Association of testicular germ cell tumor with polymorphisms in estrogen receptor and steroid metabolism genes

in Endocrine-Related Cancer

It is generally assumed that the development of testicular germ cell tumor (TGCT) is under endocrine control. In particular, unbalanced androgen/estrogen levels and/or activity are believed to represent the key events for TGCT development and progression. Furthermore, recent evidence has suggested a strong genetic component for TGCT. In this study, we analyzed whether a genetic variation in estrogen receptor (ESR) genes and steroid hormone metabolism genes is associated with TGCT. We genotyped for 17 polymorphic markers in 11 genes in 234 TGCT cases and 218 controls: ESR (ESR1 and ESR2); CYP19A1 (aromatase); 17β-hydroxysteroid dehydrogenase types 1 and 4 (HSD17B1 and HSD17B4) dehydrogenases that convert potent androgens and estrogens to weak hormones; cytochrome P450 hydroxylating enzymes CYP1A1, CYP1A2, and CYP1B1; and the metabolic enzymes COMT, SULT1A1, and SULT1E1. We observed a significant association of rs11205 in HSD17B4 with TGCT. TGCT risk was increased twofold per copy of the minor A allele at this locus (odds ratios (OR)=2.273, 95% confidence interval (CI)=1.737–2.973). Homozygous carriage of the minor A allele was associated with an over fourfold increased risk of TGCT (OR=4.561, 95% CI=2.615–7.955) compared with homozygous carriage of the major G allele. The risk was increased both for seminoma (OR=5.327, 95% CI=2.857–9.931) and for nonseminoma (OR=3.222, 95% CI=1.471–7.059). We found for the first time an association of polymorphisms in HSD17B4 gene with TGCT. Our findings expand the current knowledge on the role of genetic contribution in testicular cancer susceptibility, and support the hypothesis that variations in hormone metabolism genes might change the hormonal environment implicated in testicular carcinogenesis.

Abstract

It is generally assumed that the development of testicular germ cell tumor (TGCT) is under endocrine control. In particular, unbalanced androgen/estrogen levels and/or activity are believed to represent the key events for TGCT development and progression. Furthermore, recent evidence has suggested a strong genetic component for TGCT. In this study, we analyzed whether a genetic variation in estrogen receptor (ESR) genes and steroid hormone metabolism genes is associated with TGCT. We genotyped for 17 polymorphic markers in 11 genes in 234 TGCT cases and 218 controls: ESR (ESR1 and ESR2); CYP19A1 (aromatase); 17β-hydroxysteroid dehydrogenase types 1 and 4 (HSD17B1 and HSD17B4) dehydrogenases that convert potent androgens and estrogens to weak hormones; cytochrome P450 hydroxylating enzymes CYP1A1, CYP1A2, and CYP1B1; and the metabolic enzymes COMT, SULT1A1, and SULT1E1. We observed a significant association of rs11205 in HSD17B4 with TGCT. TGCT risk was increased twofold per copy of the minor A allele at this locus (odds ratios (OR)=2.273, 95% confidence interval (CI)=1.737–2.973). Homozygous carriage of the minor A allele was associated with an over fourfold increased risk of TGCT (OR=4.561, 95% CI=2.615–7.955) compared with homozygous carriage of the major G allele. The risk was increased both for seminoma (OR=5.327, 95% CI=2.857–9.931) and for nonseminoma (OR=3.222, 95% CI=1.471–7.059). We found for the first time an association of polymorphisms in HSD17B4 gene with TGCT. Our findings expand the current knowledge on the role of genetic contribution in testicular cancer susceptibility, and support the hypothesis that variations in hormone metabolism genes might change the hormonal environment implicated in testicular carcinogenesis.

Introduction

Testicular cancer (TC, MIM 273300) is the most common cancer in white males aged 20–40 years, with a worldwide incidence of 7.5 per 100 000. About 95% of all TCs are represented by testicular germ cell tumors (TGCTs), and seminoma accounts for about 50% of them. Epidemiological hallmarks of TGCT include a peak incidence in a very young adult age, a markedly increasing incidence worldwide ( Giwercman et al. 1993, Swerdlow et al. 1998) but with striking geographic and ethnic differences, and association with other reproductive conditions, such as cryptorchidism, testicular atrophy, and infertility ( Horwich et al. 2006, Rajpert-De Meyts 2006).

A number of lines of evidence have pointed toward a strong hereditary component for TGCT. TGCT has high familial risks compared with most other cancer types: brothers of individuals with TGCT have an 8- to 12-fold increased risk of disease, and fathers of affected individuals have a four- to six-fold increased risk ( Swerdlow et al. 1997, Hemminki & Li 2004). The proportion of TGCT susceptibility accounted for by the genetic effects is estimated at 25%, and TGCT has the third highest heritability among all cancers ( Czene et al. 2002). Early results from linkage studies identified a limited relationship with genetic factors ( Crockford et al. 2006). A linkage on Xq27 was not replicated ( Rapley et al. 2000), and the gr/gr deletion on the Y chromosome, which seems to increase TGCT risk two- to three-fold and is also associated with spermatogenic impairment, might account only for a limited number of cases because carriage frequency of this variant is low (2–3%; Nathanson et al. 2005). However, two recent genome-wide association studies have reported associations at three new loci, including two candidate genes previously implicated in testicular development, KITLG (ligand for the receptor tyrosine kinase) and sprouty 4 (SPRY4), therefore supporting the contribution of a genetic predisposition to TGCT on firmer grounds ( Kanetsky et al. 2009, Rapley et al. 2009).

Although a definitive proof is still lacking, it is generally assumed that the development of TGCT is under endocrine control and tumor-initiating events seem to occur early in life, probably during fetal development. In particular, alterations in the pituitary–testicular hormonal axis and/or alterations in gonadotropin and sex steroid action are believed to be involved in the development of this tumor and in the progression from the pre-invasive carcinoma in situ stage to invasive tumor ( Rajpert-De Meyts 2006). Probably, a combination of high gonadotropin levels coupled with unbalanced androgen/estrogen levels and/or activity is the key event for TGCT development and progression ( Garolla et al. 2005, Rajpert-De Meyts 2006). Supporting this hypothesis, recently an association of TC with maternal estrogen and androgen levels in early pregnancy has been reported ( Holl et al. 2009). In addition, exposure to endocrine-disrupting chemicals acting as either weak estrogen agonist or androgen antagonist via binding to the estrogen and androgen receptors has been suggested to be associated with the risk of TGCT and other reproductive disorders ( Wohlfahrt-Veje et al. 2009).

