Loss of Imprinting of DLK1 Due to Promoter Hypermethylation Accounts Frequently for Its Overexpression in AML.
Authors: Khoury Haytham , Wu Samantha , Suarez-Saiz Fernando , Jensen Charlotte H. , Teisner Børge , Minden Mark D. 
Published: April 2020

Abstract
DLK1 is an imprinted gene paternally expressed that encodes a transmembrane protein of the EGF superfamily. It contains an extracellular domain, a cleavage site, a transmembrane domain and a short cytoplasmic tail. The ectodomain of DLK1 may be cleaved and released into the extracellular matrix as a soluble molecule referred to as fetal antigen-1 (FA1). Due to structural similarity with the IGF2-H19 imprinted region, it has been hypothesized that DLK1-Meg3 is regulated in a similar manner. In this case loss of imprinting (LOI) of DLK1 would be caused by hypermethlation of the 3′-intergenic differentially methylated region (IG-DMR) of the maternal allele. As this is also the upstream regulatory region of Meg3, expression of maternal Meg3 would be reduced. Recently, it was reported that DLK1 is overexpressed in myeloid malignancies. To better characterize this overexpression in AML and identify its mechanism, we studied DLK1 expression by real time RT-PCR in 92 patients (pts), quanitified serum FA1 by ELISA, analyzed whether loss of imprinting (LOI) could account for its upregulation by studying the expression of 3 single nucleotide polymorphisms (SNPs) (rs#1802710, rs#2295660, rs#1058009) and their allelic expression in 16 AML pts and finally quantified by mass spectrometry the methylation pattern of 7 CpG islands located within the imprinted region on 14q32 in 3 healthy individuals and 8 AML pts. The CpG islands analyzed were located at Meg3 promoter (3 islands), the IG-DMR (1 island), DLK1 promoter (1 island) and DLK1 exons (2 islands). DLK1 overexpression was noticed in 80% of AML. Pts with trilineage dysplasia (TLD) and APL showed consistently higher DLK1 transcript. FA1 was increased in 20% of AML all of which were found to be either AML with TLD or APL. All cases with high FA1 expressed the DLK1 isoform that contains the extracellular domain and the cleavage site, whereas the cases with normal FA1 levels expressed shorter isoforms that lack the cleavage site. Informative SNPs were found in 11 cases. In 10, the SNP was at the location of rs#1802710 and in one at rs#1058009. Analysis for DLK1 transcripts revealed biallelic expression in 8 pts (72%). Cases with biallelic expression showed higher DLK1 transcripts than monoallelic ones (p=0.001). Interestingly, the quantitative methylation analysis revealed that pts with biallelic expression did not constantly have hypermethylation of the IG-DMR or Meg3 promoter but a distinctive methylation pattern of the DLK1 promoter. While monoallelic pts showed a pattern similar to healthy individuals with overall methylation around 49%, bialllelic patients had hypermethlytion of the DLK1 promoter with overall methylation of (87%). The IG-DMR was hemimethylated in all cases. Hypermethylation of Meg3 promoter was seen in 2 pts with positive correlation with Meg3 transcript level. Our results indicate DLK1 is overexpressed in a large proportion of AML. FA1 is particularly elevated in AML with TLD and APL. Whether FA1 level could be clinically useful in such cases is still to be determined. Moreover DLK1 overexpression in AML is frequently due to LOI. Finally, The DLK1-Meg3 imprinted region has a distinctive mode of regulation in which each gene is regulated through the hypermethylation of its respective promoter.