these disorders through the use of exome sequencing. Patient and Methods Diagnosis of AA, RCC, or RCMD was made on basis of the 2008 World Health Organization (WHO) classification criteria. AA patients exhibited no morphologically dysplastic changes in any of their hematopoietic cell lineages, while RCC patients had<10% dysplastic changes in two or more cell lineages or >10% in one cell lineage. Patients classified as RCMD exhibited >10% of the dysplastic changes in two or more cell lineages. Blood and BM samples were obtained from 29 children (16 boys and 13 girls) with AA (n = 8), RCC (n = 11), or RCMD (n = 10). The median age at diagnosis was 11 years (range, 2–15 years). Exome capture from paired DNA (non-T cells/CD3+ lymphocyte) was performed using SureSelect® Human All Exon V3 (Agilent Technologies, Santa Clara, CA) covering 50 Mb of the coding exons, followed by massive parallel sequencing using HiSeq 2000 (Illumina, San Diego, CA) according to the manufacturer’s protocol. Candidate somatic mutations were detected through our pipeline for whole exome sequencing (genomon: http://genomon.hgc.jp/exome/index.html). All candidate somatic nucleotide changes were validated by Sanger sequencing. Results Exome sequencing pipeline identified a total of 193 non-synonymous somatic mutations or indels candidates among the 29 patients (range, 2–15 per patient). After validation by Sanger sequencing, one nonsense, 11 missense, and two frame-shift mutations were confirmed as non-silent somatic mutations. The average numbers of mutations per sample were not significantly different when comparing morphological diagnostic groups (0.50 in AA, 0.36 in RCC, 0.60 in RCMD). Of these validated genes, BCOR (n = 2) and CSK (n = 2) mutations were recurrent genetic events. BCOR is a frequent mutational target in myelodysplastic syndrome, whereas CSK somatic mutations were not reported in human cancers. BCOR mutations were found both in AA (c.472delA:p.S158fs; patient 13) and in RCMD (c.G3856T:p.E1286X; patient 39). Both patients with CSK mutations were classified as RCC (c.G994A:p.D332N; patient 23 and 27). When comparing the clinical outcomes of patients with somatic mutations (n = 7) versus those without somatic mutations (n = 22), response rate to immunosuppressive therapy at 6 months (50% vs. 50%), 5-year clonal evolution rate (95% confidential interval) [0% (0% - 0%) vs. 6% (0% - 26%)], and the 5-year overall survival rate (95% confidential interval) [100% (100% - 100%) vs. 95% (70% - 99%)] were not significantly different. Conclusion Whole exome sequencing analysis was used for gene mutational profiling of patients with idiopathic bone marrow failure syndromes; i.e., AA, RCC, and RCMD. Although BCOR and CSK somatic mutations were recurrently identified, idiopathic bone marrow failure syndromes in children are characterized by a paucity of gene mutations, irrespective of morphological diagnosis. These findings suggest that morphological diagnosis based on WHO classification system does not discriminate the mutational profile and pathogenesis of bone marrow failure in children. Disclosures: No relevant conflicts of interest to declare.

Diagnostic Whole Exome Sequencing for 166 Patients with Inherited Bone Marrow Failure Syndrome

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 9-9
Author(s):  
Motoharu Hamada ◽  
Hideki Muramatsu ◽  
Yusuke Okuno ◽  
Ayako Yamamori ◽  
Taro Yoshida ◽  
...  
Keyword(s):  
United States ◽  
Bone Marrow ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Genetic Diagnosis ◽  
Primary Immune Deficiency ◽  
Whole Exome

