High throughput gene sequencing reveals altered landscape in DNA damage responses and chromatin remodeling in sporadic pancreatic neuroendocrine tumors

Purpose

The main objectives of this retrospective study were to survey the genetic landscape of PNETs in a clinical cohort by using the high throughput gene sequencing method and to determine cellular signaling networks affected by the uncovered gene mutations.

Sequence variation in the Fanconi anemia gene FAA

Fanconi anemia (FA) is a genetically heterogeneous autosomal recessive syndrome associated with chromosomal instability, hypersensitivity to DNA crosslinking agents, and predisposition to malignancy. The gene for FA complementation group A (FAA) recently has been cloned. The cDNA is predicted to encode a polypeptide of 1,455 amino acids, with no homologies to any known protein that might suggest a function for FAA. We have used single-strand conformational polymorphism analysis to screen genomic DNA from a panel of 97 racially and ethnically diverse FA patients from the International Fanconi Anemia Registry for mutations in the FAA gene. A total of 85 variant bands were detected. Forty-five of the variants are probably benign polymorphisms, of which nine are common and can be used for various applications, including mapping studies for other genes in this region of chromosome 16q. Amplification refractory mutation system assays were developed to simplify their detection. Forty variants are likely to be pathogenic mutations. Seventeen of these are microdeletions/microinsertions associated with short direct repeats or homonucleotide tracts, a type of mutation thought to be generated by a mechanism of slipped-strand mispairing during DNA replication. A screening of 350 FA probands from the International Fanconi Anemia Registry for two of these deletions (1115–1118del and 3788–3790del) revealed that they are carried on about 2% and 5% of the FA alleles, respectively. 3788–3790del appears in a variety of ethnic groups and is found on at least two different haplotypes. We suggest that FAA is hypermutable, and that slipped-strand mispairing, a mutational mechanism recognized as important for the generation of germ-line and somatic mutations in a variety of cancer-related genes, including p53, APC, RB1, WT1, and BRCA1, may be a major mechanism for FAA mutagenesis.     

Telomere length in inherited bone marrow failure syndromes

Telomeres are long DNA repeats and a protein complex at chromosome ends that are essential for genome integrity. Telomeres are very short in patients with dyskeratosis congenita due to germline mutations in telomere biology genes. We compared telomere length in patients with Fanconi anemia, Diamond-Blackfan anemia and Shwachman-Diamond syndrome with telomere length in dyskeratosis congenita. Telomere length was measured in six leukocyte subsets by automated multicolor flow fluorescence in situ hybridization, and age-adjusted using Z-scores (−2.326 = 1^st percentile) were created. We examined individual data, and used canonical variate analysis for group comparisons and outlier detection. Most dyskeratosis congenita telomere lengths were below the 1^st percentile, while only 2 Fanconi anemia and one each Diamond-Blackfan anemia and Shwachman-Diamond syndrome were that low. However, Fanconi anemia, Diamond-Blackfan anemia and Shwachman-Diamond syndrome clustered in the bottom half of the normal range. Canonical variate analysis separated dyskeratosis congenita widely from the other three syndromes by the first canonical variable (89.7% of the variance); the second variable (10.0%) separated Diamond-Blackfan anemia, Shwachman-Diamond syndrome, and Fanconi anemia from each other. Overall, unlike in dyskeratosis congenita, telomere lengths in patients with non-dyskeratosis congenita inherited bone marrow failure syndromes were usually in the normal range, albeit shorter than in unaffected individuals. clinicaltrials.gov identifier: 00027274     

MDS/MPN-RS-T justified inclusion as a unique disease entity?

Myelodysplastic syndrome/myeloproliferative neoplasm with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T) is a disease entity characterized by anemia, bone marrow dysplasia with ring sideroblasts and persistent thrombocytosis ≥450 × 10⁹/L with proliferation of large and morphologically atypical megakaryocytes. Although initially recognized by the World Health Organization only as a provisional entity, next generation sequencing has identified recurrent somatic mutations in SF3B1, JAK2 and other genes providing further evidence of the clonal nature of this disease and the need to recognize it as a separate entity. Despite its overlapping features with MDS with ring sideroblasts and essential thrombocythemia, MDS/MPN-RS-T is characterized by specific clinical features and distinct survival outcomes. In the current review we will describe the morphological and genomic features of MDS-RS-T and the potential diagnostic challenges and distinction from other possible conditions. We will also review how the current evidence supports its recognition as an independent disorder.     

