A genome-wide association study of men with symptoms of testicular dysgenesis syndrome and its network biology interpretation

Marlene D Dalgaard, Nils Weinhold, Daniel Edsgärd, Jeremy D Silver, Tune H Pers, John E Nielsen, Niels Jørgensen, Anders Juul, Thomas A Gerds, Aleksander Giwercman, Yvonne L Giwercman, Gabriella Cohn-Cedermark, Helena E Virtanen, Jorma Toppari, Gedske Daugaard, Thomas S Jensen, Søren Brunak, Ewa Rajpert-De Meyts, Niels E Skakkebæk, Henrik Leffers, Ramneek Gupta, Marlene D Dalgaard, Nils Weinhold, Daniel Edsgärd, Jeremy D Silver, Tune H Pers, John E Nielsen, Niels Jørgensen, Anders Juul, Thomas A Gerds, Aleksander Giwercman, Yvonne L Giwercman, Gabriella Cohn-Cedermark, Helena E Virtanen, Jorma Toppari, Gedske Daugaard, Thomas S Jensen, Søren Brunak, Ewa Rajpert-De Meyts, Niels E Skakkebæk, Henrik Leffers, Ramneek Gupta

Abstract

Background: Testicular dysgenesis syndrome (TDS) is a common disease that links testicular germ cell cancer, cryptorchidism and some cases of hypospadias and male infertility with impaired development of the testis. The incidence of these disorders has increased over the last few decades, and testicular cancer now affects 1% of the Danish and Norwegian male population.

Methods: To identify genetic variants that span the four TDS phenotypes, the authors performed a genome-wide association study (GWAS) using Affymetrix Human SNP Array 6.0 to screen 488 patients with symptoms of TDS and 439 selected controls with excellent reproductive health. Furthermore, they developed a novel integrative method that combines GWAS data with other TDS-relevant data types and identified additional TDS markers. The most significant findings were replicated in an independent cohort of 671 Nordic men.

Results: Markers located in the region of TGFBR3 and BMP7 showed association with all TDS phenotypes in both the discovery and replication cohorts. An immunohistochemistry investigation confirmed the presence of transforming growth factor β receptor type III (TGFBR3) in peritubular and Leydig cells, in both fetal and adult testis. Single-nucleotide polymorphisms in the KITLG gene showed significant associations, but only with testicular cancer.

Conclusions: The association of single-nucleotide polymorphisms in the TGFBR3 and BMP7 genes, which belong to the transforming growth factor β signalling pathway, suggests a role for this pathway in the pathogenesis of TDS. Integrating data from multiple layers can highlight findings in GWAS that are biologically relevant despite having border significance at currently accepted statistical levels.

Conflict of interest statement

Competing interests: None.

Figures

Figure 1
Figure 1
Manhattan plots showing the association between all single-nucleotide polymorphisms and (A) testicular dysgenesis syndrome and (B) the subset of cases of testicular germ cell cancer.
Figure 2
Figure 2
Integration of genome-wide association study (GWAS) data with heterogeneous data types. The rationale behind the method is to prioritise genes if several data types show mildly pronounced associations with the phenotype, thereby identifying genes that would not have been found with GWAS analysis alone. TGFBR3, which was selected by this method and whose single-nucleotide polymorphism markers were further validated, is shown in red. The position of TGFBR3 is marked by red circles in the distributions of p values, and by red squares in the ranking of each data type layer. Three data types were used: (1) single-marker GWAS results of this study (grey); (2) targeted mutations from the Mouse Genome Informatics (MGI) database, filtered for testicular dysgenesis syndrome phenotypes, and ranked by their enrichment in protein–protein complexes (pink); (3) differential expression in the fetal testis of mouse and human (green). (A) A distribution of the p values of all human genes is shown for each of the data types. The p value ranges from 0 to 1, with 0 at the top. The p value associated with each gene is converted into a rank, such that each human gene is assigned a rank. The vertical line to the right of each distribution represents the ranking of all genes, where the top corresponds to rank 1. (B) The gene ranks of each individual data type are combined to a final meta-rank of each gene. As an example, TGFBR3 had the ranks 238, 42 and 408 among all human genes in the three data types: GWAS, targeted mutations with protein–protein interaction enrichment, and differential expression in the developing testis, respectively. After combination of these data type-specific ranks, TGFBR3 was ranked 3rd among all genes. PPI, protein-protein interaction.
Figure 3
Figure 3
Regional association plots and linkage disequilibrium structure. The −log10 of the p values for the association of discovery markers are shown, and the markers are coloured in a white to red scale according to the strength of the pairwise linkage disequilibrium (r2) to the most significant discovery marker at each locus. The blue marker represents the association in the replication stage. Light blue peaks indicate recombination rates from the CEU HapMap population. r2-based linkage disequilibrium structures from the genome-wide association study data are displayed at the bottom. (A) The strongest association in the discovery stage was found at 2q31.1, close to the cluster of HOXD genes. (B) The most significant markers in the replication stage were found at the KITLG region. The genes TGFBR3 (C) and PBM7 (D) had single-nucleotide polymorphisms with mild association in the discovery stage, but were top-ranked by the integrative data analysis, and were further validated in the replication stage.
Figure 4
Figure 4
Localisation of transforming growth factor β receptor type III (TGFBR3) protein in normal fetal testis (A), adult testis with complete spermatogenesis (B), and dysgenetic adult testis with carcinoma in situ and Leydig cell hyperplasia (C). Leydig cells are marked by arrowheads (A, B, C), gonocytes (A) and CIS cells (C) by large arrows and peritubular cells by small arrows (B & C). Inserts (lower right) are without primary antibody. Bars indicate 100 μm. TGFBR3 is seen to be expressed in Leydig and peritubular cells.