BACKGROUND: Inherited bone marrow failure syndromes (IBMFSs) are a heterogeneous group of genetic disorders characterized by bone marrow failure, physical anomalies, and various kinds of organ complications. In addition to classical IBMFSs, such as Fanconi anemia, Diamond-Blackfan anemia, Dyskeratosis congenita, Shwachman-Diamond syndrome, and familial platelet disorders, many types of unclassified IBMFSs are reported. Over 100 genes are considered causative genes; however, the precise genetic diagnosis of IBMFSs remains challenging. We developed a capture-based target sequencing method for IBMFSs that covers more than 180 associated genes. Our system achieved genetic diagnosis for 225 (35%) of 738 patients between 2013 and 2018. However, the causative gene remained unknown for 513 (65%) patients, and further genetic analysis of these "target-negative" cases was necessary to achieve a precise diagnosis. METHODS: We performed whole exome sequencing (WES) for patients who were "target-negative" but strongly suspected of having IBMFS based on the following clinical characteristics: physical or organ anomalies (skin, nail, hair, skeletal, growth, cardiac, lung, liver, or genitourinary), family history of hematological disorder, young age (≤2 years), short telomere length (&lt;-2.0 SD), and hyper sensitivity to the chromosome breakage test. A sequencing library was prepared using the SureSelect Human All Exon 50Mb kit (Agilent Technologies, Santa Clara, CA, USA) and it was sequenced using the HiSeq2000 platform (Illumina, San Diego, CA, USA), according to manufacturers' instructions. The candidate germline variants were detected through our Genomon-exome analysis pipeline. With mean coverage of 100×, ≥ 85% of all protein coding bases were covered at 20× or more. RESULTS: Among the 513 "target-negative" cases, 166 patients were evaluated, of whom 17 patients' parents were also analyzed in a trio-based analysis. New pathogenic variants were identified in 18 of the 166 (11%) patients according to the American College of Medical Genetics (ACMG) guidelines, of which 5 variants were revealed to be de novo. Diagnostic variants were identified in FANCF, SRP54, RPL19, RPL5, RTEL1, RUNX1, MECOM, CDC42, GNE, SLNF14 (all n = 1). In addition to IBMFS-associated genes, causative genes for congenital hemolytic anemia (G6PD, PKLR), inborn error of metabolism (SLC46A1), and primary immune deficiency (NFKB2, LRBA) are also identified (all n = 1). Moreover, loss-of-function mutation of ADH5 gene are identified in three patients that seems to be associated to novel IBMFSs. On the other hand, no pathogenic variant in GATA2, ERCC6L2, LIG4, and SAMD9/SAMD9L genes that are reported as unclassified IBMFSs in Europe and United States are identified in our cohort. CONCLUSION: Our findings support the utility of WES (especially trio-based analysis) as a diagnostic tool for IBMFSs. Furthermore, genetic background of IBMFSs in East Asia seems to be different from that of Europe and United States. Disclosures No relevant conflicts of interest to declare.

Mutational Status of Splenic Diffuse Red Pulp Small B-Cell Lymphoma Revealed By Whole Exome Sequencing

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1448-1448
Author(s):  
Nerea Martinez ◽  
Carmen Almaraz ◽  
Manuela Mollejo ◽  
Yolanda Campos-Martin ◽  
Sophia Derdak ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cell ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Tissue Samples ◽  
Mutational Status ◽  
Whole Exome ◽  
Red Pulp

Abstract Background: Splenic diffuse red pulp lymphoma (SDRPL) is a rare small B cell neoplasm provisionally included in a category of unclassifiable splenic B-cell lymphoma/leukemias in the 2008 WHO classification. SDRPL is characterized by a diffuse pattern of involvement of the splenic red pulp by small monomorphous B lymphocytes. Patients are normally diagnosed at stage IV when spleen, bone marrow and peripheral blood are involved. This indolent but incurable disease is more common in aged males and it shows with splenomegaly and moderate lymphocytosis. The differential diagnosis with other splenic lymphomas such as marginal zone lymphoma, hairy cell lymphoma and its variant is not always easy, due to the similar clinical presentation and the absence of specific molecular markers. Here we studied the mutational status of 15 SDRPL patients using Whole Exome Next Generation Sequencing. Methods: Genomic DNA was extracted from FFPE/FF splenic tumor or bone marrow samples. When available, DNA from oral mucosa was obtained as the corresponding non-tumor control. Whole exome sequencing was performed at CNAG (Barcelona, Spain) following standard protocols for high-throughput paired-end sequencing on the Illumina HiSeq2000 instruments (Illumina Inc., San Diego, CA). Validation of variants was performed by PCR based targeted resequencing using a MiSeq instrument (Illumina Inc., San Diego, CA). We performed paired-end-76pb whole exome sequencing on DNA from 15 SDRPL patients. The corresponding normal counterpart from 3 of the patients was sequenced. From one patient FFPE and bone marrow DNA was available for comparison. In total 9 FFPE tissue samples, 3 FF tissue samples, and 4 bone marrow samples were sequenced. Almost 95% of the selected variants were validated by PCR based resequencing in 9 of the patients, while from 6 of the patients no tissue was available for validation. Results: 290 substitutions and 26 indels were obtained after filtering. Whole exome sequencing permitted us to identify variations in several genes of relevant pathways in lymphomas, such as NFkB pathway (IkBKB, TRAF, TANK, SYK), Apoptosis (BAD, DCPS, BCLAF1), MAPK (CXCR4, TCF3, NF1, MAP3K5), Cell cycle (CCND3, POLD3, BUB1), Chromatin (CREBBP, ARID1A, ARID1B, ARID3A, MLL3), MYC regulators (AKAP10, CTCF, EP400) or WNT signaling (SALL1, WNT5B, GPC6). Moreover, CCND3 and MLL3 were recurrently mutated in 2 different patients. Genes specifically found mutated in other splenic malignancies, such as NOTCH2, BRAF, MAP2K1, and KLF2 were not found mutated in this series of SDRPL patients. Conclusions: SDRPL samples contain somatic mutations involving genes regulating relevant pathways for cell survival, such as NFkB, apoptosis, cell cycle, chromatin, or WNT. The mutational signature of the series studied here may indicate that SDRPL is a distinct entity with specific molecular features different to other lymphoid splenic malignancies. Disclosures No relevant conflicts of interest to declare.