Investigating Genetic Mutations in a Large Cohort of Iranian Patients with Congenital Hyperinsulinism

Objective:Congenital hyperinsulinism (CHI) is the most frequent cause of severe and persistent hypoglycaemia from birth. Understanding the pathophysiology and genetic defects behind hyperinsulinism and its complications provides clues to timely diagnosis and management. The aim of this study was to evaluate the underlying genetic aetiology of a specific Iranian pediatric cohort with CHI.Methods:A total of 44 unrelated children, 20 girls and 24 boys, with an initial diagnosis or history of CHI from all regions of Iran were recruited between 2016 and 2019. Targeted next generation sequencing (tNGS) was performed for the genes found in about half of CHI patients.Results:Mutations were identified in 24 cases (55%). Patients with a confirmed genetic cause were mainly diagnosed below age of one year old (p=0.01), had fewer other syndromic features, excluding seizure, (p=0.03), were less diazoxide responsive (p=0.04) and were more diazoxide unresponsive leading to pancreatectomy (p=0.007) compared to those with no identified mutations. Among 24 patients with identified genetic mutations, 17 (71%) had a mutation in ABCC8, 3 (12%) in KCNJ11, 3 (12%) in HADH, and 1 patient had a mutation in KMT2D. These included five novel mutations in ABCC8, KCNJ11, and KMT2D.Conclusion:This is the biggest genetic study of CHI in Iran. A high frequency of recessive forms of CHI, especially HADH mutations, in our study could be due to a high rate of consanguineous marriage. We recommend tNGS to screen for all the CHI genes.     

Genomic analysis of bone marrow failure and myelodysplastic syndromes reveals phenotypic and diagnostic complexity

Accurate and timely diagnosis of inherited bone marrow failure and inherited myelodysplastic syndromes is essential to guide clinical management. Distinguishing inherited from acquired bone marrow failure/myelodysplastic syndrome poses a significant clinical challenge. At present, diagnostic genetic testing for inherited bone marrow failure/myelodysplastic syndrome is performed gene-by-gene, guided by clinical and laboratory evaluation. We hypothesized that standard clinically-directed genetic testing misses patients with cryptic or atypical presentations of inherited bone marrow failure/myelodysplastic syndrome. In order to screen simultaneously for mutations of all classes in bone marrow failure/myelodysplastic syndrome genes, we developed and validated a panel of 85 genes for targeted capture and multiplexed massively parallel sequencing. In patients with clinical diagnoses of Fanconi anemia, genomic analysis resolved subtype assignment, including those of patients with inconclusive complementation test results. Eight out of 71 patients with idiopathic bone marrow failure or myelodysplastic syndrome were found to harbor damaging germline mutations in GATA2, RUNX1, DKC1, or LIG4. All 8 of these patients lacked classical clinical stigmata or laboratory findings of these syndromes and only 4 had a family history suggestive of inherited disease. These results reflect the extensive genetic heterogeneity and phenotypic complexity of bone marrow failure/myelodysplastic syndrome phenotypes. This study supports the integration of broad unbiased genetic screening into the diagnostic workup of children and young adults with bone marrow failure and myelodysplastic syndromes.     

Characterization of gene mutations and copy number changes in acute myeloid leukemia using a rapid target enrichment protocol

Prognostic stratification is critical for making therapeutic decisions and maximizing survival of patients with acute myeloid leukemia. Advances in the genomics of acute myeloid leukemia have identified several recurrent gene mutations whose prognostic impact is being deciphered. We used HaloPlex target enrichment and Illumina-based next generation sequencing to study 24 recurrently mutated genes in 42 samples of acute myeloid leukemia with a normal karyotype. Read depth varied between and within genes for the same sample, but was predictable and highly consistent across samples. Consequently, we were able to detect copy number changes, such as an interstitial deletion of BCOR, three MLL partial tandem duplications, and a novel KRAS amplification. With regards to coding mutations, we identified likely oncogenic variants in 41 of 42 samples. NPM1 mutations were the most frequent, followed by FLT3, DNMT3A and TET2. NPM1 and FLT3 indels were reported with good efficiency. We also showed that DNMT3A mutations can persist post-chemotherapy and in 2 cases studied at diagnosis and relapse, we were able to delineate the dynamics of tumor evolution and give insights into order of acquisition of variants. HaloPlex is a quick and reliable target enrichment method that can aid diagnosis and prognostic stratification of acute myeloid leukemia patients.     