Exposure to exogenous and/or endogenous hormones could be modulated by genetic variation in hormone receptor and hormone metabolism genes. Recent evidence supports an association of TGCT with polymorphisms in these genes: we and others have reported an association with CAG/GGN repeat length of the androgen receptor gene ( Giwercman et al. 2004, Garolla et al. 2005) and with FSH receptor gene polymorphisms ( Ferlin et al. 2008a), and an association with some maternal and/or offspring hormone-metabolizing genes of the cytochrome P450 family (CYP1A1, CYP1A2, CYP3A4, and CYP3A5) has been suggested ( Starr et al. 2005, Figueroa et al. 2008).

In this study, we sought to expand this knowledge to determine whether a genetic variation in estrogen receptor (ESR) genes and steroid hormone metabolism genes is associated with TGCT. Genotyping included 11 genes: ESR (ESR1 and ESR2); CYP19A1 (aromatase, which converts testosterone and androstenedione to estradiol and estrone respectively); 17β-hydroxysteroid dehydrogenase types 1 and 4 (HSD17B1 and HSD17B4) dehydrogenases that convert androstenedione to testosterone and estrone to estradiol and vice versa; cytochrome P450 hydroxylating enzymes CYP1A1, CYP1A2, and CYP1B1 that metabolize estradiol and estrone to catechol metabolites; COMT that degrades the catechol metabolites to methylated compounds; and SULT1A1 and SULT1E1 that degrade the catechol metabolites to sulfated compounds ( Fig. 1).

Figure 1
Figure 1

Metabolism of estrogens. The effect of P450 cytochrome enzymes (CYP) is shown together with the action of COMT that degrades the catechol metabolites to methylated compounds, SULT enzymes that degrade the catechol metabolites to sulfated compounds, and UGT enzymes that degrade the catechol metabolites to glucuronate compounds. Estrone is metabolized in the same way as estradiol. Genes included in the genotyping are underlined. ESR, estrogen receptor; AR, androgen receptor.

Citation: Endocrine-Related Cancer 17, 1; 10.1677/ERC-09-0176

Materials and methods

Subjects and clinical analyses

Patients and controls were prospectively recruited for this study from January 2005 to December 2008 with the approval of the Hospital Ethical Committee, and informed consent was obtained from each subject after full explanation of the purpose and nature of all the procedures used. The study has been conducted in accordance with the guidelines in the Declaration of Helsinki.

We evaluated 234 consecutive subjects (mean age 30.2±8.2 years) orchiectomized for TGCT, who consulted our Center for semen cryobanking before initiating chemo- and/or radiotherapy. Totally, 173 men were affected by seminoma and 61 by nonseminoma (29 by embryonal carcinoma, 19 by teratoma, 8 by choriocarcinoma, and 5 by yolk sac tumor). Only those with a primary disease in the testis were included. A complete medical history, including the information on the familiarity for TGCT, type of TGCT, age at diagnosis, the presence of a history of cryptorchidism and laterality of disease, and physical examination, was undertaken. Karyotype analysis, Y chromosome microdeletion analysis ( Ferlin et al. 2007a), and androgen receptor gene mutation analysis ( Ferlin et al. 2006, 2008b) were performed in all the subjects to exclude the potential genetic causes of testicular damage, but no alterations were found among the 234 subjects. Male controls were represented by 218 disease-free men (mean age 37.3±11.8 years) recruited among the blood donors representing the general population. More than 40% of the controls were older than 40 years and had already passed the peak of TGCT development. Prior to inclusion, all the controls underwent andrological examination including testicular ultrasound. All cases and controls were from the northeast of Italy.

Table 1 reports the main clinical characteristics of the cases and controls.

Table 1

Age, family history of testicular germ cell tumor (TGCT), history of cryptorchidism, and tumor type for cases and controls

Cases (n=234)Controls (n=218)
No.%No.%
Age (mean±s.d.)30.2±8.237.3±11.8
Family history of TGCT
 No22194.420895.4
 Yes83.431.4
 Unknown52.273.2
Personal history of cryptorchidism
 No20587.620895.4
 Yes239.820.9
 Unknown62.683.7
Tumor type
 Seminoma17373.9
 Nonseminoma6126.1

Single nucleotide polymorphism selection

After DNA isolation from peripheral leukocytes (QIAamp DNA blood kit, Qiagen), genotyping was performed with the GenomeLab SNPStream DNA genotyping platform (Beckman Coulter, Fullerton, CA, USA) and its accompanying SNPstream software suite after optimal design of the pairs of primers for the multiplex PCR and the single-base extension primers.

We studied 11 genes involved in steroid metabolism and the ESR. Single nucleotide polymorphisms (SNPs) were selected based on published evidence of a correlation with functional activity, estradiol level, or estrogen-related outcomes (such as breast cancer). Furthermore, only SNPs with a minor allele frequency (MAF) ≥0.05 (dbSNP, www.ncbi.nlm.nih.gov) were selected. Finally, we selected only A/G or C/T base substitutions to simplify the allele-specific tag extension reaction. At the end of this process, we selected 17 polymorphisms ( Table 2). DNA sequences encompassing the SNP sites were analyzed by a Blast search (http://www.ncbi.nlm.nih.gov/BLAST/) to reveal highly homologous and repetitive elements. Then, a specific software (http://www.autoprimer.com, Beckman Coulter) was used for the design of the 17 pairs of primers (amplifying a short stretch of DNA of 90–150 bp that encompasses the SNP of interest) for the multiplex PCR and of the corresponding tagged extension primers.