Discovery of the First Pathogenic Human EPO Mutation Provides Mechanistic Insight into Cytokine Signaling

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 331-331
Author(s):  
Ah Ram Kim ◽  
Jacob C Ulirsch ◽  
Stephan Wilmes ◽  
Ekrem Unal ◽  
Ignacio Moraga ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Fluorescent Imaging ◽  
Binding Kinetics ◽  
Wild Type ◽  
Sequencing Data ◽  
Exome Sequencing Data ◽  
Whole Exome ◽  
Whole Exome Sequencing Data

Abstract Congenital hypoplastic or Diamond-Blackfan anemia (DBA) is a rare bone marrow failure disorder characterized by a paucity of red blood cells and their precursors in the bone marrow. The majority of cases of DBA are due to haploinsufficient mutations in ribosomal protein genes and in rare cases result from GATA1 mutations. However, nearly half of the DBA cases do not have an identified genetic etiology. While analyzing whole exome sequencing data from a cohort of over 450 patients with a clinical diagnosis of DBA, we encountered the case of a male child of a first cousin consanguineous union who was diagnosed with DBA as an infant and remained transfusion dependent. The patient responded to corticosteroid therapy for a year as a toddler, but this treatment was discontinued due to side effects. The patient subsequently remained transfusion dependent and at 6 years of age an allogeneic bone marrow transplant from a matched maternal aunt was performed. Surprisingly, despite achievement of robust donor chimerism, the patient remained transfusion dependent. Unfortunately the patient developed severe graft-versus-host disease and died of resultant complications. Both the potential recessive nature of the mutation, given parental consanguinity, and the lack of anemia correction following transplant made this case extremely unusual. Thus we evaluated this patient's whole exome sequencing data. We identified a homozygous recessive mutation in the erythropoietin gene (EPO), which resulted in an R150Q substitution in the mature EPO protein. This mutation was absent from a cohort of 60,706 individuals depleted for Mendelian disease and fit the model of complete penetrance in the family. The R150Q mutation was expected to disrupt the high-affinity binding site to the EPO receptor (EPOR). However, we found by producing recombinant proteins that the EPO R150Q mutation only reduced the EPOR binding affinity by 3-fold. Surprisingly, the patient had an over 100-fold elevation in their serum EPO levels, suggesting that this mutation did not cause disease through altered affinity. Rather we observed altered EPOR binding kinetics by this mutant ligand. There was a slightly increased on-rate with a much faster dissociation rate (t1/2 of 10 seconds for the mutant vs. 6 minutes for the wild type). Using human erythroid cells and primary hematopoietic stem and progenitor cells, we could show that this mutant ligand never reached the same efficacy as the wild type (WT) EPO in promoting erythroid differentiation and proliferation. To better characterize this abnormal activity, we examined downstream signaling responses. We found identical phosphorylation of STAT5 at maximally potent concentrations of the WT (1 nM) and R150Q mutant (100 nM) EPO. By surveying a broad array of >120 phosphorylation events using intracellular flow cytometry, we demonstrated that maximal levels of STAT3 and STAT1 phosphorylation were reduced by 30% and 25%, respectively, with the R150Q (100 nM) compared to WT (1 nM) EPO. To determine the mechanistic basis for variation in downstream effector activation by the R150Q mutant ligand, we used inhibitors of both the JAK2 kinase and the SHP1/2 phosphatases that are respectively up- and downstream of STAT phosphorylation. While SHP1/2 inhibition did not alter STAT phosphorylation, JAK2 inhibition by ruxolitinib more potently inhibited STAT1/3 phosphorylation compared to STAT5. Interestingly, treatment with a low dose of ruxolitinib (40 nM) reduced erythroid proliferation to the same extent at maximally potent concentrations of the WT or R150Q EPO, demonstrating that the impairment in signaling by the R150Q EPO was primarily due to reduced JAK2 activity. Finally, we utilized single molecule fluorescent imaging of EPOR dimerization at the intact cell surface to directly show that the kinetically-biased R150Q EPO has a reduced ability to promote productive dimerization as compared to the WT EPO, even at maximally potent concentrations. Collectively, our results demonstrate how the R150Q mutant EPO - the first pathogenic mutation in EPO identified in humans - results in biased agonism of EPOR signaling through reduced receptor dimerization and consequently impaired JAK2 activation. More broadly our findings reveal how variation of cytokine-receptor binding kinetics can be used to tune downstream responses, which has broad implications for modulating the activity of numerous hematopoietic cytokines. Disclosures No relevant conflicts of interest to declare.