References

    1. Schnack TH, Poulsen G, Myrup C, Wohlfahrt J, Melbye M. Familial coaggregation of cryptorchidism, hypospadias, and testicular germ cell cancer: a nationwide cohort study. J Natl Cancer Inst 2010;102:187–92
    1. Skakkebaek NE, Rajpert-De Meyts E, Main KM. Testicular dysgenesis syndrome: an increasingly common developmental disorder with environmental aspects. Hum Reprod 2001;16:972–8
    1. Purdue MP, Devesa SS, Sigurdson AJ, McGlynn KA. International patterns and trends in testis cancer incidence. Int J Cancer 2005;115:822–7
    1. Engholm G, Ferlay J, Christensen N, Bray F, Gjerstorff ML, Klint A, Køtlum JE, Olafsdóttir E, Pukkala E, Storm HH. NORDCAN–a Nordic tool for cancer information, planning, quality control and research. Acta Oncol 2010;49:725–36
    1. Asklund C, Jensen TK, Main KM, Sobotka T, Skakkebaek NE, Jørgensen N. Semen quality, reproductive hormones and fertility of men operated for hypospadias. Int J Androl 2010;33:80–7
    1. Rajpert-De Meyts E. Developmental model for the pathogenesis of testicular carcinoma in situ: genetic and environmental aspects. Hum Reprod Update 2006;12:303–23
    1. Sonne SB, Almstrup K, Dalgaard M, Juncker AS, Edsgard D, Ruban L, Harrison NJ, Schwager C, Abdollahi A, Huber PE, Brunak S, Gjerdrum LM, Moore HD, Andrews PW, Skakkebaek NE, Rajpert-De Meyts E, Leffers H. Analysis of gene expression profiles of microdissected cell populations indicates that testicular carcinoma in situ is an arrested gonocyte. Cancer Res 2009;69:5241–50
    1. Skakkebaek NE, Berthelsen JG, Giwercman A, Müller J. Carcinoma-in-situ of the testis: possible origin from gonocytes and precursor of all types of germ cell tumours except spermatocytoma. Int J Androl 1987;10:19–28
    1. Sharpe RM, Skakkebaek NE. Testicular dysgenesis syndrome: mechanistic insights and potential new downstream effects. Fertil Steril 2008;89:e33–8
    1. Boisen KA, Kaleva M, Main KM, Virtanen HE, Haavisto AM, Schmidt IM, Chellakooty M, Damgaard IN, Mau C, Reunanen M, Skakkebaek NE, Toppari J. Difference in prevalence of congenital cryptorchidism in infants between two Nordic countries. Lancet 2004;363:1264–9
    1. Jørgensen N, Carlsen E, Nermoen I, Punab M, Suominen J, Andersen AG, Andersson AM, Haugen TB, Horte A, Jensen TK, Magnus Ø, Petersen JH, Vierula M, Toppari J, Skakkebaek NE. East-West gradient in semen quality in the Nordic-Baltic area: a study of men from the general population in Denmark, Norway, Estonia and Finland. Hum Reprod 2002;17:2199–208
    1. Hemminki K, Li X. Familial risk in testicular cancer as a clue to a heritable and environmental aetiology. Br J Cancer 2004;90:1765–70
    1. Rapley EA, Turnbull C, Al Olama AA, Dermitzakis ET, Linger R, Huddart RA, Renwick A, Hughes D, Hines S, Seal S, Morrison J, Nsengimana J, Deloukas P, Rahman N, Bishop DT, Easton DF, Stratton MR. A genome-wide association study of testicular germ cell tumor. Nat Genet 2009;41:807–10
    1. Kanetsky PA, Mitra N, Vardhanabhuti S, Li M, Vaughn DJ, Letrero R, Ciosek SL, Doody DR, Smith LM, Weaver J, Albano A, Chen C, Starr JR, Rader DJ, Godwin AK, Reilly MP, Hakonarson H, Schwartz SM, Nathanson KL. Common variation in KITLG and at 5q31.3 predisposes to testicular germ cell cancer. Nat Genet 2009;41:811–15