Table 2

Single nucleotide polymorphisms (SNPs) evaluated with the minor allele frequency

GeneGene nameChromosomal locationNucleotide changedbSNP IDMinor allele frequency
ESR1Estrogen receptor 16q25.1IVS1−397T>Crs22346930.45
Ex8+1264T>Crs37985770.44
IVS1−351A>Grs93407990.36
ESR2Estrogen receptor 214q23.2IVS2−299C>Trs12560300.42
Ex6+32G>Ars12560490.06
CYP1A1Cytochrome P450, family 1, subfamily A, polypeptide 115q24.1Ex7+131A>Grs10489430.04
CYP1A2Cytochrome P450, family 1, subfamily A, polypeptide 215q24.1IVS4+43A>Grs24723040.40
CYP1B1Cytochrome P450, family 1, subfamily B, polypeptide 12p21−4977A>Grs1625550.22
Ex3+1564G>Ars28556580.39
CYP19A1Cytochrome P450, family 19, subfamily A, polypeptide 1 (aromatase)15q21.1Ex8+47C>Trs7005190.05
HSD17B1Hydroxysteroid (17β) dehydrogenase 117q11−q21Ex6+220G>A rs605059
HSD17B4Hydroxysteroid (17β) dehydrogenase 45q23Ex6+15G>Ars256400.46
Ex19−6G>Ars112050.46
Ex24+61A>Grs289435940.00
COMTCatechol-O-methyltransferase22q11.21Ex4−12G>Ars46800.43
SULT1A1Sulfotransferase family 1A, phenol-preferring, member 116p12.1Ex10−145C>Trs1042157
SULT1E1Sulfotransferase family 1E, estrogen-preferring, member 1 4q13.1Ex1+50G>A rs37365990.08

Genotyping

Multiplex PCR was performed in an MJ thermal cycler (MJ Research, Waltham, MA, USA) under the following conditions: one denaturation cycle at 94 °C for 1 min and 40 cycles with denaturation at 94 °C for 1 min, annealing at 55 °C for 30 s, and extension at 72 °C for 1 min, followed by a final extension at 72 °C for 1 min. The reaction was performed in a final volume of 25 μl with 10 ng genomic DNA, 75 μM dNTP mix, 50 nM primer pool, 5 mM MgCl2, and 0.5 U Taq polymerase (Amplitaq Gold, Applera, Milano, Italy) in PCR 1× buffer. PCR products were purified by Exo-Sap reaction (0.67 U exonuclease I and 0.33 U shrimp alkaline phosphatase in 3 μl mix volume), which was performed in an Applied Biosystem thermal cycler to degrade the unincorporated PCR primers and dNTPs (USB Corp., Cleveland, OH, USA), following the recommended conditions.

Extension reaction was performed in a mix containing TAMRA- and Bodipy–fluorescein-labeled dideoxynucleotides (ddNTPs) and a pool of allele-specific tagged extension primers. The tagged extension primers were directed toward a specific location in each well (containing a 4×4 oligonucleotide array) of the 384 SNPware plate because of the presence of a tag at the 5′ terminus of the extension primer, which was complementary to the arrayed oligonucleotides. The 3′ terminus of the tagged extension primers was complementary to the sequence adjacent to the SNP site. The extension reaction permits the incorporation of the dye-labeled dideoxynucleotides at the 3′ terminus of the tagged extension primer. In each 4×4 array, three positive controls and one negative control were included to ensure accuracy in the genotype determination. The extension reaction was performed in an MJ thermal cycler under the following conditions: one denaturation cycle at 96 °C for 3 min and 44 cycles with a first step at 94 °C for 20 s and a second step at 40 °C for 11 s, and a final hold at 4 °C. The reaction was performed in a final volume of 15 μl with 0.04 μl SNP extension mix, 5.6 μl SNPware extension dilution buffer, 0.3 μl SNP 20× extension mix, and 0.03 μl SNPware DNA polymerase (Beckman Coulter). The SNP 20× extension mix contains the dye-labeled dideoxynucleotides, which are used in the primer extension reaction, and self-annealing oligonucleotides that are extended with dye-labeled terminators, which serve as positive controls in each well.

The extension products were transferred to the SNPware plate so that each extended primer could hybridize in a specific way to one of the unique probes arrayed in each well. A 12 μl volume of the hybridization mix (Beckman Coulter) was added to each well. The SNPware plate was incubated at 42 °C for 2 h (±15 min) in a humid chamber and was then washed thrice to remove the probes that were not arrayed.

The SNPware plate was analyzed with the GenomeLab SNPStream array imager that detects the fluorescent signal of the extended base for each spot. GetGenos software (Beckman Coulter, Fullerton, CA, USA) assigns the genotype to each SNP spot on the basis of the fluorescence intensity. Self-annealing oligonucleotides are used to get a correct grid alignment based on four fixed controls composed by oligonucleotides in homozygosis in blue and green and in heterozygosis and a blank control.

From the analysis, we obtained three possible calls for each spot in the 384 SNPware plate and these were collected into three clusters, one representing the homozygous XX genotype, one representing the heterozygous XY genotype, and one representing the homozygous YY genotype. Plot data were finally converted into an Excel format by the software itself, registering the genotype observed for each SNP in the samples analyzed.

Statistical analysis

Tests for Hardy–Weinberg equilibrium (HWE) were performed for each SNP for controls and cases with the use of the χ2 Goodness-of-Fit test. The extent of linkage disequilibrium (LD) among SNPs was quantified using Lewontin's D′ value and the correlation coefficient r2. A pair of SNPs was defined as being in high LD if they had D′ >0.8 and r2>0.5. Differences in allele and genotype distribution between cases and controls were tested with the use of Cochran–Armitage trend test. Odds ratios (OR) and 95% confidence interval (95% CI) were estimated to assess the effect of each SNP on cancer risk using a logistic regression model. Allelic ORs and 95% CIs were estimated on the basis of a multiplicative model. All reported P values were corrected to account for multiple comparisons using the false discovery rate Benjamini–Hochberg method. Two-sided adjusted P values are reported, and P of <0.05 was considered to indicate statistical significance. Statistical analyses were conducted with SAS Version 9.1.1 (SAS Institute Inc., Cary, NC, USA).

Results

The genes and SNPs analyzed with minor allele frequencies among controls are shown in Table 2. Out of the 17 markers selected in 11 genes, 2 (rs605059 in HSD17B1 and rs1042157 in SULT1A1) had high missing data rates, and were therefore not considered for further analysis. The other 15 SNPs were all in HWE equilibrium both in controls and cases, but 2 (rs1048943 in CYP1A1 and rs28943594 in HSD17B4) had a MAF <0.05. LD analysis showed no linkage between the markers of the same gene.

