Whole exome sequencing of radiation-induced thymic lymphoma in mouse models identifies Notch1 activation as a driver of p53 wild-type lymphoma

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.

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Whole Exome Sequencing Uncovers Germline Variants of Cancer-Related Genes in Sporadic Pheochromocytoma

International Journal of Genomics ◽

10.1155/2018/6582014 ◽

2018 ◽

Vol 2018 ◽

pp. 1-9 ◽

Cited By ~ 1

Author(s):

Milena Urbini ◽

Margherita Nannini ◽

Annalisa Astolfi ◽

Valentina Indio ◽

Valentina Vicennati ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Susceptibility Genes ◽

Wild Type ◽

Germline Variants ◽

Whole Exome ◽

Sporadic Pheochromocytoma ◽

Passenger Mutations ◽

Tumor Dna

Background. Pheochromocytomas (PCCs) show the highest degree of heritability in human neoplasms. However, despite the wide number of alterations until now reported in PCCs, it is likely that other susceptibility genes remain still unknown, especially for those PCCs not clearly syndromic. Methods. Whole exome sequencing of tumor DNA was performed on a set of twelve PCCs clinically defined as sporadic. Results. About 50% of PCCs examined had somatic mutations on the known susceptibility VHL, NF1, and RET genes. In addition to these driver events, mutations on SYNE1, ABCC10, and RAD54B genes were also detected. Moreover, extremely rare germline variants were present in half of the sporadic PCC samples analyzed, in particular variants of MAX and SAMD9L were detected in the germline of cases wild-type for mutations in the known susceptibility genes. Conclusions. Additional somatic passenger mutations can be associated with known susceptibility VHL, NF1, and RET genes in PCCs, and a wide number of germline variants with still unknown clinical significance can be detected in these patients. Therefore, many efforts should be aimed to better define the pathogenetic role of all these germline variants for discovering novel potential therapeutic targets for this disease still orphan of effective treatments.

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Functional Characterization of a Novel GFI1B Mutation Causing Congenital Macrothrombocytopenia

Blood ◽

10.1182/blood.v126.23.75.75 ◽

2015 ◽

Vol 126 (23) ◽

pp. 75-75

Author(s):

Katsumasa Kitamura ◽

Yusuke Okuno ◽

Kenichi Yoshida ◽

Masashi Sanada ◽

Yuichi Shiraishi ◽

...

Keyword(s):

Platelet Count ◽

Dna Binding ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Functional Characterization ◽

Dominant Negative ◽

Red Cell ◽

Wild Type ◽

Whole Exome ◽

Proplatelet Formation

Abstract Congenital macrothrombocytopenia (CMTP) is a heterogeneous group of disorders characterized by abnormally large platelets and low platelet counts. So far, genes responsible for CMTP have been identified in approximately half of the cases. The genes currently known are MYH9, GP1BA, GP1BB, GP9, ITGA2B, ITGB3, ACTN1 and other less frequent genes. However, in the remaining half, CMTP-causing genes are unknown. To identify genes causing CMTP, we first performed whole exome sequencing and target sequencing in 46 Japanese CMTP families and identified 1 family with a heterozygous mutation in GFI1B gene. Several groups have recently reported GFI1B as a causative gene for CMTP with alpha-granule deficiency and red cell anisopoikilocytosis. Here, we report on the identification of a novel mutation in GFI1B gene and its functional characterization. We performed whole exome sequencing and additional targeted sequencing in genetically undefined 46 CMTP families and identified 1 family with GFI1B variant (Figure), which co-segregated with CMTP. The proband (arrow in the Figure) of this family was a 15 years old boy with a history of easy bruising and platelet count of 70-115x109/L. His mother also had low platelet count, although details of her bleeding complications are unknown. Morphologic analysis of peripheral blood smear from the affected family members showed gray appeared enlarged platelets and red cell anisocytosis. Subsequent sequencing analysis confirmed a heterozygous point mutation (c.G814+1C, p.G272fsX274) at the splice donor site flanking exon 6 of GFI1B. GFI1B contains six zinc fingers (Znfs), of which Znf 3 to 5 are essential for DNA binding. The mutated transcript predicted a truncation of 58 C-terminal amino acids, which resulted in complete deletion of Znf 5. Since previous reports on GFI1B mutations have also described mutations of Znf 5 (p.H294fsX307 and p.Q287X mutations by Stevenson et al and Monteferrario et al, respectively), we performed biological analyses of these 3 patient-derived mutations affecting essential DNA-binding domain. GFI1B is an essential transcriptional factor for megakaryocyte and erythroid development. Therefore, we tested the effects of the GFI1b mutations on the transcriptional repression of firefly luciferase reporter constructs containing GFI1B promoter as a validated GFI1B target. Whereas wild-type GFI1B repressed the expression of reporter gene, the G272fsX274, H294fsX307 and Q287X mutants were unable to repress the expression. To test whether these patient-derived mutations acted in dominant-negative manner, we measured the effect of increasing the amount of mutant GFI1B cDNA, cotransfected into cells with a set amount of wild-type GFI1B cDNA. All 3 mutants inhibited gene repression mediated by wild-type GFI1B in a dose-dependent manner. To investigate biological effects of mutant GFI1B on proplatelet formation from megakaryocytes, we expressed the mutant GFI1B in mouse fetal liver cells (FLCs) by retrovirus-mediated gene transfer. FLCs harvested from embryonic day 13.5 embryos were transfected with pGCDNsamIRES/EGFP vector containing wild-type or the 3 patient-derived mutant GFI1B and were cultured with thrombopoietin. And we observed proplatelet formation of EGFP positive megakaryocytes. The number of proplatelet-producing megakaryocytes was reduced in mutant GFI1B transduced FLCs on day 3 and 4 after transfection, suggesting that mutant GFI1B inhibited the rate of proplatelet production from megakaryocytes. Next, we investigated the size and number of proplatelet tips from megakaryocytes, and found that proplatelet tips produced from G272fsX274, H294fsX307 and Q287X-transduced megakaryocytes were significantly reduced in numbers, but were increased in the size of the tips. This was compatible with decreased platelet count and enlarged platelet seen in GFI1B mutated patients. These findings indicated that patient-derived mutant GFI1B disrupted proplatelet formation of megakaryocyte. In summary, we have identified a novel GFI1B mutation that causes autosomal dominant macrothrombocytopenia with gray platelets and red cell anisocytosis. The mutant GFI1B acted in a dominant-negative manner over wild-type GFI1B. In addition, we first showed that the mutant-transduced FLCs produced abnormally enlarged proplatelet tips, which was reduced in numbers. Disclosures No relevant conflicts of interest to declare.