High frequency of ribosomal protein gene deletions in Italian Diamond-Blackfan anemia patients detected by multiplex ligation-dependent probe amplification assay

Diamond-Blackfan anemia is an autosomal dominant disease due to mutations in nine ribosomal protein encoding genes. Because most mutations are loss of function and detected by direct sequencing of coding exons, we reasoned that part of the approximately 50% mutation negative patients may have carried a copy number variant of ribosomal protein genes. As a proof of concept, we designed a multiplex ligation-dependent probe amplification assay targeted to screen the six genes that are most frequently mutated in Diamond-Blackfan anemia patients: RPS17, RPS19, RPS26, RPL5, RPL11, and RPL35A. Using this assay we showed that deletions represent approximately 20% of all mutations. The combination of sequencing and multiplex ligation-dependent probe amplification analysis of these six genes allows the genetic characterization of approximately 65% of patients, showing that Diamond-Blackfan anemia is indisputably a ribosomopathy.Key words: Diamond-Blackfan anemia, multiplex ligation-dependent probe amplification, ribosomal protein gene deletion     

Molecular diagnostic update in hereditary hemolytic anemia and neonatal hyperbilirubinemia

Hereditary hemolytic anemia (HHA) is a group of genetically and phenotypically heterogeneous disorders characterized by premature destruction of red blood cells (RBCs) with clinical manifestations ranging from asymptomatic to marked hemolytic anemia. There are three main categories of HHA: (a) RBC membrane defects; (b) hemoglobinopathies/thalassemias; and (c) RBC enzyme deficiencies. Hyperbilirubinemia is a frequent consequence of hemolytic anemia and can lead to bilirubin‐associated neurotoxicity in neonates and to jaundice, and formation of gall stones in adults. Hyperbilirubinemia can also be caused by impaired bilirubin conjugation due to polymorphisms and mutations in genes involved in bilirubin metabolism (eg, UGT1A1). Neonates with HHA and co‐inherited variants impairing bilirubin conjugation are at increased risk of bilirubin‐associated toxicity. Prior to the advent of next‐generation sequencing (NGS), molecular diagnosis of these disorders was limited to targeted single gene Sanger sequencing. However, NGS is making its way into the standard diagnostic workup of complex and multigene disorders like HHA. This review will focus on the molecular updates of HHA with particular focus on the neonatal and pediatric population.     

Functional analysis of human mismatch repair gene mutations identifies weak alleles and polymorphisms capable of polygenic interactions

Many of the mutations reported as potentially causing Lynch syndrome are missense mutations in human mismatch repair (MMR) genes. Here, we used a Saccharomyces cerevisiae-based system to study polymorphisms and suspected missense mutations in human MMR genes by modeling them at the appropriate S. cerevisiae chromosomal locus and determining their effect on mutation rates. We identified a number of weak alleles of MMR genes and MMR gene polymorphisms that are capable of interacting with other weak alleles of MMR genes to produce strong polygenic MMR defects. We also identified a number of alleles of MSH2 that act as if they inactivate the Msh2-Msh3 mispair recognition complex thus causing weak MMR defects that interact with an msh6Δ mutation to result in complete MMR defects. These results indicate that weak MMR gene alleles capable of polygenic interactions with other MMR gene alleles may be relatively common.     

CCDC40 mutation as a cause of infertility in a Chinese family with primary ciliary dyskinesia