scholarly journals Mesenchymal Stromal Cells (MSCs) from Myelodysplastic Syndromes (MDS) Are Not Clonally Mutated I n Vivo

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1514-1514
Author(s):  
Johann-Christoph Jann ◽  
Maximilian Mossner ◽  
Vladimir Riabov ◽  
Eva Altrock ◽  
Nanni Schmitt ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Myelodysplastic Syndromes ◽  
Research Funding ◽  
Ex Vivo ◽  
Mutational Burden ◽  
Bm Niche ◽  
Whole Exome ◽  
Validation Experiments

Abstract Introduction There is increasing evidence for an active role of the bone marrow (BM) microenvironment in the pathogenesis of Myelodysplastic Syndromes (MDS). Genetically engineered murine models have shown that isolated mutations in the BM niche can disrupt the non-mutated hematopoietic compartment and induce MDS-like phenotypes. However, it is still unclear whether primary MDS in humans may possibly be associated with acquired mutations non-hematopoietic BM stroma cells. Although chromosomal aberrations and mutations have been described in in ex vivo expanded MSC cultures from MDS and AML patients, little validation has been performed to address whether such molecular lesions were not clonal outgrowths resulting from the strenuous and massively expansive cell culture procedures. Materials and Methods We performed whole exome sequencing on paired ex vivo expanded MSCs and native BM samples of n=98 MDS and associated myeloid neoplasia cases treated at the Department of Hematology and Oncology of the Medical Faculty Mannheim, Heidelberg University, Germany (median age 73 years, range 44-86). As controls, we included a cohort of n=28 samples from healthy subjects (median age 75 years, range 36-84). MSCs were expanded adherently on plastic dishes by seeding 5x10e6 mononuclear cells in StemMACS MSC Expansion Medium XF (Miltenyi Biotec) for a median of 34 days, (95% confidence interval 22-50d). Whole exome sequencing was carried out using Nextera DNA Flex Tagmentation kit (Illumina) with IDT xGene Research probe v1 at a median coverage at 88x with BM MNC as germline control accounting for possible LOH in the BM sample. Validation experiments were performed by deep re-sequencing of single CFU-F colonies (n=4 patients), sequencing of serial cultures (n=7 patients) and re-sequencing of primary sorted native bone marrow MSCs from n=9 patients. Results In the exome sequencing analyses of ex vivo expanded MSCs we discovered multiple recurrent mutations in MSCs of MDS patients including but not limited to genes such as ZFX (n=8/98) and RANK (n=5/98). MSCs from MDS patients displayed an overall higher mutational burden and increased replicative stress as determined by gH2AX and RPA staining, which correlated with the mutational burden and shorter telomeres as compared to healthy controls. The analysis of mutational signatures revealed that MDS MSCs were distinct compared to healthy MSCs. Furthermore, we found that MDS MSCs displayed increased senescence assessed by flow bGAL staining and associated inflammatory gene expression determined by IL6 qPCR/ELISA for n=32 cases. To investigate whether acquired mutations in MSCs were driven by the ex vivo expansion we performed individualized amplicon based deep re-sequencing of serial culture passages and different BM aspirations for n=7 patients as well as single colony re-sequencing in n=4 patient cases. Furthermore, we re-sequenced primary sorted CD45-,CD235a-,CD31+/-,CD271+/- BM cells of n=9 cases. All of these validation experiments indicated that the discovered mutations were associated with expansion in culture and but not present in clonally relevant cell populations in the primary BM in vivo. Discussion Together with previously published data of the BM niche of myeloid neoplasms, our results add to the notion that MSCs in MDS are molecularly and functionally altered. Nevertheless, our current comprehensive sequencing analyses leave little doubt that if acquired mutations in the stroma of MDS patients play a role in MDS disease initiation at all, then at such a low clonal and possibly locally confined level, that they are not detectable with currently feasible sample acquisition and methodology. In our current study, we discovered no evidence for acquired mutations in BM derived MSCs in MDS. Disclosures Schmitt: Affimed GmbH: Research Funding. Flach: Gilead: Current Employment. Hofmann: BMS: Honoraria; Amgen: Honoraria; Novartis: Honoraria. Nowak: Pharmaxis: Current holder of individual stocks in a privately-held company, Research Funding; Celgene: Honoraria; AbbVie: Other: Investigator on funded clinical trial; Tolero Pharma, Pharmaxis, Apogenix: Research Funding; Affimed: Research Funding; Takeda: Honoraria.

