Subphenotype meta-analysis of testicular cancer genome-wide association study (GWAS) data suggests a potential role for Fox family RNA-binding proteins in cryptorchidism susceptibility
Yanping Wang, PhD1, Erin Crowgey, PhD1, Marcella Devoto, PhD2, Katharine Nathanson, MD3, Katherine McGlynn, PhD4, Clare Turnbull, MD, PhD, MA, MSc, MRCP5, Zhaoming Wang, PhD6, Stephen J. Chanock, MD4, Julia Barthold, MD7.
1Nemours Biomedical Research/Alfred I. duPont Hospital for Children, Wilmington, DE, USA, 2Division of Genetics, Children's Hospital of Philadelphia and Departments of Biostatistics and Epidemiology, and Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA, 3Department of Medicine, Division of Translational Medicine and Human Genetics,Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA, 4Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Department of Health and Human Services, Bethesda, MD, USA, 5Division of Genetics and Epidemiology, Institute of Cancer Research, London, United Kingdom, 6St. Jude Children's Research Hospital, Department of Computational Biology, Memphis, TN, USA, 7Nemours Biomedical Research and Division of Urology, Alfred I. duPont Hospital for Children, Wilmington, DE, USA.
BACKGROUND: Cryptorchidism (undescended testis, UDT) is a strong risk factor for testicular germ cell tumor (TGCT). To better define UDT genetic susceptibility, we performed a subphenotype analysis of existing genome-wide association study (GWAS) data from the Testicular Cancer Consortium (TECAC). We compared these TECAC GWAS data with our previously published clinical (UDT GWAS) and animal model (UDT rat gubernaculum transcriptome) data.
METHODS: The TGCT GWAS results came from studies conducted by the National Cancer Institute (NCI), United Kingdom (UK) and University of Pennsylvania (Penn). We completed UDT subphenotype case-case (TGCT/UDT vs. TGCT/non-UDT) association analyses for each of these 3 cohorts followed by a meta-analysis comprising a total of 129 TGCT/UDT and 1771 TGCT/non-UDT cases. We then compared these TECAC meta-analysis data with our previously published UDT GWAS meta-analysis comprising 844 UDT and 2718 unaffected boys. We mapped suggestive signals (p<0.001) to genes using Ingenuity Pathway Analysis (IPA®), after excluding those with minor allele frequency (MAF) <0.05 from further analysis to avoid biased effect estimates for rarer SNPs due to the small TGCT/UDT sample size. We compared associated pathways and enriched gene categories common to both the TECAC and UDT analyses after Benjamini-Hochberg multiple testing correction.
RESULTS: We found suggestive intragenic signals in 76 genes common to both the TECAC and UDT analyses. Two of these are the paralogs RBFOX1 and RBFOX3, which encode RNA-binding proteins (RBPs) targeting (U)GCATG-containing transcripts. Of note, multiple stronger signals (p=e-09) within RBFOX1 that emerged in the TECAC analysis were excluded due to low (<0.02) MAF. Experimentally or computationally predicted RBFOX target genes are highly overrepresented among suggestive intragenic signals for both the UDT (165 of 628 (26%), p=2e-35) and TECAC (174 of 711 (24%), p=3e-35) analyses, and a majority of the genes common to both analyses (42 of 76 (55%), p=1e-20) are predicted RBFOX targets. Interestingly, lists of differentially expressed genes that we identified in prior developmental transcriptomic studies of the fetal rat gubernaculum during normal development (p=1e-05) and in the cryptorchid LE/orl rat strain (p=2e-13) are also enriched in predicted RBFOX targets.
CONCLUSIONS: Although the number of TGCT/UDT cases was small in the TECAC analysis, the overlap that we observed with our more well-powered UDT GWAS provides suggestive evidence of association of Fox family RBPs with UDT. Moreover, we found that RBFOX targets are highly enriched among suggestive signals from these two independent UDT and TECAC GWASs, and among transcripts that are differentially expressed during development in normal and cryptorchid rat fetal gubernaculum. Notably, UDT is associated with a 16p13.2 deletion involving only RBFOX1, and the RBFOX1 protein regulates sex determination in c. elegans and production of calcitonin gene-related peptide (CGRP), an alternatively spliced protein product linked to testicular descent. These complementary human and animal model data support a potential regulatory role for Fox family proteins in UDT susceptibility.
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