University of Groningen Genetic predisposition to testicular cancer Lutke Holzik, Martijn Frederik IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2007 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Lutke Holzik, M. F. (2007). Genetic predisposition to testicular cancer s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 25-11-2018
Chapter Absence of constitutional Y chromosome AZF deletions in patients with Testicular Germ Cell Tumors MF Lutke Holzik 1, K Storm 2, RH Sijmons 3, M D Hollander 2, EGJM Arts 4, ML Verstraaten 1, DTh Sleijfer 5, HJ Hoekstra 1 Departments of : 1 Surgical oncology, 3 Genetics, 4 Obstetrics and Gynecology, 5 Medical oncology. University Medical Center Groningen, the Netherlands Department of : 2 Medical Genetics, University of Antwerp, Belgium Urology 2005; 5:19-201 75
Absence of constitutional Y chromosome AZF deletions in patients with Testicular Germ Cell Tumors Introduction Although testicular germ cell tumors (TGCT) constitute the most common malignancy in men aged 15 to 40 years, their etiology is still poorly understood. (244) In the past few years a decrease in fertility and an increase in TGCT has been reported. (4) This could suggest that fertility and TGCT share a common etiological factor. A typical illustration is the East-West semen quality gradient in the Nordic Baltic area and the incidence of TGCT. Finland and Estonia have only one third of the TGCT incidence compared with Denmark and Norway, which is inversely related to the lower sperm counts observed in Danish and Norwegian men compared with men from Finland and Estonia. (245) A retrospective cohort study of more than 30,000 men from infertile couples found an association between infertility and a subsequent risk of TGCT. (4) Men with infertility were 1. times more likely to develop TGCT. The greatest risk of TGCT was in the first 2 years (standardized incidence ratio 1,8) after the first semen analysis. At 2 to 11 years after the first semen analyses, the standardized incidence ratio was 1,5 to 1,. This is a relatively constant risk for TGCT, and impaired spermatogenesis may, therefore, have been present many years before TGCT was diagnosed. (4) These results are in line with our previous results showing that TGCT patients have already impaired spermatogenesis before orchiectomy was performed. (24) In the model postulated by Skakkebaek et al., (12) TGCT and poor semen quality are symptoms of one underlying entity, the testicular dysgenesis syndrome (TDS). Endogenous, as well as exogenous, risk factors, including inherited predisposing gene mutations may result in TDS. Past research has traditionally focused on the range of possible exogenous risk factors for TDS and TGCT, including prenatal exposure to maternal hormones. Although TGCT susceptibility genes remain to be identified, (244) more light has recently been shed on the genetics of infertility. Up to approximately 8% of infertility in the general male population can be explained by the presence of constitutional deletions of part of the long arm of the Y chromosome (Yq11), referred to as the azoospermia factor (AZF) region (subdivided into AZFa to AZFd). (247-249) AZFc deletions are the most commonly found (0%). Although most of the AZF deletions observed in infertile men are new (de novo) mutations, they have been inherited in some cases from (apparently fertile) fathers, and 0.4% of fertile men in the general population appear to carry an AZF deletion. (248) Foresta et al. recently observed a particularly high percentage of 27.5% AZF (a-c) deletions in patients with low sperm counts as well as unilateral cryptorchism. (250) Cryptorchism is an acknowledged risk factor for TGCT and is one of the postulated other possible manifestations of the TDS. (12;81;251) Taken together, these observations suggests that, at least from a theoretical point of view, constitutional AZF deletions might be one of the genetic contributors to the 7 Absence of constitutional Y chromosome AZF deletions in patients with Testicular Germ Cell Tumors 77
development of TDS and thereby of TGCT and other TDS manifestations. This possibility would be in line with tumor cytogenetic studies that have demonstrated a nonconstitutional loss of Y chromosome material in adult TGCT, as well as in their precursor carcinoma in situ, which suggests that loss of Y chromosome material may indeed play a role in TGCT development. (252) Bianchi et al. (253) have demonstrated that, noninherited mosaic AZF deletions can be observed in tumor as well as nontumor, tissues from some patients with TGCT. Altogether, this might point at a role for the loss of Y chromosome material, inculding AZF, in TGCT development. Given that constitutional AZF deletions have been observed in fertile men in the general population and TDS does not necessarily present with infertility, the possibility of constitutional AZF deletions causing TDS and thereby TGCT in fertile men has not be ruled out. In the present study, we investigated the frequency of Y chromosome deletions in the AZF region in a series of fertile, as well as infertile, patients with TGCT. Patients and Methods Patients selection A total of 112 patients with TGCT treated at the University Medical Center Groningen (UMCG) in the Netherlands were randomly selected for initial analysis. Patient characteristics are listed in Table 1. Familial TGCT was defined as more than one case in the family. Histologic