    1. Turnbull C, Rapley EA, Seal S, Pernet D, Renwick A, Hughes D, Ricketts M, Linger R, Nsengimana J, Deloukas P, Huddart RA, Bishop DT, Easton DF, Stratton MR, Rahman N. Variants near DMRT1, TERT and ATF7IP are associated with testicular germ cell cancer. Nat Genet 2010;42:604–7
    1. Kratz CP, Han SS, Rosenberg PS, Berndt SI, Burdett L, Yeager M, Korde LA, Mai PL, Pfeiffer R, Greene MH. Variants in or near KITLG, BAK1, DMRT1, and TERT-CLPTM1L predispose to familial testicular germ cell tumour. J Med Genet 2011;48:473–6
    1. Zhong H, Yang X, Kaplan LM, Molony C, Schadt EE. Integrating pathway analysis and genetics of gene expression for genome-wide association studies. Am J Hum Genet 2010;86:581–91
    1. Naukkarinen J, Surakka I, Pietiläinen KH, Rissanen A, Salomaa V, Ripatti S, Yki-Järvinen H, van Duijn CM, Wichmann HE, Kaprio J, Taskinen MR, Peltonen L. Use of genome-wide expression data to mine the “Grey Zone” of GWA studies leads to novel candidate obesity genes. PLoS Genet 2010;6:e1000976.
    1. Hsu YH, Zillikens MC, Wilson SG, Farber CR, Demissie S, Soranzo N, Bianchi EN, Grundberg E, Liang L, Richards JB, Estrada K, Zhou Y, van Nas A, Moffatt MF, Zhai G, Hofman A, van Meurs JB, Pols HAP, Price RI, Nilsson O, Pastinen T, Cupples LA, Lusis AJ, Schadt EE, Ferrari S, Uitterlinden AG, Rivadeneira F, Spector TD, Karasik D, Kiel DP. An integration of genome-wide association study and gene expression profiling to prioritise the discovery of novel susceptibility loci for osteoporosis-related traits. PLoS Genet 2010;6:e1000977.
    1. Pers TH, Hansen NT, Lage K, Koefoed P, Dworzynski P, Miller ML, Flint TJ, Mellerup E, Dam H, Andreassen O, Djurovic S, Melle I, Børglum A, Werge T, Purcell S, Ferreira M, Kouskoumvekaki I, Workman C, Hansen T, Mors O, Brunak S. Meta-analysis of heterogeneous data sources for genome-scale identification of risk genes in complex phenotypes. Genet Epidemiol 2011;35:318–32
    1. Jensen MK, Pers TH, Dworzynski P, Girman CJ, Brunak S, Rimm EB. Protein interaction-based genome-wide analysis of incident coronary heart disease. Circ Cardiovasc Genet 2011;4:549–56
    1. Segrè AV, Groop L, Mootha VK, Daly MJ, Altshuler D. Common inherited variation in mitochondrial genes is not enriched for associations with type 2 diabetes or related glycemic traits. PLoS Genet 2010;6:e1001058.
    1. Wang K, Li M, Hakonarson H. Analysing biological pathways in genome-wide association studies. Nat Rev Genet 2010;11:843–54
    1. Li Q, Zheng G, Li Z, Yu K. Efficient approximation of P-value of the maximum of correlated tests, with applications to genome-wide association studies. Ann Hum Genet 2008;72:397–406
    1. Novotny GW, Nielsen JE, Sonne SB, Skakkebaek NE, Rajpert-De Meyts E, Leffers H. Analysis of gene expression in normal and neoplastic human testis: new roles of RNA. Int J Androl 2007;30:316–26; discussion 326–7.
    1. Kristensen DM, Nielsen JE, Skakkebaek NE, Graem N, Jacobsen GK, Rajpert-De Meyts E, Leffers H. Presumed pluripotency markers UTF-1 and REX-1 are expressed in human adult testes and germ cell neoplasms. Hum Reprod 2008;23:775–82
    1. Sidak Z. On multivariate normal probabilities of rectangles: their dependence on correlations. Ann Math Statist 1968;39:1425–34