Whole-Exome Sequencing In Myelodysplastic Syndromes With 5q- and Normal Karyotype

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1551-1551
Author(s):  
Vera Adema ◽  
Mar Mallo ◽  
Leonor Arenillas ◽  
María Díez-Campelo ◽  
Elisa Luño ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Exome Sequencing ◽  
Board Of Directors ◽  
Whole Exome Sequencing ◽  
Research Funding ◽  
Somatic Mutations ◽  
Normal Karyotype ◽  
Advisory Committees ◽  
Whole Exome ◽  
5Q Deletion

Abstract Introduction Myelodysplastic Syndromes (MDS) are a heterogeneous group of clonal myeloid stem cells disorders with high prevalence in the elderly characterized by inefficient hematopoiesis, peripheral blood (PB) cytopenias, and an increased risk of transformation to acute myeloid leukemia (AML). The karyotype is the clinical parameter with the strongest prognostic impact according the IPSS-R (Greenberg et al., 2012). The most frequent cytogenetic alteration is the chromosome 5q deletion (del[5q]) which as a single anomaly, confers a good prognosis and predicts an excellent response to lenalidomide. Whether other genetic abnormalities routinely cooperate with del(5q) is not known. Whole-exome sequencing (WES) is a powerful tool to identify somatic mutations in protein coding genes that might cooperate with del(5q). In order to better understand the genetic basis of MDS with del(5q), we performed whole-exome sequencing (assessing 334,378 exons) of tumor-normal paired samples from 21 MDS patients. Herein we describe the preliminary findings. The analysis is ongoing and the complete results will be presented in the meeting. Methods A total of 21 patients with MDS (16 with del(5q) as a sole abnormality, 3 with del(5q) and additional alterations and 2 with normal karyotype) were included in our study. We examined a total of 25 tumor samples (21 diagnostic bone marrow (BM) samples with matched CD3+ cells as a controls, additional BM samples from 3 patients during lenalidomide treatment and 1 bone marrow sample from a del(5q) patient after AML progression). DNA was extracted from BM samples and from isolated peripheral blood CD3+ cells (magnetic-activated cell sorting (MACS), MiltenyiBiotec GmbH, Germany). The purity of CD3+ cells was assessed by FC 500 flow cytometer (Beckman Coulter, Hialeah, Fl, USA). Only DNA that fulfilled quality controls required by WES were submitted. For each diagnostic sample, we performed Conventional G-banding cytogenetics and fluorescence in situ hybridization (FISH, to confirm or dismiss 5q deletions). Whole-exome targeted capture was carried out on 3 μg of genomic DNA, using the SureSelect Human Exome Kit 51Mb version 4 (Agilent Technologies, Inc., Santa Clara, CA, USA). The captured and amplified exome library was sequenced with 100 bp paired-end reads on an Illumina HiSeq2000. Whole-exome sequencing data were analyzed using an in-house bioinformatics pipeline as previously reported. Somatic mutations identified as alterations present in tumor but not in the matched CD3+ sample were validated by Sanger sequencing. Results In our preliminary analysis of WES from 12 patients (10 patients with 5q- and 2 patients with normal karyotype), a total of 249 non-silent somatic variant candidates were identified, of which 146 were confirmed as somatic mutations. Recurrent mutations were observed in three genes (ASXL1, NBPF10 and SF3B1) in 3 different patients. Seven genes (HRNR, JAK2, POTEG, MUC5B, PHLDA, TTN, ZNF717) were mutated in two patients. Mutations in several genes known to be mutated in MDS (ASXL1, JAK2, RUNX1, SF3B1, SRSF2 and TET2) were also identified. Patients with the 5q deletion had an average of 11 mutations whereas patients with normal karyotype had a higher mean (14.5). Mutated genes identified in both groups were HRNR, JAK2, MUC5B, NBPF10 and SF3B1. No mutations in TP53 were detected in this subset. Pathway analysis of the complete list of somatically mutated genes will be carried out once all 21 patients are analyzed. The four in-treatment samples will be examined from their matched diagnostic samples. Conclusions Whole-exome sequencing of largely del(5q) MDS patient samples identified both known and previously unreported somatic mutations. Analysis of additional samples will allow a more complete description of the genes and pathways that may cooperate with del(5q) in the development and progression of MDS. Acknowledgments Financial support: This work has been supported (in part) by a grant from Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain (PI 11/02010); by Red Temática de Investigación Cooperativa en Cáncer (RTICC, FEDER) (RD07/0020/2004; RD12/0036/0044); Acción COST BM0801: European Genomics and Epigenomics Study on MDS and AML; Sociedad Española de Hematología y Hemoterapia (SEHH) and MDS Celgene. Footnotes Rafael Bejar and Francesc Sole contributed equally. Disclosures: Díez-Campelo: Novartis and Celgene: Honoraria, Research Funding. Cañizo:Celgene Jansen-Cilag Arry Novartis: Membership on an entity’s Board of Directors or advisory committees, Research Funding. Sanchez:Celgene: Honoraria, Research Funding. Bejar:Genoptix: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Solé:Celgene: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

