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Int J Mol Epidemiol Genet 2012;3(3):213-227

Original Article
Promoter methylation of candidate genes associated with familial testicular

Lisa Mirabello, Christian P Kratz, Sharon A Savage, Mark H Greene

Clinical Genetics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of
Health, Department of Health and Human Services, Bethesda, Maryland, USA. All authors have contributed to, read and
approved the final manuscript for submission.

Received July 10, 2012; accepted July 29, 2012; Epub August 31, 2012; Published September 15, 2012

Abstract: Recent genomic studies have identified risk SNPs in or near eight genes associated with testicular germ cell tumors
(TGCT). Mouse models suggest a role for Dnd1 epigenetics in TGCT susceptibility, and we have recently reported that
transgenerational inheritance of epigenetic events may be associated with familial TGCT risk. We now investigate whether
aberrant promoter methylation of selected candidate genes is associated with familial TGCT risk. Pyrosequencing assays
were designed to evaluate CpG methylation in the promoters of selected genes in peripheral blood DNA from 153 TGCT
affecteds and 116 healthy male relatives from 101 multiple-case families. Wilcoxon rank-sum tests and logistic regression
models were used to investigate associations between promoter methylation and TGCT. We also quantified gene product
expression of these genes, using quantitative PCR. We observed increased PDE11A, SPRY4 and BAK1 promoter methylation,
and decreased KITLG promoter methylation, in familial TGCT cases versus healthy male family controls. A significant upward
risk trend was observed for PDE11A when comparing the middle and highest tertiles of methylation to the lowest [odds ratio
(OR) =1.55, 95% confidence intervals (CI) 0.82-2.93, and 1.94, 95% CI 1.03-3.66], respectively; Ptrend=0.042). A significant
inverse association was observed for KITLG when comparing the middle and lowest tertiles to the highest (OR=2.15, 95% CI
1.12-4.11, and 2.15, 95% CI 1.12-4.14, respectively; Ptrend=0.031). There was a weak inverse correlation between promoter
methylation and KITLG expression. Our results suggest that familial TGCT susceptibility may be associated with promoter
methylation of previously-identified TGCT risk-modifying genes. Larger studies are warranted. (IJMEG1207002).

Keywords: Promoter methylation, testicular germ cell tumors, familial testicular cancer, epidemiology, candidate gen

Address all correspondence to:
Dr. Mark H Greene
Clinical Genetics Branch
Division of Cancer Epidemiology and Genetics
National Cancer Institute, NIH
6120 Executive Blvd., EPS/7032
Rockville, MD 20892.
Tel: 301.594.7641; Fax: 301.496.1854
E-mail: greenem@mail.nih.gov