Table 3 shows the allele and genotype distribution in TGCT cases (overall, seminoma, and nonseminoma) and controls of the 15 informative markers of nine genes. We observed a significant association of rs11205 in HSD17B4 with TGCT, with a P for trend <0.0001 for TGCT considered as a whole and for seminoma, and 0.0157 for nonseminoma. TGCT risk was increased twofold per copy of the minor A allele in HSD17B4 rs11205 (OR=2.273, 95% CI=1.737–2.973; Table 4). Homozygous carriage of the minor A allele at this locus was associated with an over fourfold increased risk of TGCT (OR=4.561, 95% CI=2.615–7.955) compared with homozygous carriage of the major G allele ( Table 4). The increased risk was higher for seminoma (OR=5.327, 95% CI=2.857–9.931) than for nonseminoma (OR=3.222, 95% CI=1.471–7.059). Heterozygous carriage of the A/G allele did not increase the risk of TGCT.

Table 3

Allele and genotype distribution for controls and cases (overall testicular germ cell tumor (TGCT), seminoma, and nonseminoma) for the 15 informative single nucleotide polymorphisms (SNPs) in nine genes

TGCT
GeneSNPControls (n=218)Overall (n=234)Seminoma (n=173)Nonseminoma (n=61)
ESR1rs2234693Allele C/T197 (0.45)/239 (0.55)200 (0.43)/268 (0.57)147 (0.42)/199 (0.58)53 (0.43)/69 (0.57)
Genotype CC/CT/TT39 (0.18)/119 (0.55)/60 (0.27)47 (0.2)/106 (0.45)/81 (0.35)33 (0.19)/81 (0.47)/59 (0.34)14 (0.23)/25 (0.41)/22 (0.36)
P for trend0.71690.71690.9405
rs3798577Allele C/T191 (0.44)/245 (0.56)184 (0.39)/284 (0.61)132 (0.38)/214 (0.62)52 (0.43)/70 (0.57)
Genotype CC/CT/TT41 (0.19)/109 (0.50)/68 (0.31)40 (0.17)/104 (0.44)/90 (0.38)27 (0.16)/78 (0.45)/68 (0.39)13 (0.21)/26 (0.43)/22 (0.36)
P for trend0.42650.37010.9987
rs9340799Allele A/G279 (0.64)/157 (0.36)320 (0.68)/148 (0.32)242 (0.70)/104 (0.30)78 (0.64)/44 (0.36)
Genotype AA/AG/GG83 (0.38)/113 (0.52)/22 (0.1)116 (0.49)/88 (0.38)/30 (0.13)89 (0.52)/64 (0.37)/20 (0.12)27 (0.44)/24 (0.39)/10 (0.16)
P for trend0.42650.33850.9987
ESR2rs1256030Allele C/T252 (0.58)/184 (0.42)270 (0.58)/198 (0.42)200 (0.58)/146 (0.42)70 (0.57)/52 (0.43)
Genotype CC/CT/TT67 (0.31)/118 (0.49)/33 (0.15)71 (0.30)/128 (0.55)/35 (0.15)53 (0.31)/94 (0.54)/26 (0.15)18 (0.29)/34 (0.56)/9 (0.15)
P for trend0.99870.99870.9987
rs1256049Allele A/G25 (0.06)/411 (0.94)10 (0.02)/458 (0.98)9 (0.03)/337 (0.97)1 (0.01)/121 (0.99)
Genotype AA/AG/GG0/25 (0.11)/193 (0.89)0/10 (0.04)/224 (0.96)0/9 (0.05)/164 (0.95)0/1 (0.02)/60 (0.98)
P for trend0.04670.21250.1724
CYP1A1rs1048943Allele A/G420 (0.96)/16 (0.04)438 (0.94)/30 (0.06)325 (0.94)/21 (0.06)113 (0.93)/9 (0.07)
Genotype AA/AG/GG202 (0.93)/16 (0.07)/0206 (0.88)/26 (0.11)/2 (0.01)153 (0.88)/19 (0.11)/1 (0.01)53 (0.87)/7 (0.11)/1 (0.02)
P for trend0.32230.37010.3427
CYP1A2rs2472304Allele A/G262 (0.60)/174 (0.40)261 (0.56)/207 (0.44)200 (0.58)/146 (0.42)61 (0.50)/61 (0.50)
Genotype AA/AG/GG84 (0.39)/94 (0.43)/40 (0.18)70 (0.30)/121 (0.52)/43 (0.18)55 (0.32)/90 (052)/28 (0.16)15 (0.25)/31 (0.51)/15 (0.25)
P for trend0.42650.74070.3022
CYP1B1rs162555Allele A/G340 (0.78)/96 (0.22)356 (0.76)/112 (0.24)264 (0.76)/82 (0.24)92 (0.75)/30 (0.25)
Genotype AA/AG/GG130 (0.60)/80 (0.37)/8 (0.04)133 (0.57)/90 (0.38)/11 (0.05)98 (0.57)/68 (0.39)/7 (0.04)35 (0.57)/22 (0.36)/4 (0.07)
P for trend0.72950.74970.7407
rs2855658Allele A/G168 (0.39)/268 (0.61)198 (0.42)/270 (0.58)150 (0.43)/196 (0.57)48 (0.39)/74 (0.61)
Genotype AA/AG/GG38 (0.17)/92 (0.42)/88 (0.40)37 (0.16)/124 (0.53)/73 (0.31)27 (0.16)/96 (0.55)/50 (0.29)10 (0.16)/28 (0.46)/23 (0.38)
P for trend0.49960.42650.9987
CYP19A1rs700519Allele C/T416 (0.95)/20 (0.05)454 (0.97)/14 (0.03)334 (0.97)/12 (0.03)120 (0.98)/2 (0.02)
Genotype CC/CT/TT204 (0.94)/8 (0.04)/6 (0.03)222 (0.95)/10 (0.04)/2 (0.01)163 (0.94)/8 (0.04)/2 (0.01)59 (0.97)/2 (0.03)/0
P for trend0.51980.74070.4847
HSD17B4rs25640Allele A/G188 (0.43)/218 (0.57)197 (0.42)/271 (0.58)147 (0.42)/199 (0.58)50 (0.41)/72 (0.59)
Genotype AA/AG/GG40 (0.18)/108 (0.49)/70 (0.32)43 (0.18)/111 (0.47)/80 (0.34)32 (0.19)/83 (0.48)/58 (0.33)11 (0.18)/28 (0.46)/22 (0.36)
P for trend0.42650.51370.5137
rs11205Allele A/G202 (0.46)/234 (0.54)310 (0.66)/158 (0.34)233 (0.67)/113 (0.33)77 (0.63)/45 (0.37)
Genotype AA/AG/GG42 (0.19)/118 (0.54)/58 (0.27)109 (0.47)/92 (0.39)/33 (0.14)81 (0.47)/71 (0.41)/21 (0.12)28 (0.46)/21 (0.34)/12 (0.20)
P for trend<0.0001<0.00010.0157
rs28943594Allele A/G436 (1.0)/0465 (0.99)/3 (0.01)343 (0.99)/3 (0.01)122 (1.00)/0
Genotype AA/AG/GG218 (1.0)/0/0231 (0.99)/3 (0.01)/0170 (0.98)/3 (0.02)/061 (1.00)/0/0
P for trend0.34270.3022
COMTrs4680Allele A/G186 (0.43)/250 (0.57)200 (0.43)/268 (0.57)148 (0.43)/198 (0.57)52 (0.43)/70 (0.57)
Genotype AA/AG/AA2 (0.01)/182 (0.83)/34 (0.16)0/200 (0.85)/34 (0.15)0/148 (0.86)/25 (0.14)0/52 (0.85)/9 (0.15)
P for trend0.99870.99870.9987
SULT1E1rs3736599Allele A/G33 (0.08)/403 (0.92)35 (0.07)/433 (0.93)21 (0.06)/325 (0.94)14 (0.11)/108 (0.89)
Genotype AA/AG/GG1 (0.01)/31 (0.14)/186 (0.85)2 (0.01)/31 (0.13)/201 (0.86)2 (0.01)/17 (0.10)/154 (0.89)0/14 (0.23)/47 (0.77)
P for trend0.99870.71370.4265

