Molecular Genetics of Myeloproliferative Disorders

Prof. Dr. med. Konstanze Döhner
Dr. med. Frank Stegelmann

Department of Internal Medicine III

University Hospital of Ulm

Albert-Einstein-Allee 23

89081 Ulm

Germany

E-Mail: konstanze.doehner[at]uniklinik-ulm.de

E-Mail: frank.stegelmann[at]uniklinik-ulm.de
 

 
Project Leader

Dr. Frank Stegelmann

Technicians

Susanne Kuhn
Anita Szmaragowska

(A)
Molecular diagnosis of Myeloproliferative Disorders (MPD)

Myleoproliferative Disorders (MPD) comprise a large variety of chronic leukemias with myeloid origin. Among them, the molecular cause of chronic myeloid leukemia (CML) is defined most precisely. Typical CML is represented by the activating fusion protein BCR/ABL that results from the recurrent chromosome translocation t(9;22). At diagnosis, the most frequent BCR/ABL fusion transcripts that occur in CML (e1a2, b2a2 and b3a2) are detectable by Reverse transcriptase- (RT-) Multiplex-PCR. In the course of disease, Minimal residual disease- (MRD-) monitoring is predictive for the clinical outcome. Therefore, different PCR-based techniques were established in our lab, allowing both primary diagnosis and monitoring of treatment response in BCR/ABL positive CML patients.

More recently, genetic alterations were also identified in BCR/ABL negative MPDs. In particular, the activating JAK2 V617F mutation has improved the understanding of BCR/ABL negative MPDs. JAK2 V617F has been discovered as a single-site, clonal, gain-of-function mutation in myeloid cells from the majority of patients with Polycythemia vera (PV, >95%), Essential thrombocythemia (ET, 50-60%) and Idiopathic myelofibrosis (MF, 60%). Though less frequent, JAK2 Exon 12 mutations (e.g JAK2 K539L) occur in V617F negative PV patients, and the MPL W515L point mutation is present in about 5% of MF and ET patients, respectively. Additionally, the FIP1L1/PDGFRA fusion gene and the KIT D816V are detectable in a subset of patients suffering from Hypereosinophilic syndrome (HES) and Systemic mastocytosis (SM), respectively. For all these genetic alterations, molecular assays based on PCR, DNA sequencing and DHPLC technique were established in our lab. Moleculargenetic results help clinicians in terms of diagnosis, classification and treatment of MPD patients.

(B)
Prognostic impact of the JAK2 V617F mutation

In the mouse model, the JAK2 V617F mutation leads to a myeloproliferative phenotype resembling PV because of markedly elevated erythrocytes and granulocytes. To date, little is known on the pathogenetic and prognostic value of JAK2 V617F in patients with MPD. It is still unclear, whether the mutation increases the risk of vascular events, myelofibrosis and transormation into acute myeloid leukemia. The aim of our study is to analyze a large cohort of BCR/ABL negative MPD patients for the presence of JAK2 V617F. Moreover, the proportion of the mutant allele (homozygous vs. heterozygous) is going to be determined in JAK2 V617F mutated patients by quantitative Real-time PCR technique. Samples from primary diagnosis and during follow-up (3-6 months interval) will be analysed with the intention to explore the prognostic impact of JAK2 V617F in ET, PV and MF by correlating clinical and molecular data.

(C)
Identification of genomic imbalances in BCR/ABL negative MPDs using DNA microarrays

Recent findings show that the proportion of clonal cells in ET, PV and MF exceed the percentage of JAK2 V617F positive cells in mutated patients. This finding is in line with the fact that approximately one third of the patients lack mutations in the JAK2 and MPL gene. Therefore, mutations in so far unknown genes are supposed to contribute to leukemogenesis. The technique of Comparative genomic hybridization to microarrays (Array CGH or Matrix CGH) is one approach that allows genome-wide screening for genomic imbalances at high resolution and thus facilitating the identification of new chromosomal regions harboring potential disease-related genes and gene mutations. Using an 8.0 k platform consisting of 8051 different bacterial artificial chomosome (BAC) or P1-derived artificial chromosome (PAC) vectors with an average resolution of about 1 Mb, we are currently investigating DNA from ET, PV and MF patients. Moreover, we have established the high resolution SNP chip technology (Affymetrix® platform) for genome-wide exploration of gains, losses and uniparental disomies (UPD) in the pathogeneis of BCR/ABL negative MPDs. Newly identified hot spot regions serve as a starting point for further studies to identify disease-relevant genes.

• Cell isolation techniques (Ficoll Paque® centrifugation, MACS® Cell Separation)
• DNA/RNA preparation
• PCR techniques including quantitative Real-time PCR (TaqMan®, LightCycler®)
• DNA sequencing
• Sequence analysis
• DHPLC- (Wave®-) based mutation screening
• Array-CGH
• LOH analysis (Affymetrix® SNP microarrays)
• Gene expression profiling using different platforms (cDNA, Affymetrix®)

• Else Kröner Fresenius Stiftung

Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ, Boggon TJ, Wlodarska I, Clark JJ, Moore S, Adelsperger J, Koo S, Lee JC, Gabriel S, Mercher T, D'Andrea A, Fröhling S, Döhner K, Marynen P, Vandenberghe P, Mesa RA, Tefferi A, Griffin JD, Eck MJ, Sellers WR, Meyerson M, Golub TR, Lee SJ, Gilliland DG:
Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis.
Cancer Cell. 2005 Apr;7(4):387-97.

Campbell PJ, Griesshammer M, Döhner K, Döhner H, Kusec R, Hasselbalch HC, Larsen TS, Pallisgaard N, Giraudier S, Le Bousse-Kerdiles MC, Desterke C, Guerton B, Dupriez B, Bordessoule D, Fenaux P, Kiladjian JJ, Viallard JF, Briere J, Harrison CN, Green AR, Reilly JT:
V617F mutation in JAK2 is associated with poorer survival in idiopathic myelofibrosis.
Blood. 2006 Mar 1;107(5):2098-100.

Fröhling S, Lipka DB, Kayser S, Scholl C, Schlenk RF, Döhner H, Gilliland DG, Levine RL, Döhner K:
Rare occurrence of the JAK2 V617F mutation in AML subtypes M5, M6, and M7.
Blood. 2006 Feb 1;107(3):1242-3.

Döhner K, Du J, Corbacioglu A, Scholl C, Schlenk RF, Döhner H:
JAK2V617F mutations as cooperative genetic lesions in t(8;21)-positive acute myeloid leukemia.
Haematologica. 2006 Oct 17; [Epub ahead of print]

Prof. Dr. Gary Gilliland

Harvard Medical School, Boston, USA

Prof. Dr. Heinz Gisslinger / Prof. Dr. Robert Kralovics

Department of Internal Medicine / Molecular Genetics, AKH Vienna, Austria

Prof. Dr. Andreas Reiter

Department of Internal Medicine, University of Heidelberg-Mannheim, Germany