DNA Methylation Differences in Twins Discordant for Adolescent/Young Adult Hodgkin Lymphoma
Authors: Wang Jun , Hwang Amie , Weisenberger Dan , Van Den Berg David J. , Cortessis Victoria K. , Epeldegui Marta , Martinez-Maza Otoniel , Triche Timothy Junius , Hamilton Ann S. , Nathwani Bharat N. , Conti David V. , Horvath Steve , Mack Thomas M. , Cozen Wendy 
Published: October 2019

Abstract
Hodgkin lymphoma (HL) is derived from germinal center B-lymphocytes, has a peak incidence in adolescents and young adults (AYA) in developed countries and results in substantial treatment-related late effects. We previously identified a SNP (rs1860661G) in the TCF3 gene, inversely associated with HL risk, that is also mutated in tumor cells. The protein encoded by TCF3 is responsible for maintaining the B-cell program, which is downregulated in HRS cells; therefore higher expression levels may be protective.1 DNA methylation is associated with repression of gene expression and may thereby play a role in HL pathogenesis.
We extracted DNA from blood samples obtained from HL survivors and their unaffected monozygotic MZ twins (4 female and 5 male twin pairs discordant for AYA HL). Average age at blood draw was 62 years, and average time since diagnosis of the case was 34 years. We also extracted DNA from blood samples from 25 unaffected MZ twin pairs as control pairs. DNAs were bisulfite converted and DNA methylation quantified using the Illumina Infinium HumanMethylation27 BeadChip array, which scores methylation levels as β values. We compared average DNA methylation β value differences at the TCF3 locus cg16524139 (near rs1860661) in HL survivors and their unaffected co-twins using a paired t-test. We also examined the TCF3 germline genotype by DNA methylation level rank in HL-discordant pairs. We also computed DNA methylation age, a global measure of physiological aging, using a validated algorithm2 and compared the result in the HL survivor to that of each corresponding unaffected co-twin using a paired t-test.
We found a small but significantly higher DNA methylation level at the TCF3 locus in HL survivors compared to their unaffected co-twins (p=0.05), but no difference between the members of healthy MZ twin pairs (p=0.81). The protective allele (G) was more prevalent in the 6 pairs with higher DNA methylation level in HL survivors (33%), compared to the 3 pairs with higher levels in unaffected co-twins (17%). This pattern would be expected if methylation at the cg16524139 were to diminish TCF3 expression related to a protective rs1860661G variant.
HL survivors had an average DNA methylation age of 64.1 years compared to 61.3 years in their unaffected MZ co-twins (p=0.04).
Differential DNA methylation in HL survivors and their unaffected twins suggests a role for DNA methylation in HL, possibly as a result of treatment or disease.
1. Cozen W, Timofeeva MN, Li D, Diepstra A, Hazelett D, Delahaye-Sourdeix M, et al.: A meta-analysis of Hodgkin lymphoma reveals 19p13.3 TCF3 as a novel susceptibility locus. Nat Comm 25:3856. doi: 10.1038/ ncomms 4856, 2014
2. Horvath S: DNA methylation age of human tissues and cell types. Genome Biol, 14:R115 doi:10.1186/gb-2013-14-10-r115, 2013.

Disclosures
No relevant conflicts of interest to declare.