In myelodysplastic syndrome (MDS) chromosomal abnormalities play an important diagnostic and prognostic role, pointing towards possible disease-causing pathways. However, using traditional metaphase cytogenetics (MC), chromosomal lesions can be found in only about 50% of patients;higher resolution methods may allow for detection of cryptic chromosomal lesions in patients with normal and abnormal MC. Detection of such defects could facilitate identification of novel therapeutic targets, aid diagnosis and improve current prognostic schemes by explaining phenotypic heterogeneity within established MDS subtypes. Single nucleotide polymorphism arrays (SNP-A) are a new method for high resolution karyotyping and detection of unbalanced DNA defects including somatic uniparental disomy (UPD). We have demonstrated that somatic UPD is very frequent in MDS and hypothesized that shared regions of UPD may point towards mutations of genes involved in the development of MDS. In particular areas of somatic UPD have been linked to the presence of biallelic mutations, such as Jak2 (UPD9p) and Ftl3 (UPD13q). Based on this theory we mapped invariant UPD and deletions in a large group of patients with MDS and found that UPD11q was particularly frequent in patients with chronic myelomonocytic leukemia. By sequencing the c-Cbl gene, an E3 ubiquitin ligase in this chromosomal region, a mutation involving a critical part of this gene was identified. In this proposal we plan to test the clinical utility of SNP-A karyotyping and establish its impact on prognosis, clinical phenotype and the discovery of new molecular targets in MDS. In SA1 we will analyze paired samples (bone marrow and sorted CD3+ cells as a clonal control) from patients with MDS using the Affymetrix 6.0 SNP array. New invariant clonal lesions, in particular somatic UPD, will be identified, mapped and their impact on clinical phenotypes, survival and progression analyzed. The influence of newly detected lesions will be assessed in the context of IPSS to improve prognostic accuracy. In SA2, as a proof of concept that recurrent somatic UPD points towards important mutations, we will analyze in detail patients with UPD11q and c-Cbl mutations and determine the frequency and clinical outcomes of this mutation associated with this lesion. We will perform in vitro studies to delineate the effect of c-Cbl mutations on tyrosine kinase receptors and Src family kinases and explain whether pathologic mechanisms are mediated by mutant c-Cbl. For example, we will compare functional consequences of ring finger mutant knock-in vs. c-Cbl protein knockdown. Improved cytogenetic diagnostics using SNP-A karyotyping may contribute to a better understanding of the molecular pathogenesis of MDS and has the potential of identification of genes involved in the pathogenesis of MDS, perhaps pointing towards therapeutic targets for development of molecular therapies. Clinically, SNP-A-based karyotyping may complement cytogenetic diagnosis and, through the detection of previously cryptic chromosomal lesions (including UPD), may allow for better prognostic assignment.

Public Health Relevance

Myelodysplastic syndromes (MDS) is a heterogeneous group of bone marrow failure states characterized by dysplastic hematopoiesis, deficient blood cell production and a propensity to progression to acute myelogenous leukemia (AML);this heterogeneity has greatly impeded investigations into the molecular pathogenesis and potential therapies for these diseases. We propose that single nucleotide polymorphisms arrays (SNP-A) can be applied, complementary to metaphase cytogenetics for the identification of chromosomal abnormalities, including a newly recognized class of lesion, somatic uniparental disomy. We will precisely map these lesions, identifying genes that may play a role in the disease and potentially act as targets of therapy.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL098511-03
Application #
8197800
Study Section
Hematopoiesis Study Section (HP)
Program Officer
Di Fronzo, Nancy L
Project Start
2010-01-29
Project End
2013-11-30
Budget Start
2011-12-01
Budget End
2012-11-30
Support Year
3
Fiscal Year
2012
Total Cost
$392,500
Indirect Cost
$142,500
Name
Cleveland Clinic Lerner
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
135781701
City
Cleveland
State
OH
Country
United States
Zip Code
44195
Kurtovic-Kozaric, A; Przychodzen, B; Singh, J et al. (2015) PRPF8 defects cause missplicing in myeloid malignancies. Leukemia 29:126-36
Jankowska, Anna M; Makishima, Hideki; Tiu, Ramon V et al. (2011) Mutational spectrum analysis of chronic myelomonocytic leukemia includes genes associated with epigenetic regulation: UTX, EZH2, and DNMT3A. Blood 118:3932-41
Huh, Jungwon; Tiu, Ramon V; Gondek, Lukasz P et al. (2010) Characterization of chromosome arm 20q abnormalities in myeloid malignancies using genome-wide single nucleotide polymorphism array analysis. Genes Chromosomes Cancer 49:390-9