Characterization of Chromosome 20q Deletions In Myeloproliferative Neoplasms Using Microarray Karyotyping and Next-Generation Sequencing

Abstract 4099

Deletions on chromosome 20q (del20q) are among the most frequent cytogenetic aberrations in myeloid disorders. Mapping of common deleted regions (CDRs) has the potential to link often large deletions to single gene defects. In the past years 20q CDRs from several patient cohorts have been reported, most of them combining patients with different myeloid malignancies, such as myeloproliferative neoplasms (MPN), chronic myeloid leukemia and myelodysplastic syndrome. However, it is not clear whether the same genes within del20q are relevant for pathogenesis in the different myeloid diseases. We aimed to delineate a common deleted region exclusively based on a substantial number of MPN patients. Using a copy number assay, we screened for del20q in a total of 822 MPN patients combined from three independent MPN patient cohorts. Del20q was present in granulocyte DNA from 11 patients (1.1%). We consequently mapped the 11 deletions using Affymetrix 6.0 microarray karyotyping, resulting in a common deleted region of 6.4 Mb size, comprising 82 transcribed genes. In a next step, we aimed to investigate the remaining undeleted allele for the presence of somatic mutations. We applied a next generation sequencing approach and sequenced 774 coding exons within our del20q CDR in 11 patients with del20q. We prepared a DNA pool containing equal amounts of granulocyte DNA from each patient and amplified 774 exons in separate PCR reactions. The PCR reactions were pooled, concatemerized by ligation, refragmented, and prepared for a single-end 36bp sequencing read on a Genome Analyzer IIx (Illumina). The single-end read delivered a coverage between 1000- and 5000-fold per exonic base. We identified five putative variants in the genes SGK2, SEMG1, SEMG2, WFDC9 and SLC13A3 that were non-synonymous and were not annotated in any of the public SNP databases. Further validation in the single patients using classical capillary sequencing identified heterozygous calls in SGK2, SEMG1 and SEMG2 as false positives. The putative variants in WFDC9 (position chr20:43670771; G/A; NCBI36/hg18) and SLC13A3 (position chr20:44654462; C/G; NCBI36/hg18) could be confirmed in granulocyte DNA from a subset of the del20q patients. However, the variants could also be detected in DNA from a control non-myeloid tissue indicating their germ line origin. The variants were present in healthy individuals at variable frequencies, identifying them as novel SNPs presumably not involved in MPN pathogenesis. From the absence of mutations on the undeleted allele in patients with del20q we can conclude that haploinsufficiency is the most likely functional consequence of tumor suppressor inactivation within the del20q CDR in myeloproliferative neoplasms.