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Whole-exome sequencing in tumor samples from sequenom-wild-type, ALK negative stage IV lung adenocarcinoma (ADC) patients (p).

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.8070 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. 8070-8070

Author(s):

Enriqueta Felip ◽

Ginevra Caratu ◽

Daniel Silberschmidt ◽

Pablo Martinez ◽

Alex Martinez Marti ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Transmembrane Domain ◽

Lung Tumor ◽

Stage Iv ◽

Genetic Alterations ◽

Wild Type ◽

Kras Mutations ◽

Tissue Samples ◽

Whole Exome

8070 Background: The majority of lung ADC tumors are characterized by specific genetic features with KRAS mutations (mut) seen in 20-30%, EGFR mut in 15%, EML4-ALK translocations in 5%, and ERBB2 mut in 2%, among others. Some of these genetic alterations are already being used for selecting targeted therapies. However, identification of additional genomic alterations is required. Methods: In the present ongoing study we perform whole-exome sequencing in paraffin-embedded tumor samples from OncoCarta v1.0 panel wild-type (no mut in hotspots of KRAS, EGFR, ERBB2, AKT1, BRAF, PIK3CA genes) and ALK negative (by FISH) stage IV ADC lung cancer p, and in their matched normal tissue samples. Results: To date, a total of 7 tumors and matched normal tissues have been successfully analyzed. We have detected mut in previously identified ADC genes, such as ERBB2, CTNNB1, TP53, SMAD4 or APC. Interestingly, mut were found in genes belonging to the proposed new cancer hallmark ‘epigenetic and RNA regulation’, such as BRD3, EPC1, PHF1 in almost every p included in our study. Regarding alterations that could be considered relevant for lung tumor pathogenesis/growth or as potential targets for treatment therapies, we were able to identify candidates in 4 of the 7 p. In one p, a case of a transmembrane domain ERBB2mut in exon 20 (p.E770delinsEAYVM) not previously detected by the OncoCarta v1.0 panel (that interrogates L755P, G776S/L/V/C, A775_G776insYVMA, P780_Y781insGSP, S779_P780insVGS mut) was found. In another p, three somatic mut in the BRCA1/2 genes were detected. Additionally, one p had an ALK point mut (p.P336K), for which no functional information is available, together with an APC mutation. In the remaining p, a non-hotspot mutation (although previously detected in a colorectal tumor) was found in CTNNB1. Conclusions: In this limited experience of whole-exome sequencing of a subgroup of stage IV lung ADC p, potentially targetable alterations not formerly detected by other techniques were found. We believe that genomic approaches to detecting alterations may be useful in clinical practice and will hopefully provide assistance in making treatment decisions.

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Whole exome sequencing identifies a germline MET mutation in two siblings with hereditary wild-type RET medullary thyroid cancer

Human Mutation ◽

10.1002/humu.23378 ◽

2017 ◽

Vol 39 (3) ◽

pp. 371-377 ◽

Cited By ~ 12

Author(s):

Marialuisa Sponziello ◽

Silvia Benvenuti ◽

Alessandra Gentile ◽

Valeria Pecce ◽

Francesca Rosignolo ◽

...