diagnosis was established in all patients by the Department of Pathology of the UMCG. Owing to the position of our medical center (academic referral hospital for the northern part of The Netherlands) most patients had undergone orchiectomy (before diagnosis) in the referring hospitals without prior semen analysis and preservation. Therefore, in the current series of patients no data were available on semen quality before orchiectomy; however, semen data were available for 25 patients after orchiectomy. We stratified these 25 patients into four groups according to semen concentration (Table 2). All participants gave their written informed consent and the ethical committee of the UMCG approved the study. Table 1: Characteristics of the 112 TGCT patients Table 2: Distribution of semen concentration in 25 patients after orchiectomy Sperm Concentration Normozoospermia (>20 10 /ml) 13* Moderate oligozoospermia (5 20 10 /ml) 5 Severe oligozoospermia (<5 10 /ml) 2 Azoospermia 5* * Normozoospermia and azoospermia groups both included 1 patient with cryptorchism. Genotyping Patients (n) High-molecular-weight genomic DNA was extracted from peripheral blood lymphocytes according to standard protocols. (254) After DNA extraction screening for AZF deletions was performed by multiplex polymerase chain reaction (PCR) analysis using the Y Chromosome Deletion Detection System, version 1.1 (Promega), and the addition of Multiplex Master Mix E (Promega). Version 1.1 has been extensively described by Aknin-Seifer et al. (255) and the addition of mix E has improved accuracy. Currently, the system that includes mix E is known as the Y Chromosome Deletion Detection System, version 2.0. (25) In the current study the system consisted of 24 primer pairs, of which 20 primer pairs are homologous to previously identified and mapped sequenced tag sites (STSs) within the AZF regions on the Y chromosome (locations provided in Fig. 1 and Table 3). All the loci analyzed in this study have been recommended by the European Quality Monitoring Network Group (EQMNG) for detection of Yq11 deletions associated with male infertility. (25) Primers were combined into five primer sets to use in five parallel PCR Figure 1: Diagram of the Y chromosome with AZF regions, previously cloned genes and pseudogenes, and STSs. Reprinted form Technical Manual No 248. (25) Used with permission of Promega Corporation Type of Neoplasia Patients/total(%) Patients with nonseminoma 94/112 (84%) Patients with pure seminoma 18/112 (1%) Other characteristics Patients with bilateral TGCT 4/112 (3,5%) Patients with cryptorchism 21/112 (18,8%) Patients with familial TGCT 10/112 (9%) TGCT = testicular germ cell tumor 78 Absence of constitutional Y chromosome AZF deletions in patients with Testicular Germ Cell Tumors 79
Table 3: Overview of the 24 STSs amplified STS Locus PCR Fragment (bp) Multiplex PCR Position sy81 DYS271 209 A distal to AZFa sy8 DYS148 232 E AZFa sy84 DYS273 177 E AZFa sy182 KAL-Y 125 A proximal to AZFa sy121 DYS212 190 C AZFb SYPR3 SMCY 350 B AZFb sy124 DYS215 109 D AZFb sy127 DYS218 274 B AZFb sy128 DYS219 228 C AZFb sy130 DYS221 173 A AZFb sy133 DYS223 177 D AZFb sy134 DYS224 303 E AZFb sy145 DYF51S1 143 C proximal to AZFc = AZFd sy152 DYS23 285 D proximal to AZFc = AZFd sy153 DYS237 139 D AZFd (nonpathogenic) sy242 DAZ 233 B AZFc sy239 DAZ 200 B AZFc sy208 DAZ 140 B AZFc sy254 DAZ 370 A AZFc sy255 DAZ 12 C AZFc sy157 DYS240 285 A distal to AZFc sy14 SRY 400 E SRY gene SMCX 83 A-D X chromosome (control) ZFX/ZFY 49 E X/Y chromosome (control) STSs = sequenced tag sites; PCR = polymerase chain reaction; AZF = azoospermia factor. Overview of the 24 STSs amplified in five multiplex PCR amplifications (A-E), including 20 STSs for detection of AZF deletions associated with male infertility, one STS sy153 which seems to be polymorphic or in multiple copies and 3 control STSs (SMCX, ZFX/ZFY and sy14). amplifications (multiplex PCR A through E; Table 3). The slight modifications to the protocol (25) provided by the manufacturer were: 500 ng DNA in a final volume of 25 µl multiplex Master Mix, amplification in 35 cycles on a Perkin-Elmer GeneAMP System 9700 thermal cycler (Applied Biosystems), and annealing at 58 C for 1 minute 30 seconds. The control samples analyzed in each multiplex PCR were a male genomic DNA control, a female genomic DNA control and a blank (no-dna) control. The separation and visualization of the PCR products were performed by electrophoresis in 4% NuSieve 3:1 Plus agarose gels (Cambrex Bio Science), stained with Ethidium Bromide (Fig. 2). The multiplex primer sets A through D contained a control primer pair that amplifies a fragment of the X-linked SMCX locus. Multiplex E contains a control primer pair that amplifies a unique region in both male and female DNA (ZFX/ZFY). Both control primer pairs are internal controls for the amplification reaction and the integrity of the genomic DNA sample. In addition multiplex E contains a primer pair that amplifies a region of the SRY gene that is a control for the presence of the testis determining factor on the short arm of the Y chromosome (Yp) and allows XX males (arising from Y to X translocations) to be detected. The Y chromosome deletion detection system (25) is the standard procedure in the laboratory of the Department of Medical Genetics, University of Antwerp, to detect AZF deletions in men analyzed for infertility and subfertility. We previously found