Abstract
Recently, the identification of the gain of function mutation JAK2V617F delivered important insights into the pathogenesis of BCR/ABL negative myeloproliferative disorders (MPD). JAK2V617F is detectable in more than 90% of polycythemia vera (PV) patients (pts) and in approximately 50% of pts with essential thrombocythemia (ET) or primary myelofibrosis (PMF), representing the genetic hallmark of BCR/ABL negative disease. However, about 30% of MPD pts lack the JAK2V617F mutation and previous studies on ET and PV demonstrated that clonality exceeds the percentage of V617F mutated cells. These findings suggest that additional genetic alterations are involved in the pathogenesis of MPD, in both JAK2 mutated and unmutated pts. To identify novel genetic aberrations and to determine whether specific lesions are associated with disease phenotype, genomic DNA from granulocytes of 72 MPD pts classified according to the WHO criteria was analyzed using high-resolution, genome-wide microarray techniques [disease, number analyzed, JAK2 mutation status: PMF, n=14, 9/14; post-ET MF, n=5, 3/5; post-PV MF, n=5, 5/5; PV, n=37, 37/37; ET, n=11, 11/11]. In a first approach, all cases were investigated by comparative genomic hybridization to 8k arrays (array CGH) with an average probe spacing of less than 1 Mb. While no genomic imbalances were found in ET, 11% of PV pts (n=4) exhibited large (>10 Mb) deletions on 20q (n=2) or gains on 9p and 1q (n=1, each). In addition, small (<1 Mb) recurrent gains in 1q21.1 (n=2) and 22q11.23 (n=2) were identified. In MF pts the incidence of large genomic imbalances was 25% (n=6) with trisomy 9 (n=3) being the most frequent aberration followed by loss of 20q, 5q, and 13q in single cases. Furthermore, in one pt with post-PV MF small genomic losses in 17q11.2 (2 Mb) and 17p13.2 (0.8 Mb) were identified harbouring NF1 but not TP53. Deletion of the NF1 allele without concomitant loss of TP53 was confirmed by FISH. To further increase resolution and to investigate the role of uniparental disomy (UPD), single nucleotide polymorphism (SNP) analysis using the Affymetrix 250k Nsp SNP array was performed in all MF cases. Copy number estimation and loss of heterozygosity probability were analyzed using a set of 117 remission samples from acute myeloid leukemia pts as a common reference. SNP analysis confirmed all anomalies detected by array CGH. In addition, SNP analysis revealed small genomic losses (1.6–2.6 Mb) in 1q21.2 (n=3), 5q13.2, and 3p13 (n=1, each), and in one secondary MF pt another microdeletion in 17q11.2 (1.2 Mb). UPDs recurrently affected 9p (n=5) in a region harbouring the JAK2 locus. In single cases, large UPDs of 1q (25 Mb), 2p (14 Mb), 5q (4 Mb), 6p (11 Mb), and 7q (11 Mb) were identified. Of note, all JAK2V617F mutated post-PV and post-ET MF cases exhibited 9p abnormalities represented either by trisomy 9 or UPD of 9p. In conclusion, using a combined microarray approach we were able to detect novel submicroscopic alterations in addition to known abnormalities. Parallel analysis of both techniques clearly demonstrated the superiority of array-SNP mapping. Further analyses on larger pt populations and correlation with global gene expression data will facilitate the identification of disease-related genes that are involved in the pathogenesis of BCR/ABL negative MPD.
A case report and mechanism analysis of a normal phenotype mosaic 47, XXY complicated by paternal iUPD (9) who had a normal PGD result