The corrected P for trend is shown. Significant differences (P<0.05) are in bold. The numbers and frequency are shown in parentheses.

Table 4

Associations of testicular germ cell tumor (TGCT) with single nucleotide polymorphism (SNP) marker rs11205 in HSD17B4 gene (risk allele A)

Genotype countaOR (95% CI)
Tumor typeControlsCasesPer alleleHeterozygote bHomozygote c
All TGCTs42/118/58109/92/332.273 (1.737–2.973)1.370 (0.825–2.275)4.561 (2.615–7.955)
Seminoma42/118/5881/71/212.388 (1.782–3.202)1.662 (0.931–2.967)5.327 (2.857–9.931)
Nonseminoma42/118/5828/21/121.982 (1.311–2.996)0.860 (0.396–1.869)3.222 (1.471–7.059)

Number of individuals genotyped as homozygous for the risk allele/heterozygous for the risk allele/homozygous for the nonrisk allele.

OR for heterozygous carriage of risk allele compared with that for homozygous carriage of nonrisk allele.

OR for homozygous carriage of risk allele compared with that for homozygous carriage of nonrisk allele.

A weak association was also found with rs1256049 in ESR2 only for TGCT considered as a whole (P for trend was 0.0467). However, no homozygotes for the minor A allele were found in cases and controls, and OR calculation showed no increased risk.

Discussion

This study analyzed the possible association between polymorphisms in ESR and estrogen metabolism genes and TGCT. By studying 17 SNPs in 11 genes, we found that one polymorphism in the HSD17B4 gene is associated with risk of TGCT. In particular, the TGCT risk was increased twofold per copy of the minor A allele in HSD17B4 and more than fourfold for A homozygotes compared with homozygous carriage of the major G allele. The risk was increased for both types of TGCTs, but it was higher for seminoma (OR=5.327) than for nonseminoma (OR=3.222).

HSD17B4 gene encodes for the HSD17B4, the enzyme that inactivates estradiol to estrone and converts testosterone to androstenedione. Although there is no functional information for the HSD17B4 SNP (rs11205) with which we found an association in the current study, some evidence suggests that it might alter the enzyme activity. The G>A polymorphism of rs11205 is a missense mutation located in exon 19 that leads to amino acid change (Val>Ile). Although these two amino acids have similar physico-chemical properties and are both medium sized and hydrophobic, this residue is highly conserved among the species and it is part of a conserved domain composing the substrate binding site. Therefore, change in functional activity of the HSD17B4 enzyme could be hypothesized, leading to an alteration in the levels of sex hormones and therefore changing susceptibility to TGCT. Several lines of evidence support our findings and this hypothesis.

Unbalanced androgen/estrogen levels and/or activity are believed to represent the key events for TGCT development and progression ( Garolla et al. 2005, Rajpert-De Meyts 2006). The frequent association of TGCT with other disorders of the male genital tract suggested the existence of a ‘testicular dysgenesis syndrome’ (TDS; Skakkebaek et al. 2001) caused by genetic and environmental factors altering gonadal development in utero leading to clinical manifestations at birth (cryptorchidism and hypospadias) or in adulthood (spermatogenic failure and TGCT). This hypothesis also includes a possible role for endogenous and exogenous hormones or hormone-like substances (endocrine disruptors) mainly acting as estrogens or anti-androgens. Although direct evidence in humans is lacking, numerous animal models support this notion ( Sharpe 2003, Sharpe & Skakkebaek 2008), and associations of TGCT with maternal estrogen and androgen levels in early pregnancy have been reported ( Holl et al. 2009).