    1. Workman C, Jensen LJ, Jarmer H, Berka R, Gautier L, Nielser HB, Saxild H-H, Nielsen C, Brunak S, Knudsen S. A new non-linear normalization method for reducing variability in DNA microarray experiments. Genome Biol 2002;3(9):research0048.1–research0048.16
    1. Houmard B, Small C, Yang L, Naluai-Cecchini T, Cheng E, Hassold T, Griswold M. Global gene expression in the human fetal testis and ovary. Biol Reprod 2009;81:438–43
    1. Small CL, Shima JE, Uzumcu M, Skinner MK, Griswold MD. Profiling gene expression during the differentiation and development of the murine embryonic gonad. Biol Reprod 2005;72:492–501
    1. McMahon AP, Aronow BJ, Davidson DR, Davies JA, Gaido KW, Grimmond S, Lessard JL, Little MH, Potter SS, Wilder EL, Zhang P. GUDMAP: the genitourinary developmental molecular anatomy project. J Am Soc Nephrol 2008;19:667–71
    1. Lage K, Karlberg EO, Størling ZM, Olason PI, Pedersen AG, Rigina O, Hinsby AM, Tümer Z, Pociot F, Tommerup N, Moreau Y, Brunak S. A human phenome-interactome network of protein complexes implicated in genetic disorders. Nat Biotechnol 2007;25:309–16
    1. Dias V, Meachem S, Rajpert-De Meyts E, McLachlan R, Manuelpillai U, Loveland KL. Activin receptor subunits in normal and dysfunctional adult human testis. Hum Reprod 2008;23:412–20
    1. Thorup J, McLachlan R, Cortes D, Nation TR, Balic A, Southwell BR, Hutson JM. What is new in cryptorchidism and hypospadias–a critical review on the testicular dysgenesis hypothesis. J Pediatr Surg 2010;45:2074–86
    1. Veyrieras JB, Kudaravalli S, Kim SY, Dermitzakis ET, Gilad Y, Stephens M, Pritchard JK. High-resolution mapping of expression-QTLs yields insight into human gene regulation. PLoS Genet 2008;4(10):e1000214.
    1. Dias VL, Rajpert-De Meyts E, McLachlan R, Loveland KL. Analysis of activin/TGFB-signaling modulators within the normal and dysfunctional adult human testis reveals evidence of altered signaling capacity in a subset of seminomas. Reproduction 2009;138:801–11
    1. Sarraj MA, Escalona RM, Umbers A, Chua HK, Small C, Griswold M, Loveland K, Findlay JK, Stenvers KL. Fetal testis dysgenesis and compromised Leydig cell function in Tgfbr3 (beta glycan) knockout mice. Biol Reprod 2010;82:153–62
    1. Anderson RA, Cambray N, Hartley PS, McNeilly AS. Expression and localization of inhibin alpha, inhibin/activin betaA and betaB and the activin type II and inhibin beta-glycan receptors in the developing human testis. Reproduction 2002;123:779–88
    1. Cobellis L, Cataldi P, Reis FM, De Palo G, Raspagliesi F, Pilotti S, Arcuri F, Petraglia F. Gonadal malignant germ cell tumors express immunoreactive inhibin/activin subunits. Eur J Endocrinol 2001;145:779–84
    1. Galan JJ, De Felici M, Buch B, Rivero MC, Segura A, Royo JL, Cruz N, Real LM, Ruiz A. Association of genetic markers within the KIT and KITLG genes with human male infertility. Hum Reprod 2006;21:3185–92
    1. Kondo T, Zákány J, Innis JW, Duboule D. Of fingers, toes and penises. Nature 1997;390:29.
    1. Wang Y, Barthold J, Kanetsky PA, Casalunovo T, Pearson E, Manson J. Allelic variants in HOX genes in cryptorchidism. Birth Defects Res Part A Clin Mol Teratol 2007;79:269–75

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