Definition of Commonly Deleted Regions and Identification of Candidate Genes of Chromosome 20 in Myeloid Malignancies Using Genome-Wide Single Nucleotide Polymorphism (SNP) Array.
Authors: Huh Jungwon , Tiu Ramon V. , Jiang Ying , Gondek Lukasz P , O’Keefe Christine , Theil Karl S. , Maciejewski Jaroslaw P 
Published: April 2020

Abstract
Deletion of the long arm of chromosome 20 (20q) is one of the more common recurring abnormalities associated with myeloid malignancies. The previous studies utilizing FISH and microsatellite PCR attempted to define the commonly deleted regions (CDR) of chromosome 20, but due to the limited resolution of conventional technologies, the precise delineation of such a region was difficult. Among our cohort of 1162 patients with myeloid malignancies, 3% of the patients (34/1162) showed 20q deletion (27 MDS, 2 AML, 5 MDS/MPD). The incidence of 20q deletion as a sole cytogenetic abnormality was 44% (15/34). To decrease the possible size of CDR and better delineate the boundaries of areas of copy-neutral LOH, we analyzed a subcohort of 532 of the patients using high density single nucleotide polymorphism microarray (SNP-A) with 250K and SNP 6.0 including 1.8x106 markers. As a result, we were able to identify chromosome 20q deletion in 21 patients (2 RARS, 3 RCMD, 6 RAEB, 4 MDS-U, 2 AML, 2 MPD, 2 MDS/MPD), while metaphase cytogenetics in the same cohort of patient revealed only 14 cases with 20q deletion. Although the size and breakpoints of the deleted regions varied, they showed considerable overlap. We have defined 2 CDRs on 20q shared by all patients with deletion of chromosome 20q. CDR1 (present 18/21 patients) spanned 2.5Mb and is located between bands 20q11.23 and 20q12 (34,968,632-37,417,814), while CDR2 (present in 21/21 patients) encompassed 1.8Mb within bands 20q13.12 (43,636,105-45,417,399). We found 3 patients (1 RAEB and 2 MDS-U) whose lesions included CDR2 only and without CDR1. Two patients with MDS-U had 20q deletion as a sole abnormality and belong to the low and intermediate-1 risk group, respectively. One RAEB patient who only had CDR2 had duplication of the short arm of chromosome 20 and progressed to AML. As most of the lesions were interstitial, the terminal regions of chromosome 20q and the short arms of chromosome 20 were retained or even duplicated in 8 cases. For RARS and MPD, MDS/MPD patients, there were no patients that have duplicated regions. CDR1 and CDR2 spanned 31 and 33 genes known to be expressed in hematopoietic tissues. We selected 3 candidate genes (retinoblastoma like protein 1), CTNNBL1 (beta-catenine like protein 1) and TP53RK (TP53 regulating kinase) and we did not find any inactivating mutations when the genes were sequenced. Theoretically, deletions result in haploinsufficiency but underexpression may be due to methylation silencing which can result in events similar to a functional knockout if it affects genes in the deleted area. Using 27K methylation arrays, we studied methylation of genes corresponding to CDR1 and CDR2 (10 patients with 20q deletion and 53 patients without 20q deletion). CpG islands that were hypermethylated within these CDR regions relative to controls were studied and there was no hypermethylated CpG site in &gt;25% of patients for CDR1. There were 4 hypermethylated CpG sites (MMP9, SLC12A5, SLC13A3 and CHD22) in &gt;25% of patients for CDR2. It is still unclear if SLC12A5, SLC13A3 and CDH22 are associated with cancer. Among 10 patients with 20q CDR2 deletions, we found 3 patients with hypermethylated CpG sites within MMP9 gene (1 AML, 2 MDS-U). Based on the assumption that methylation can results in deficient gene expression analogous to haploinsufficiency and hypermethylation of genes corresponding to the CDRs in patients without deletion could result in a similar phenotype. Among 53 patients without deletion of 20q, we found hypermethylated MMP9 genes in 42% (22/53) of patients (13 AML, 1 RA, 4 RCMD, 1 RAEB, 1 5q- syndrome, 1 MDS/MPD). Among 10 patients with 20q deletion, there was no evidence of hypermethylation in our candidate genes of RBL1, CTNNBL1 and TP53RK genes. For CTNNBL1 gene, 25% (13/53) of patients without 20q deletion showed hypermethylation (8 AML, 1 RAEB, 3 RCMD, 1 MDS-U). In conclusion, our results suggest that CDR1 and CDR2 regions of chromosome 20 which were delineated by SNP-A may be associated with pathogenesis of myeloid malignancies and candidate genes of RBL1, CTNNBL1, TP53RK and MMP9 need to be further studied.