High Resolution SNP Arrays and Correlation with Expression Arrays Identify Novel Genomic Aberrations and Potential Disease Genes in Polycythemia Vera and Other Myeloproliferative Disorders.

Polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF) are all associated with the JAK2V617F gain of function mutation as well as with gross cytogenetic anomalies. To determine whether specific genomic copy number alterations are associated with disease phenotype we subjected genomic DNA from the granulocytes of 24 patients with the JAK2V617F mutation with PV, ET or IMF, to analysis using high resolution Affymetrix 250K Nsp SNP arrays with an average probe spacing of 12Kb. Firstly this analysis confirmed previously reported anomalies in MPD patients such as trisomy 9 (5/24), trisomy 8 (1/24), and deletion of 20q (2/24) indicating the robustness of the technique. Amplification of 9p in a region harboring the JAK2 locus and 17q12.31 were the most common shared alterations in the MPDs while 20q deletion was only seen in ET patients (p<0.05 vs PV and IMF). We further identified large alterations (>1Mb) found only in single patients including (+1q, del 2p13.3, del 5q23.1-q23.2, +5p, several deletions of chromosome 6, del 7q35, del 9p21.1-q32), suggesting sporadic genomic instability. This analysis also identified several recurrent large alterations including del1p21.3 (3/24 patients), +3q21.3-q22.1 (3/24), +9p24 (6/24), +10q22.3-q23.1 (3/24), and +11p15.5 (4/24). By removing genomic Copy Number Polymorphisms (CNPs) obtained from a set of 35 normal people from the HapMap project, we detected a number of novel small recurrent alterations (<1Mb): a 588Kb gain on 4p16.1 (4/24 patients); a 50Kb deletion on 6q14.2 (6/24); a 700Kb gain on 10q23.31 (4/24); and a 180Kb gain on 17q12.31 (9/24). The boundaries of altered regions were precisely defined by the SNP data and the genes within these segments were curated and used to query our separate dataset of expression array data from CD34+ cells of 9 PV patients and 8 normal controls to determine whether regions of chromosome gain or loss may contain disease-associated genes. 21 genes in recurrently amplified regions showed a significantly elevated expression level in PV patients, including NFIB (nuclear factor I/B) on 9p24 where JAK2 resides and the VAV2 oncogene on 9q34. 7 genes in recurrently deleted regions had significantly depressed expression levels in PV including TOP1 on 20q. The large recurrent gain on 11p identified by SNP arrays correlated with a consistent upregulation of SIRT3 at 11p15.5 in the PV dataset. Similarly, the large recurrent DNA deletion on 1p correlated with a consistent decreased expression of the DPH5 gene. DPH5 is 1 of 5 genes (SAMHD1, ADA, ASS, CRAT, DPH5) deregulated both in copy number and expression involved in nucleic acid metabolism. Moreover, there were 6 genes in these regions (METTL4, DHX35, TOP1,SIRT3, ZBP1, NFIB) involved in DNA replication or transcriptional regulation. The correlation of amplified or lost genomic segments determined from the study of one group of patients with deregulated gene expression from an independent data set suggests there may be selective pressure in MPD to alter expression of a subset of genes other than JAK2. In addition loss of specific chromosome segments as in ET could play a role in modulating the phenotype of JAK2V617F associated disease.