Previous studies on polymorphisms in ESR, androgen receptor, and estrogen metabolism genes have documented an association with TGCT and/or other signs of TDS, in particular cryptorchidism and male infertility ( Ferlin et al. 2007b, Foresta et al. 2008, Krausz & Looijenga 2008 for review). Associations with TGCT have been found with mutations in the androgen receptor gene ( Garolla et al. 2005), different lengths of the CAG and GGC repeats in exon 1 of the androgen receptor gene which modulate the sensitivity of the receptor to androgens ( Giwercman et al. 2004, Garolla et al. 2005), polymorphisms of the FSH receptor gene which modulate FSH sensitivity ( Ferlin et al. 2008a), and polymorphisms in cytochrome P450 genes involved in estrogen metabolism ( Starr et al. 2005, Figueroa et al. 2008). With regards to these latter genes, an association between TGCT and CYP1A2 (rs762551), CYP3A4 (rs2740574), and CYP3A5 (rs776746) gene polymorphisms was found by one study ( Starr et al. 2005), whereas an association with polymorphisms in CYP1A1 (rs4886605 and rs2606345) gene was found by another one ( Figueroa et al. 2008). We did not include CYP3A4 and CYP3A5 genes in our screening, nor we included SNPs rs4886605 and rs2606345 in CYP1A1 and rs762551 in CYP1A2; therefore, we were unable to confirm such associations. On the contrary, Figueroa et al. (2008) also studied the HSD17B4 gene and they found no association with TGCT; however, SNP rs11205 that we found differently distributed between cases and controls was not included in their study. Taken together these results, although not definitive, strongly suggest that steroid hormone metabolism genes might modulate the susceptibility to TC. Supporting this idea, it is interesting to note that one polymorphism (rs2830) in the gene encoding for HSD17B1, the enzyme that catalyzes the same reaction of HSD17B4 converting weak estrogen estrone to potent estradiol, has been associated with nonseminoma risk ( Figueroa et al. 2008). Interestingly, studies on breast cancer showed that other polymorphisms in this gene have been found to be associated with increased estrogen levels ( Setiawan et al. 2004). Finally, the importance of variation in steroid hormone metabolism genes is also highlighted by the finding that polymorphisms in these genes, including HSD17B4 that we found to be associated with TGCT, have been associated with the efficacy of the androgen-deprivation therapy in men with prostate cancer ( Ross et al. 2008), a well-known hormone-dependent tumor.

Location of a disease-associated SNP in a gene does not necessarily implicate the gene in disease susceptibility. Therefore, an alternative hypothesis is that the association of SNP rs11205 in HSD17B4 with TGCT merely reflects functional alterations elsewhere near this SNP. However, this hypothesis is not supported by the two recent genome-wide association studies performed on a high number of TGCT cases ( Kanetsky et al. 2009, Rapley et al. 2009), which found different chromosomal regions linked to the tumor but not in the vicinity of HSD17B4 gene. In fact, a strong association was found for chromosome 5q (where HSD17B4 map) in both the studies, but the associated linkage block was about 23 Mb far from rs11205 with which we found an association in the current study.

Although the sample size of our study is relatively large, the association between HSD17B4 gene polymorphisms and TGCT should be replicated in other studies with a larger number of patients and with additional polymorphisms in these loci. Therefore, our study although highly suggestive of a role for HSD17B4 gene polymorphisms in modulating the risk of TGCT should be considered as preliminary.

In conclusions, we found for the first time an association of polymorphisms in HSD17B4 gene with TGCT. Our findings expand the current knowledge on the role of genetic contribution in TC susceptibility, and support the hypothesis that variations in hormone metabolism genes might change the hormonal environment implicated in testicular carcinogenesis.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported in part by grants from the University of Padova (to A Ferlin) and the Italian Ministry of University and Research (grant number 2007TKYYJR) (to C Foresta).

References

  • CrockfordGPLingerRHockleySDudakiaDJohnsonLHuddartRTuckerKFriedlanderMPhillipsKAHoggD2006Genome-wide linkage screen for testicular germ cell tumour susceptibility loci. Human Molecular Genetics15443451.

    • Search Google Scholar
    • Export Citation
  • CzeneKLichtensteinPHemminkiK2002Environmental and heritable causes of cancer among 9.6 million individuals in the Swedish Family-Cancer Database. International Journal of Cancer99260266.

    • Search Google Scholar
    • Export Citation
  • FerlinAVinanziCGarollaASeliceRZuccarelloDCazzadoreCForestaC2006Male infertility and androgen receptor gene mutations: clinical features and identification of seven novel mutations. Clinical Endocrinology65606610.

    • Search Google Scholar
    • Export Citation
  • FerlinAArrediBSpeltraECazzadoreCSeliceRGarollaAForestaC2007aMolecular and clinical characterization of Y chromosome microdeletions in infertile men: ten years experience in Italy. Journal of Clinical Endocrinology and Metabolism92762770.

    • Search Google Scholar
    • Export Citation
  • FerlinARaicuFGattaVZuccarelloDPalkaGForestaC2007bMale infertility: role of genetic background. Reproductive Biomedicine Online14734745.

    • Search Google Scholar
    • Export Citation
  • FerlinAPengoMSeliceRSalmasoLGarollaAForestaC2008aAnalysis of single nucleotide polymorphisms of FSH receptor gene suggests association with testicular cancer susceptibility. Endocrine-Related Cancer15429437.

    • Search Google Scholar
    • Export Citation
  • FerlinAZuccarelloDZuccarelloBChiricoMRZanonGFForestaC2008bGenetic alterations associated with cryptorchidism. Journal of the American Medical Association30022712276.

    • Search Google Scholar
    • Export Citation
  • FigueroaJDSakodaLCGraubardBIChanockSRubertoneMVEricksonRLMcGlynnKA2008Genetic variation in hormone metabolizing genes and risk of testicular germ cell tumors. Cancer Causes & Control19917929.

    • Search Google Scholar
    • Export Citation
  • ForestaCZuccarelloDGarollaAFerlinA2008Role of hormones, genes, and environment in human cryptorchidism. Endocrine Reviews29560580.

  • GarollaAFerlinAVinanziCRoveratoASottiGArtibaniWForestaC2005Molecular analysis of the androgen receptor gene in testicular cancer. Endocrine-Related Cancer12645655.

    • Search Google Scholar
    • Export Citation
  • GiwercmanACarlsenEKeidingNSkakkebaekNE1993Evidence for increasing incidence of abnormalities of the human testis: a review. Environmental Health Perspectives1016571.

    • Search Google Scholar
    • Export Citation
  • GiwercmanALundinKBEberhardJStahlOCwikielMCavallin-StahlEGiwercmanYL2004Linkage between androgen receptor gene CAG trinucleotide repeat length and testicular germ cell cancer histological type and clinical stage. European Journal of Cancer4021522158.