Trial design:Primary ciliary dyskinesia (PCD) is a genetical disease that inherited in an autosomal-recessive way. Its clinical manifestations (such as male infertility) are mainly caused by defects of motion-related cilia that encoded by mutated genes. Although some mutation has been verified, a number of mutations of PCD remain elusive. The main purpose of this study is to identify mutant genes in a Chinese family with PCD, and to verify the safety and effectiveness of intracytoplasmic sperm injection (ICSI) of infertility caused by PCD.Methods:Imaging examination was used to exclude pulmonary inflammation and visceral translocation. Semen analysis was used to assess the quality of the proband''s sperm. Transmission electron microscopy (TEM) was conducted to assess the ultrastructure of flagella and cilia. Targeted next generation sequencing and Sanger sequencing and qPCR (real-time quantitative polymerase chain reaction detecting system) were applied to identified mutation of Chinese Family suspected of having PCD. Viable sperm were selected by hypo-osmotic swelling test (HOST) for ICSI.Results:We report 2 novel mutations in CCDC40 gene (c.1259delA and EX17_20 deletion) resulted in immobility of sperm and infertility of the proband. These mutations were confirmed in the proband''s sister (heterozygous) and his parents (recessive carrier) by Sanger sequencing and qPCR. All the spermatozoa from the proband were immotile. Ultrastructural defects were found in flagella and cilia of proband and his sister. Viable sperms were selected by HOST for ICSI and fertilized 9 of 21 eggs. Two frozen embryos were transplanted and a healthy 3500 g boy was delivered at 40 + 4 weeks’ gestation. And then, we summarized the genes related to PCD and the mutant sites of CCDC40 gene.Conclusion:We reported 2 novel mutants in CCDC40 gene (c.1259delA and EX17_20 deletion), which could be candidates for genetic diagnosis in PCD patients. The combination of targeted next generation sequencing and Sanger sequencing may be a useful tool to diagnose PCD. ICSI is a considerable method in treatment of infertility caused by PCD.     

Functional analysis of patient-derived mutations in the Fanconi anemia gene, FANCG/XRCC9.

OBJECTIVE: Fanconi anemia (FA) is an autosomal-recessive cancer susceptibility syndrome with seven complementation groups. Six of the FA genes have been cloned (corresponding to subtypes A, C, D2, E, F, and G) and the encoded proteins interact in a common pathway. Patient-derived mutations in FA genes have been helpful in delineating functional domains of FA proteins. The purpose of this work was to subtype FA patient-derived cell lines in our repository and to identify FA gene mutations. METHODS: We subtyped 62 FA patients as type A, G, C, or non-ACG by using a combination of retroviral gene transfer and immunoblot analysis. Among these FA patients, we identified six FA-G patients for further analysis. We used a strategy involving amplification of FANCG/XRCC9 exons and direct sequencing to identify novel FANCG mutations in cell lines derived from these FA-G patients. We functionally analyzed FANCG mutant alleles by transducing the corresponding cDNAs into a known FA-G indicator cell line and scoring correction of MMC sensitivity. RESULTS: Our results demonstrate a wide range of mutations in the FANCG gene (splice, nonsense, and missense mutations). Based on this mutational screen, a carboxy terminal functional domain of the FANCG protein appears to be required for complementation of FA-G cells and for normal assembly of the FANCA/FANCG/FANCC protein complex. CONCLUSION: The identification of patient-derived mutant alleles of FA genes can provide important insights to the function of FA proteins. FA subtyping is also a necessary precondition for gene therapy.     

Lynch syndrome and Lynch syndrome mimics: The growing complex landscape of hereditary colon cancer

Hereditary non-polyposis colorectal cancer (HNPCC) was previously synonymous with Lynch syndrome; however, identification of the role of germline mutations in the DNA mismatch repair (MMR) genes has made it possible to differentiate Lynch syndrome from other conditions associated with familial colorectal cancer (CRC). Broadly, HNPCC may be dichotomized into conditions that demonstrate defective DNA MMR and microsatellite instability (MSI) vs those conditions that demonstrate intact DNA MMR. Conditions characterized by MMR deficient CRCs include Lynch syndrome (germline MMR mutation), Lynch-like syndrome (biallelic somatic MMR mutations), constitutional MMR deficiency syndrome (biallelic germline MMR mutations), and sporadic MSI CRC (somatic biallelic methylation of MLH1). HNPCC conditions with intact DNA MMR associated with familial CRC include polymerase proofreading associated polyposis and familial colorectal cancer type X. Although next generation sequencing technologies have elucidated the genetic cause for some HNPCC conditions, others remain genetically undefined. Differentiating between Lynch syndrome and the other HNPCC disorders has profound implications for cancer risk assessment and surveillance of affected patients and their at-risk relatives. Clinical suspicion coupled with molecular tumor analysis and testing for germline mutations can help differentiate the clinical mimicry within HNPCC and facilitate diagnosis and management.     

