Whole exome sequencing identifies functional classes of gene mutations associated with bone marrow failure in pediatric Fanconi Anemia patients

2021 ◽  
Author(s):  
Shiyu Wang ◽  
Natalia H. Zbib ◽  
Alyza Skaist ◽  
Jiang Gui ◽  
Rafael Madero‐Marroquin ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
Gene Mutations ◽  
Whole Exome ◽  
Functional Classes

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 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 Reveals a Paucity of Somatic Gene Mutations in Aplastic Anemia and Refractory Cytopenia of Childhood

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4388-4388
Author(s):  
Yusuke Okuno ◽  
Atsushi Narita ◽  
Hideki Muramatsu ◽  
Kenichi Yoshida ◽  
Asahito Hama ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Who Classification ◽  
Somatic Mutations ◽  
Gene Mutations ◽  
Confidential Interval ◽  
Cell Lineages ◽  
Whole Exome ◽  
Somatic Gene

Abstract Introduction: The appropriate classification of bone marrow (BM) failure syndromes in children is challenging, particularly in relation to histological distinction between aplastic anemia (AA), refractory cytopenia of childhood (RCC), and refractory cytopenia with multilineage dysplasia (RCMD). The goal of this study is to characterize the molecular pathogenesis of these conditions by identifying the full spectrum of gene mutations in 29 children with three diseases using whole-exome sequencing. Patients and Methods: Wediagnosed AA, RCC, or RCMD on the basis of morphology and histological findings of bone marrow (BM) according to the 2008 World Health Organization (WHO) classification criteria. Patients with AA exhibited hypocellular BM and no morphologically dysplastic changes in any of three hematopoietic cell lineages, while patients with RCC had <10% dysplastic changes in two or more cell lineages or >10% in one cell lineage. Patients with RCMD exhibited >10% dysplastic changes in two or more cell lineages. We obtained peripheral blood and BM samples 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). We performed exome capture from paired DNA (non-T cells/CD3+ lymphocytes) using SureSelect® Human All Exon V4 kit (Agilent Technologies, Santa Clara, CA), which covered all part of the coding exons, followed by massively-parallel sequencing using HiSeq 2000 (Illumina, San Diego, CA) according to the manufacturer’s protocol. Candidate somatic mutations and germline variants were detected through our pipeline for whole-exome sequencing (Genomon-exome). All candidate somatic nucleotide changes were validated by Sanger sequencing. The ethics committee of Nagoya University Graduate School of Medicine approved this study. Results: Whole-exome sequencing pipeline identified a total of 14 non-synonymous somatic (one nonsense, 11 missense, and two frameshift) changes among the 29 patients, which resulted in only 0.48 mutations per patient. The average numbers of somatic mutations per sample were not significantly different among these groups (0.50 in AA, 0.36 in RCC, and 0.60 in RCMD). As a whole, childhood AA, RCC, and RCMD were characterized by a paucity of somatic mutations compared with adult myelodysplastic syndromes (MDS) in which 10 or more mutations per exome were detected on average. Among the mutated genes, BCOR-inactivating mutations in two patients (p.S158fs in AA and p.E1286X in RCMD) were considered significant genetic events based on previous reports that it is a driver gene in MDS. With regard to germline events, we did not detect any germline mutations of inherited BM failure syndromes. Moreover, we did not identifiy significantly frequent germline events in the entire cohort or any genetic hallmarks to be able to discriminate between these three diseases. 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%) 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: We usedwhole-exome sequencing analysis for gene mutational profiling of children with AA, RCC, and RCMD. Idiopathic bone marrow failure syndromes in children are characterized by a paucity of somatic gene mutations, irrespective of histological diagnosis. These findings suggest that histological diagnosis based on the WHO classification system does not discriminate the mutational profile of idiopathic BM failure syndromes 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.

scholarly journals The identification of novel gene mutations for degenerative lumbar spinal stenosis using whole-exome sequencing in a Chinese cohort

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Xin Jiang ◽  
Dong Chen
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Spinal Stenosis ◽  
Lumbar Spinal Stenosis ◽  
Gene Mutations ◽  
Specific Gene ◽  
Single Nucleotide ◽  
Degenerative Lumbar Spinal Stenosis ◽  
Whole Exome ◽  
Lumbar Spinal

Abstract Background Degenerative lumbar spinal stenosis (DLSS) is a common lumbar disease that requires surgery. Previous studies have indicated that genetic mutations are implicated in DLSS. However, studies on specific gene mutations are scarce. Whole-exome sequencing (WES) is a valuable research tool that identifies disease-causing genes and could become an effective strategy to investigate DLSS pathogenesis. Methods From January 2016 to December 2017, we recruited 50 unrelated patients with symptoms consistent with DLSS and 25 unrelated healthy controls. We conducted WES and exome data analysis to identify susceptible genes. Allele mutations firstly identified potential DLSS variants in controls to the patients’ group. We conducted a site-based association analysis to identify pathogenic variants using PolyPhen2, SIFT, Mutation Taster, Combined Annotation Dependent Depletion, and Phenolyzer algorithms. Potential variants were further confirmed using manual curation and validated using Sanger sequencing. Results In this cohort, the major classification variant was missense_mutation, the major variant type was single nucleotide polymorphism (SNP), and the major single nucleotide variation was C > T. Multiple SNPs in 34 genes were identified when filtered allele mutations in controls to retain only patient mutations. Pathway enrichment analyses revealed that mutated genes were mainly enriched for immune response-related signaling pathways. Using the Novegene database, site-based associations revealed several novel variants, including HLA-DRB1, PARK2, ACTR8, AOAH, BCORL1, MKRN2, NRG4, NUP205 genes, etc., were DLSS related. Conclusions Our study revealed that deleterious mutations in several genes might contribute to DLSS etiology. By screening and confirming susceptibility genes using WES, we provided more information on disease pathogenesis. Further WES studies incorporating larger DLSS patient cohorts are required to comprehend the genetic landscape of DLSS pathophysiology fully.