    • Search Google Scholar
    • Export Citation
  • HemminkiKLiX2004Familial risk in testicular cancer as a clue to a heritable and environmental aetiology. British Journal of Cancer9017651770.

    • Search Google Scholar
    • Export Citation
  • HollKLundinESurcelHMGrankvistKKoskelaPDillnerJHallmansGWadellGOlafsdottirGHOgmundsdottirHM2009Endogenous steroid hormone levels in early pregnancy and risk of testicular cancer in the offspring: a nested case-referent study. International Journal of Cancer12429232928.

    • Search Google Scholar
    • Export Citation
  • HorwichAShipleyJHuddartR2006Testicular germ-cell cancer. Lancet367754765.

  • KanetskyPAMitraNVardhanabhutiSLiMVaughnDJLetreroRCiosekSLDoodyDRSmithLMWeaverJ2009Common variation in KITLG and at 5q31.3 predisposes to testicular germ cell cancer. Nature Genetics41811815.

    • Search Google Scholar
    • Export Citation
  • KrauszCLooijengaLH2008Genetic aspects of testicular germ cell tumors. Cell Cycle735193524.

  • NathansonKLKanetskyPAHawesRVaughnDJLetreroRTuckerKFriedlanderMPhillipsKAHoggDJewettMA2005The Y deletion gr/gr and susceptibility to testicular germ cell tumor. American Journal of Human Genetics7710341043.

    • Search Google Scholar
    • Export Citation
  • Rajpert-De MeytsE2006Developmental model for the pathogenesis of testicular carcinoma in situ: genetic and environmental aspects. Human Reproduction Update12303323.

    • Search Google Scholar
    • Export Citation
  • RapleyEACrockfordGPTeareDBiggsPSealSBarfootREdwardsSHamoudiRHeimdalKFossâSD2000Localization to Xq27 of a susceptibility gene for testicular germ-cell tumours. Nature Genetics24197200.

    • Search Google Scholar
    • Export Citation
  • RapleyEATurnbullCAl OlamaAADermitzakisETLingerRHuddartRARenwickAHughesDHinesSSealS2009A genome-wide association study of testicular germ cell tumor. Nature Genetics41807810.

    • Search Google Scholar
    • Export Citation
  • RossRWOhWKXieWPomerantzMNakabayashiMSartorOTaplinMEReganMMKantoffPWFreedmanM2008Inherited variation in the androgen pathway is associated with the efficacy of androgen-deprivation therapy in men with prostate cancer. Journal of Clinical Oncology26842847.

    • Search Google Scholar
    • Export Citation
  • SetiawanVWHankinsonSEColditzGAHunterDJDe VivoI2004HSD17B1 gene polymorphisms and risk of endometrial and breast cancer. Cancer Epidemiology Biomarkers & Prevention13213219.

    • Search Google Scholar
    • Export Citation
  • SharpeRM2003The ‘oestrogen hypothesis’– where do we stand now?International Journal of Andrology26215.

  • SharpeRMSkakkebaekNETesticular dysgenesis syndrome: mechanistic insights and potential new downstream effectsFertility and Sterility89Supplement 22008e33e38.

    • Search Google Scholar
    • Export Citation
  • SkakkebaekNERajpert-De MeytsEMainKM2001Testicular dysgenesis syndrome: an increasingly common developmental disorder with environmental aspects. Human Reproduction16972978.

    • Search Google Scholar
    • Export Citation
  • StarrJRChenCDoodyDRHsuLRicksSWeissNSSchwartzSM2005Risk of testicular germ cell cancer in relation to variation in maternal and offspring cytochrome p450 genes involved in catechol estrogen metabolism. Cancer Epidemiology Biomarkers & Prevention1421832190.

    • Search Google Scholar
    • Export Citation
  • SwerdlowAJDe StavolaBLSwanwickMAMaconochieNE1997Risks of breast and testicular cancers in young adult twins in England and Wales: evidence on prenatal and genetic aetiology. Lancet35017231728.

    • Search Google Scholar
    • Export Citation
  • SwerdlowAJdos Santos SilvaIReidAQiaoZBrewsterDHArrundaleJ1998Trends in cancer incidence and mortality in Scotland: description and possible explanations. British Journal of Cancer77154.

    • Search Google Scholar
    • Export Citation
  • Wohlfahrt-VejeCMainKMSkakkebækNE2009Testicular Dysgenesis syndrome; fetal origin of adult reproductive problems. Clinical Endocrinology71459465.

    • Search Google Scholar
    • Export Citation

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    Metabolism of estrogens. The effect of P450 cytochrome enzymes (CYP) is shown together with the action of COMT that degrades the catechol metabolites to methylated compounds, SULT enzymes that degrade the catechol metabolites to sulfated compounds, and UGT enzymes that degrade the catechol metabolites to glucuronate compounds. Estrone is metabolized in the same way as estradiol. Genes included in the genotyping are underlined. ESR, estrogen receptor; AR, androgen receptor.

References

  • CrockfordGPLingerRHockleySDudakiaDJohnsonLHuddartRTuckerKFriedlanderMPhillipsKAHoggD2006Genome-wide linkage screen for testicular germ cell tumour susceptibility loci. Human Molecular Genetics15443451.

    • Search Google Scholar
    • Export Citation
  • CzeneKLichtensteinPHemminkiK2002Environmental and heritable causes of cancer among 9.6 million individuals in the Swedish Family-Cancer Database. International Journal of Cancer99260266.

    • Search Google Scholar
    • Export Citation
  • FerlinAVinanziCGarollaASeliceRZuccarelloDCazzadoreCForestaC2006Male infertility and androgen receptor gene mutations: clinical features and identification of seven novel mutations. Clinical Endocrinology65606610.

    • Search Google Scholar
    • Export Citation
  • FerlinAArrediBSpeltraECazzadoreCSeliceRGarollaAForestaC2007aMolecular and clinical characterization of Y chromosome microdeletions in infertile men: ten years experience in Italy. Journal of Clinical Endocrinology and Metabolism92762770.

    • Search Google Scholar
    • Export Citation
  • FerlinARaicuFGattaVZuccarelloDPalkaGForestaC2007bMale infertility: role of genetic background. Reproductive Biomedicine Online14734745.