Akute myeloische Leukämie

Acute myeloid leukemia (AML) is a genetically heterogeneous disease. The genetic diagnostics have become an essential component in the initial work-up for disease classification, prognostication and prediction. More and more promising molecular targeted therapeutics are becoming available. A prerequisite for individualized treatment strategies is a fast pretherapeutic molecular screening including the fusion genes PML-RARA, RUNX1-RUNX1T1 and CBFB-MYH11 as well as mutations in the genes NPM1, FLT3 and CEBPA. Promising new therapeutic approaches include the combination of all- trans retinoic acid and arsentrioxid in acute promyelocytic leukemia, the combination of intensive chemotherapy with KIT inhibitors in core-binding factor AML and FLT3 inhibitors in AML with FLT3 mutation, as well as gemtuzumab ozogamicin therapy in patients with low and intermediate cytogenetic risk profiles. With the advent of the next generation sequencing technologies it is expected that new therapeutic targets will be identified. These insights will lead to a further individualization of AML therapy.     

The diagnostic utility of targeted gene panel sequencing in discriminating etiologies of cytopenia

The diagnostic utility of somatic mutations in the context of cytopenias is unclear: clonal hematopoiesis can be found in healthy individuals, patients with aplastic anemia (AA), clonal cytopenia of undetermined significance (CCUS) and myelodysplastic syndrome (MDS). We examined a cohort of 207 well-characterized cytopenic patients with a 640-gene next generation sequencing (NGS) panel and compared its diagnostic utility with a “virtual” 41 gene panel. The TET2, SF3B1, ASXL1, and TP53 were the most commonly mutated genes (frequency > 10%). Mutations in the 640-gene panel show high sensitivity (98.3%) but low specificity (47.6%) for diagnosis of MDS. Notably, mutations of splicing factors and genes in the RAS pathway are relatively specific to MDS. Furthermore, high variant allele frequency (VAF) predicts MDS: when the VAF is set at 20%, the positive predictive value (PPV) for MDS is 95.9%, with a specificity of 95.3%. The presence of two or more somatic mutations with ≥10% VAF showed a PPV of 95.2%. While the “virtual” 41-gene panel showed a mild decrease in sensitivity (95.7% vs 98.3%), 100% specificity was observed when either VAF was set at ≥20% (100% vs 95.3%), or two or more somatic mutations had VAFs ≥ 10%. Our study shows targeted gene panel sequencing improves the diagnostic approach and accuracy for unexplained cytopenia, with its high sensitivity and high PPV for MDS when applying VAF cutoffs. Furthermore, a 41-gene panel was shown to have at least comparable performance characteristics to the large 640-gene panel.     

FANCA D1359Y mutation in a patient with gastric polyposis and cancer susceptibility: A case report and review of literature

Gastric polyposis is a rare disease. Not all polyps progress to cancer. Monoallelic mutation in Fanconi anemia(FA) genes, unlike biallelic gene mutations that causes typical FA phenotype, can increase risks of cancers in a sporadic manner. Aberrations in the FA pathway were reported in all molecular subtypes of gastric cancer. We studied a patient with synchronous gastric cancer from gastric polyposis by conducting a 13-year long-term follow up. Via pathway-driven massive parallel genomic sequencing, a germline mutation at FANCA D1359Y was identified. We identified several recurrent mutations in DNA methylation(TET1, V873I), the β-catenin pathway(CTNNB1, S45F) and RHO signaling pathway(PLEKHG5, R203C) by comparing the genetic events between benign and malignant gastric polyps. Furthermore, we revealed gastric polyposis susceptible genes and genetic events promoting malignant transformation using pathway-driven targeted gene sequencing.     

Clinical utility of next‐generation sequencing in the diagnosis of hereditary haemolytic anaemias

Hereditary haemolytic anaemias are genetically and phenotypically heterogeneous disorders characterized by increased red cell destruction, with consequences ranging from innocuous to severe life‐threatening anaemia. Diagnostic laboratories endeavour to assist clinicians reach the exact patient diagnosis by using tests principally based on morphological and biochemical techniques. However, these routine studies may be inconclusive, particularly in newborn infants and when transfusions have recently been administered. Large numbers and size of the potentially involved genes also impose a practical challenge for molecular diagnosis using routine sequencing approaches. To overcome these diagnostic shortcomings, we have utilized next‐generation sequencing to provide a high‐throughput, highly sensitive assay. We developed a panel interrogating 28 genes encoding cytoskeletal proteins and enzymes with sequencing coverage of the coding regions, splice site junctions, deep intronic and regulatory regions. We then evaluated 19 samples, including infants with unexplained extreme hyperbilirubinaemia and patients with transfusion‐dependent haemolytic anaemia. Where possible, inheritance patterns of pathogenic mutations were determined by sequencing of immediate relatives. We conclude that this next‐generation sequencing panel could be a cost‐effective approach to molecular diagnosis of hereditary haemolytic anaemia, especially when the family history is uninformative or when routine laboratory testing fails to identify the causative haemolytic process.     