Characterization of Subclonal Changes Along Progression in Multiple Myeloma.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2924-2924
Author(s):  
Joaquin Martinez Lopez ◽  
Margarita Sanchez Beato ◽  
Martinez Nerea ◽  
Ignacio Varela ◽  
Sophia Derdak ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Massive Parallel Sequencing ◽  
Myeloma Patient ◽  
Dynamic Changes ◽  
Multiple Myeloma Patient ◽  
Parallel Sequencing ◽  
Whole Exome

Abstract Abstract 2924 Background: Clonal composition and clone dynamic changes of neoplasms are a controversial issue, whose investigation is now facilitated by the development of massive parallel sequencing. Here we have analyzed the changes in the mutational spectrum associated with progression, treatment response and relapse in a multiple myeloma patient. We sequenced exomes for the primary quiescent-tumor, the progression and the relapsed samples. M&M: Patient and samples description Samples from asymptomatic, progression and relapse phases were compared by FISH and Whole exome massive parallel sequencing in a multiple myeloma patient carrying the t(4;14)(p16.3;q32) alteration. At relapse the cytogenetic study identified the presence of two major clones, 13q14 deletion and t(4;14)(p16.3;q32) in the 60% of the cells, and 17p13 deletion in the 12% of the cells. Whole exome sequencing was performed at CNAG (Barcelona, Spain) following standard protocols for high-throughput paired-end 76pb sequencing on the Illumina HiSeq2000 instruments (Illumina Inc., San Diego, CA). The variant calling was performed using an in house written software calling potential mutations showing a minimum independent multi-aligner evidence. Results: We performed whole exome sequencing on 3 tumor samples from the same patient: the first one at the time of diagnosis correspond to bone marrow infiltrated by 7% of plasma cells. The two additional samples, at progression and relapse, were done in CD138+ bone marrow cells, at this moment the percentage of infiltration was of 84% and 64% respectively. The germinal DNA from the same patient was used as reference. The mean coverage obtained for the four samples were 93x, with around 85% of bases with at least 20X coverage. After filtering, a total of 104 single nucleotide variations (SNV) were identified, some of them in more than one sample. The variations were classified into silent (25), missense (71), nonsense (6), and essential splice (2), according to their potential functional effect. In addition to t(4;14) and del13q14, progression and relapse samples shared 36 common SNVs. There were also some variants gained and/or loss in the different time points, suggesting the presence of at least five different clones, independent but related in their evolution. The two main clones were present in progression and relapse samples, but the ratio of the mutant alleles decreased in parallel to the decrement in the percentage of cells carrying on the described cytogenetic alterations Conclusions: There is a coupling between the cytogenetic and tumor sequence changes indicating that tumor at progression was composed by a dominant clone, together with multiple minor clones. Relapse after treatment was associated with multiple changes in the clone dynamics, progressive reduction of the main clone, emerging of new subclones and lost of minor clones. Dynamic changes along progression could be facilitated/induced by the therapy received. Disclosures: No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document