    • Search Google Scholar
    • Export Citation
  • FerlinAPengoMSeliceRSalmasoLGarollaAForestaC2008aAnalysis of single nucleotide polymorphisms of FSH receptor gene suggests association with testicular cancer susceptibility. Endocrine-Related Cancer15429437.

    • Search Google Scholar
    • Export Citation
  • FerlinAZuccarelloDZuccarelloBChiricoMRZanonGFForestaC2008bGenetic alterations associated with cryptorchidism. Journal of the American Medical Association30022712276.

    • Search Google Scholar
    • Export Citation
  • FigueroaJDSakodaLCGraubardBIChanockSRubertoneMVEricksonRLMcGlynnKA2008Genetic variation in hormone metabolizing genes and risk of testicular germ cell tumors. Cancer Causes & Control19917929.

    • Search Google Scholar
    • Export Citation
  • ForestaCZuccarelloDGarollaAFerlinA2008Role of hormones, genes, and environment in human cryptorchidism. Endocrine Reviews29560580.

  • GarollaAFerlinAVinanziCRoveratoASottiGArtibaniWForestaC2005Molecular analysis of the androgen receptor gene in testicular cancer. Endocrine-Related Cancer12645655.

    • Search Google Scholar
    • Export Citation
  • GiwercmanACarlsenEKeidingNSkakkebaekNE1993Evidence for increasing incidence of abnormalities of the human testis: a review. Environmental Health Perspectives1016571.

    • Search Google Scholar
    • Export Citation
  • GiwercmanALundinKBEberhardJStahlOCwikielMCavallin-StahlEGiwercmanYL2004Linkage between androgen receptor gene CAG trinucleotide repeat length and testicular germ cell cancer histological type and clinical stage. European Journal of Cancer4021522158.

    • Search Google Scholar
    • Export Citation
  • HemminkiKLiX2004Familial risk in testicular cancer as a clue to a heritable and environmental aetiology. British Journal of Cancer9017651770.

    • Search Google Scholar
    • Export Citation
  • HollKLundinESurcelHMGrankvistKKoskelaPDillnerJHallmansGWadellGOlafsdottirGHOgmundsdottirHM2009Endogenous steroid hormone levels in early pregnancy and risk of testicular cancer in the offspring: a nested case-referent study. International Journal of Cancer12429232928.

    • Search Google Scholar
    • Export Citation
  • HorwichAShipleyJHuddartR2006Testicular germ-cell cancer. Lancet367754765.

  • KanetskyPAMitraNVardhanabhutiSLiMVaughnDJLetreroRCiosekSLDoodyDRSmithLMWeaverJ2009Common variation in KITLG and at 5q31.3 predisposes to testicular germ cell cancer. Nature Genetics41811815.

    • Search Google Scholar
    • Export Citation
  • KrauszCLooijengaLH2008Genetic aspects of testicular germ cell tumors. Cell Cycle735193524.

  • NathansonKLKanetskyPAHawesRVaughnDJLetreroRTuckerKFriedlanderMPhillipsKAHoggDJewettMA2005The Y deletion gr/gr and susceptibility to testicular germ cell tumor. American Journal of Human Genetics7710341043.

    • Search Google Scholar
    • Export Citation
  • Rajpert-De MeytsE2006Developmental model for the pathogenesis of testicular carcinoma in situ: genetic and environmental aspects. Human Reproduction Update12303323.

    • Search Google Scholar
    • Export Citation
  • RapleyEACrockfordGPTeareDBiggsPSealSBarfootREdwardsSHamoudiRHeimdalKFossâSD2000Localization to Xq27 of a susceptibility gene for testicular germ-cell tumours. Nature Genetics24197200.

    • Search Google Scholar
    • Export Citation
  • RapleyEATurnbullCAl OlamaAADermitzakisETLingerRHuddartRARenwickAHughesDHinesSSealS2009A genome-wide association study of testicular germ cell tumor. Nature Genetics41807810.

    • Search Google Scholar
    • Export Citation
  • RossRWOhWKXieWPomerantzMNakabayashiMSartorOTaplinMEReganMMKantoffPWFreedmanM2008Inherited variation in the androgen pathway is associated with the efficacy of androgen-deprivation therapy in men with prostate cancer. Journal of Clinical Oncology26842847.

    • Search Google Scholar
    • Export Citation
  • SetiawanVWHankinsonSEColditzGAHunterDJDe VivoI2004HSD17B1 gene polymorphisms and risk of endometrial and breast cancer. Cancer Epidemiology Biomarkers & Prevention13213219.

    • Search Google Scholar
    • Export Citation
  • SharpeRM2003The ‘oestrogen hypothesis’– where do we stand now?International Journal of Andrology26215.

  • SharpeRMSkakkebaekNETesticular dysgenesis syndrome: mechanistic insights and potential new downstream effectsFertility and Sterility89Supplement 22008e33e38.

    • Search Google Scholar
    • Export Citation
  • SkakkebaekNERajpert-De MeytsEMainKM2001Testicular dysgenesis syndrome: an increasingly common developmental disorder with environmental aspects. Human Reproduction16972978.

    • Search Google Scholar
    • Export Citation
  • StarrJRChenCDoodyDRHsuLRicksSWeissNSSchwartzSM2005Risk of testicular germ cell cancer in relation to variation in maternal and offspring cytochrome p450 genes involved in catechol estrogen metabolism. Cancer Epidemiology Biomarkers & Prevention1421832190.

    • Search Google Scholar
    • Export Citation
  • SwerdlowAJDe StavolaBLSwanwickMAMaconochieNE1997Risks of breast and testicular cancers in young adult twins in England and Wales: evidence on prenatal and genetic aetiology. Lancet35017231728.

    • Search Google Scholar
    • Export Citation
  • SwerdlowAJdos Santos SilvaIReidAQiaoZBrewsterDHArrundaleJ1998Trends in cancer incidence and mortality in Scotland: description and possible explanations. British Journal of Cancer77154.

    • Search Google Scholar
    • Export Citation
  • Wohlfahrt-VejeCMainKMSkakkebækNE2009Testicular Dysgenesis syndrome; fetal origin of adult reproductive problems. Clinical Endocrinology71459465.

    • Search Google Scholar
    • Export Citation

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