scholarly journals Whole-exome sequencing associates novel CSMD1 gene mutations with familial Parkinson disease

2017 ◽  
Vol 3 (5) ◽  
pp. e177 ◽  
Author(s):  
Javier Ruiz-Martínez ◽  
Luis J. Azcona ◽  
Alberto Bergareche ◽  
Jose F. Martí-Massó ◽  
Coro Paisán-Ruiz
Keyword(s):  
Parkinson Disease ◽  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Genome Wide Association Study ◽  
Single Gene ◽  
Gene Mutations ◽  
Complement Control Protein ◽  
Genome Wide ◽  
Whole Exome ◽  
A Genome

Objective:Despite the enormous advancements made in deciphering the genetic architecture of Parkinson disease (PD), the majority of PD is idiopathic, with single gene mutations explaining only a small proportion of the cases.Methods:In this study, we clinically evaluated 2 unrelated Spanish families diagnosed with PD, in which known PD genes were previously excluded, and performed whole-exome sequencing analyses in affected individuals for disease gene identification.Results:Patients were diagnosed with typical PD without relevant distinctive symptoms. Two different novel mutations were identified in the CSMD1 gene. The CSMD1 gene, which encodes a complement control protein that is known to participate in the complement activation and inflammation in the developing CNS, was previously shown to be associated with the risk of PD in a genome-wide association study.Conclusions:We conclude that the CSMD1 mutations identified in this study might be responsible for the PD phenotype observed in our examined patients. This, along with previous reported studies, may suggest the complement pathway as an important therapeutic target for PD and other neurodegenerative diseases.

Whole Exome Sequencing Reveals Spectrum of Gene Mutations in Pediatric AML

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 124-124
Author(s):  
Norio Shiba ◽  
Kenichi Yoshida ◽  
Yusuke Okuno ◽  
Yuichi Shiraishi ◽  
Yasunobu Nagata ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Conflicts Of Interest ◽  
Massively Parallel Sequencing ◽  
Gene Mutations ◽  
Pediatric Leukemia ◽  
Protein Coding ◽  
Whole Exome ◽  
Recurrent Mutations ◽  
Pediatric Aml

Abstract Abstract 124 Background Pediatric acute myeloid leukemia (AML) comprises ∼20% of pediatric leukemia, representing one of the major therapeutic challenges in pediatric oncology with the current overall survival remains to be ∼60%. As for the molecular pathogenesis of pediatric AML, it has been well established that gene fusions generated by recurrent chromosomal translocations, including t(15;17), t(8;21), inv(16) and t(9;11), play critical roles in leukemogenesis. However, they are not sufficient for leukemogenesis, indicating apparent need of additional genetic hits, and approximately 20% of pediatric AML cases lack any detectable chromosomal abnormalities (normal karyotype AML). Currently, a number of gene mutations have been implicated in the pathogenesis of both adult and pediatric AML, including mutations of RAS, KIT and FLT3, and more recently, a new class of mutational targets have been reported in adult AML, including CEBPA, NPM1, DNMT3A, IDH1/2, TET2 and EZH2. However, mutations of the latter class of gene targets seem to be rare in pediatric AML cases, whereas other abnormalities such as a NUP98-NSD1 fusion are barely found in adult cases, indicating the discrete pathogenesis between both AML at least in their subsets. Meanwhile, the recent development of massively parallel sequencing technologies has provided a new opportunity to discover genetic changes across the entire genomes or protein-coding sequences in human cancers at a single-nucleotide level, which could be successfully applied to the genetic analysis of pediatric AML to obtain a better understanding of its pathogenesis. Methods In order to reveal a complete registry of gene mutations and other genetic lesions, we performed whole exome sequencing of paired tumor-normal specimens from 23 pediatric AML cases using Illumina HiSeq 2000. Although incapable of detecting non-coding mutations and gene rearrangements, the whole-exome approach is a well-established strategy for obtaining comprehensive spectrum of protein-coding mutations. Recurrently mutated genes were further examined for mutations in an extended cohort of 200 pediatric AML samples, using deep sequencing, in which the prevalence and relative allele frequencies of mutations were investigated. Results Whole-exome sequencing of paired tumor-normal DNA from 23 patients were analyzed with a mean coverage of more than x120, and 90 % of the target sequences were analyzed at more than x20 depth on average. A total of 237 somatic mutations or 10.3 mutations per sample were identified. Many of the recurrent mutations identified in this study involved previously reported targets in adult AML, such as FLT3, CEBPA, KIT, CBL, NRAS, WT1, MLL3, BCOR, BCORL1, EZH2, and major cohesin components including XXX and ZZZ. On the other hand, several genes were newly identified in the current study, including BRAF, CUL2 and COL4A5, which were validated for the clinical significance in an extended cohort of 200 pediatric cases. Discussion Whole exome sequencing unmasked a complexity of gene mutations in pediatric AML genomes. Our results indicated that a subset of pediatric AML represents a discrete entity that could be discriminated from the adult counterpart, in terms of the spectrum of gene mutations. Disclosures: No relevant conflicts of interest to declare.

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