some AZF deletions in our laboratory amongst infertile and subfertile men, who did not have a history of TGCT (data not shown). Results Microdeletions analysis of the AZF region (Yq11) was successfully performed on genomic DNA of 112 patients with TGCT. Figure 2 includes representative examples of the electrophoresis gels, showing amplification products for multiplex A-E. No PCR products detected in the blank (no DNA) control. As expected the positive male control showed the appropriate number and sizes of bands for each multiplex master mix. The positive female control only showed amplification for the SMCX and ZFX loci. In the 112 patients with TGCT no deletions within the AZF region were detected. Discussion A detailed analysis of microdeletions on the Y chromosome was performed in 112 Dutch patients with TGCT by studying 24 STSs within the AZF regions on Yq11. These patients included bilateral cases (n=4), cases with cryptorchism (n=21), cases with a positive family history (n=10) for TGCT (Table 1) and patients with proven normal or low sperm counts (Table 2). No AZF deletions were observed in any of the patients. The distribution of nonseminomatous TGCTs and seminomatous TGCTs as shown in Table 1 is related to the referral pattern of TGCT s to the UMCG. Our data confirmed and extended the findings of another study recently published while our study was in progress. Frydelund-Larsen et al. (257) screened 10 Danish TGCT patients for microdeletions on chromosome Yq11. In 103 patients seven STSs spanning the three AZF regions (AFZa, AZFb and AZFc) (plus SRY and ZFX/ZFY) were analyzed. In 57 patients, nine additional STSs spanning AZFabc, (and TSPY on Yp) were studied. Four of the 1 STSs spanning AZFabc (sy84 in AZFa, sy134 in AZFb, and sy152 and sy254 in AZFc) were in common with those studied in our patient group. No AZF deletions were observed in their study population. Because, in the study by Foresta et al., (250) AZF deletions were only found in a group of patients with a history 80 Absence of constitutional Y chromosome AZF deletions in patients with Testicular Germ Cell Tumors 81
A C Figure 2: Multiplexes A-E. B D of cryptorchism together with azoospermia or severe oligozoospermia, it is possible that AZF deletions are only present in that minority of patients with TGCT who also have markedly reduced fertility. In the current study, fertility status (Table 2) was known in 25 patients (22%) and 5 of these had azoospermia after orchiectomy. Data on semen concentration before orchiectomy were not available because the vast majority of patients were referred for treatment after orchiectomy performed elsewhere. Furthermore, semen analyses after orchiectomy was only offered to patients treated with adjuvant chemotherapy or radiotherapy with the intention to father children in the near future. Frydelund-Larsen et al. (257) presented data on fertility for 70 of 10 of their patients before TGCT treatment. A total of 37 of these patients (23% of the total group) had severe (n = 17; less than 5x10 /ml) or very severe (n = 8; less than 0.2x10 /ml) oligozoospermia or azoospermia (n = 12). Although their study as well as the current study did not detect any AZF deletions in patients with TGCT with reduced fertility, our study did not have enough statistical power to exclude low percentages of AZF deletions in small subsets of patients E In conclusion, the data suggest that a substantial contribution of constitutional large AZF deletions to the development of TGCT, whether or not in the presence of reduced fertility, cryptorchism, previous history of TGCT or positive family history, is unlikely. The present data do not rule out the possibility of constitutional smaller deletions or other type of mutations in genes mapped to the AZF region. These genes could therefore be the subject of additional research. Given the complexity of urogenital differentiation and testicular tumor development, only the tip of the iceberg has been mapped with respect to genes involved in these processes to date. As new tools for molecular study become available and mapping efforts advance, more opportunities will undoubtedly arise to explore the molecular basis of the testicular dysgenesis model and testicular tumor development and these explorations should include interactions with environmental risk factors. Note added in proof: A very recent study performed by Nathanson et al. (258) has revealed that a small inherited or de novo deletion within the AZFc region, referred to as the gr/gr deletion appears to be associated with an increased risk to develop TGCT. This gr/gr deletion on the Y chromosome is not detected by the commonly used test for the larger AZF deletions, and was recently found to be a risk factor for spermatogenic failure by Repping et al. (259) Figure 2A-2E. Electrophoresis gel (4% NuSieve 3:1 Plus agarose) showing amplification products for (A) multiplex PCR A, (B) multiplex PCR B, (C) multiplex PCR C, and (D) multiplex PCR D, representing STSs within AZF regions and control STS (SMCX) and (E) multiplex PCR E showing amplification products for multiplex PCR E, representing STSs within AZF regions and control STSs (ZFX/ZFY and sy14). Lanes 1 to 4 = patients with TGCT; L = 50 bp DNA Step Ladder; B = blank (no-dna control), M = normal male control; F = normal female control. 82 Absence of constitutional Y chromosome AZF deletions in patients with Testicular Germ Cell Tumors 83