Keyword(s):

Thyroid Cancer ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Medullary Thyroid Cancer ◽

Wild Type ◽

Medullary Thyroid ◽

Whole Exome

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Whole exome sequencing of an asbestos-induced wild-type murine model of malignant mesothelioma

BMC Cancer ◽

10.1186/s12885-017-3382-6 ◽

2017 ◽

Vol 17 (1) ◽

Cited By ~ 15

Author(s):

Sophie Sneddon ◽

Ann-Marie Patch ◽

Ian M. Dick ◽

Stephen Kazakoff ◽

John V. Pearson ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Malignant Mesothelioma ◽

Murine Model ◽

Wild Type ◽

Whole Exome

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Mutation Screening Associated with Chromosome 7 Abnormalities Using Next Generation Whole Exome Sequencing

Blood ◽

10.1182/blood.v120.21.173.173 ◽

2012 ◽

Vol 120 (21) ◽

pp. 173-173 ◽

Cited By ~ 1

Author(s):

Naoko Hosono ◽

Hideki Makishima ◽

Andres Jerez ◽

Ines Gomez-Segui ◽

Bartlomiej Przychodzen ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Research Funding ◽

False Positives ◽

Chromosome 7 ◽

Next Generation ◽

Wild Type ◽

Monosomy 7 ◽

Whole Exome ◽

The Impact

Abstract Abstract 173 One of the most common karyotypic abnormalities identified in myelodysplastic syndromes (MDS) is monosomy 7 (del7) or deletion of the long arm of chromosome 7 (del7q). The presence of del7/del7q carries a poor prognosis in MDS, MDS/myeloproliferative neoplasms (MPN) and acute myeloid leukemia (AML); the impact of these defects appears similar. Recently, a copy-neutral type of loss of heterozygozity (LOH also referred to as a somatic UPD) has been identified on 7q. Microdeletion on 7q corresponding to the EZH2 locus led to identification of inactivating mutations in this gene, though hemizygous EZH2 mutations are only rarely found and do not fully explain del7/7q pathogenesis. We performed a comprehensive analysis of myeloid neoplasms (N=189), using next generation whole exome sequencing technology, including MDS (N=34), MDS/MPN (N=26) or MPN (N=4) and 124 with AML (both primary and secondary). Among them, LOH7, involving del7/del7q were observed in 17% of cases (N=33). To minimize false positives and focus on the most prevalent/relevant somatic events, we implemented a rational bioanalyitic filtering approach, whereby paired DNA (tumor/CD3 lymphocyte) were sequenced and results aligned using Burrows-Wheeler Aligner and variants detected using GATK pipeline (Best Practice Variant Detection from Broad Institute). We focused on searching for del7/7q linked somatic mutational events involved comparisons of mutations in the area of del7q to cases diploid for this locus. We hypothesized that there may be heterozygous mutations of 7q, which could lead to functional haploinsufficiency that is also a result of del7q (haploinsuffcient theory, heterozygous mutations). Conversely, mutations may be either unique to del7q hemizygous inactivation, or shared between 7q diploid and haploid cases. In total, we found alterations in 12 genes located on chromosome 7 (6% of all alterations found). Using filtering strategies we narrowed the focus to “tier 1” mutations to avoid false positives; 11 mutated genes were found in cases with del7/7q and 2 in UPD7q. For example, novel hemzygous (but not heterozygous mutations) of an E3 ubiquitin ligase CUL1 gene were detected only in cases with del7/7q, suggesting that the wild type allele is protective. In cases with diploid 7q, 24 heterozygous alterations were observed (10 genes shared with del7/7q). The previously described EZH2 mutations were seen in heterozygous, homozygous and hemizygous configurations, but were most common in UPD7q (100%), while only 7% of del7/7q cases were positive. Notably, 5/12 mutant genes were located in commonly deleted regions (CDRs) either 7q22, 7q34 or 7q35–36. These CDRs also contain recurrently mutated lesions, including 7q22 (CUX1:n=4; STAG3:n=2), 7q34 (a splicing factor; LUC7L2: n=3) and 7q35–36 (EZH2: n=10). When we investigated the association between haploinsufficiency and heterozygous mutations, among those on del7/7q, cases with wild type forms of corresponding genes showed decreased expression. Similarly, such mutations were occasionally present in diploid configuration; here again the wild type cases showed a decreased expression. These findings suggest that mutated genes located in CDRs can be pathogenic due to both haploinsufficiency of WT genes and heterozygous mutations. EZH2 is a good example of such a gene. We also searched accessory genetic events observed on other chromosomes along with del7/7q and UPD7. By SNP-A, there were clear differences among 3 LOH7 groups, in which del7 was more associated with accessory chromosomal defects than cases with UPD7q or del7. Similarly, mutational patterns were specific to each LOH cohort. For example, while well known frequently mutated genes, such as U2AF1, TET2 and TP53, were commonly found in all 3 LOH7 groups, some specific genes, including the CSMD family, were uniquely observed in monosomy 7, not in del7q or UPD7. Similarly, LOH7q was associated with somatic mutations in SETBP1 and RUNX1. In conclusion, we detected several candidate genes that could be associated with del7/7q and UPD7. Some mutations were heterozygous in cases with diploid 7q and correlated with CDRs on del7/7q without mutation. Certain mutations are specifically observed with del7, while others are commonly observed in all categories of LOH7, including EZH2. Moreover, some genes outside of the chromosome 7 were coincidently mutated with LOH7. Disclosures: Makishima: Scott Hamilton CARES Initiative: Research Funding. Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

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