Fanca?/? hematopoietic stem cells demonstrate a mobilization defect which can be overcome by administration of the Rac inhibitor NSC23766

Fanconi anemia is a severe bone marrow failure syndrome resulting from inactivating mutations of Fanconi anemia pathway genes. Gene and cell therapy trials using hematopoietic stem cells and progenitors have been hampered by poor mobilization of HSC to peripheral blood in response to G-CSF. Using a murine model of Fanconi anemia (Fanca^−/− mice), we found that the Fanca deficiency was associated with a profound defect in hematopoietic stem cells and progenitors mobilization in response to G-CSF in absence of bone marrow failure, which correlates with the findings of clinical trials in Fanconi anemia patients. This mobilization defect was overcome by co-administration of the Rac inhibitor NSC23766, suggesting that Rac signaling is implicated in the retention of Fanca^−/− hematopoietic stem cells and progenitors in the bone marrow. In view of these data, we propose that targeting Rac signaling may enhance G-CSF-induced HSC mobilization in Fanconi anemia.     

Rapid detection of IDH2 (R140Q and R172K) mutations in acute myeloid leukemia

NADP-dependent enzyme isocitrate dehydrogenase (IDH) mutations, IDH1 and IDH2, have been described in acute myeloid leukemia (AML) using next generation sequencing approaches. IDH2 mutations are heterozygous; they alter a single arginine residue at position 140 or 172 and have distinct prognostic significance. The current detection methods of IDH2 mutations are laborious and time consuming as they require DNA sequencing. Herein, we report a new allele-specific oligonucleotide–polymerase chain reaction (ASO-PCR) method to detect the IDH2 mutations. Analysis of leukemic DNA samples from 120 AML patients enabled to identify IDH2 mutations in 22 cases which were confirmed by direct DNA sequencing. Of these, 17 harbored IDH2 (R140Q) and 5 IDH2 (R172K) mutations. Serial dilution experiments showed that the assay enable to detect mutations in 10^?3 dilutions. Our ASO-PCR method appears useful for routine diagnostic screening of these prognostically relevant alterations in AML and may be conveniently included in the diagnostic workup.     

BRCC3 mutations in myeloid neoplasms

Next generation sequencing technologies have provided insights into the molecular heterogeneity of various myeloid neoplasms, revealing previously unknown somatic genetic events. In our cohort of 1444 cases analyzed by next generation sequencing, somatic mutations in the gene BRCA1-BRCA2-containing complex 3 (BRCC3) were identified in 28 cases (1.9%). BRCC3 is a member of the JAMM/MPN+ family of zinc metalloproteases capable of cleaving Lys-63 linked polyubiquitin chains, and is implicated in DNA repair. The mutations were located throughout its coding region. The average variant allelic frequency of BRCC3 mutations was 30.1%, and by a serial sample analysis at two different time points a BRCC3 mutation was already identified in the initial stage of a myelodysplastic syndrome. BRCC3 mutations commonly occurred in nonsense (n=12), frameshift (n=4), and splice site (n=5) configurations. Due to the marginal male dominance (odds ratio; 2.00, 0.84–4.73) of BRCC3 mutations, the majority of mutations (n=23; 82%) were hemizygous. Phenotypically, BRCC3 mutations were frequently observed in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms and associated with -Y abnormality (odds ratio; 3.70, 1.25–11.0). Clinically, BRCC3 mutations were also related to higher age (P=0.01), although prognosis was not affected. Knockdown of Brcc3 gene expression in murine bone marrow lineage negative, Sca1 positive, c-kit positive cells resulted in 2-fold more colony formation and modest differentiation defect. Thus, BRCC3 likely plays a role as tumor-associated gene in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms.