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Absence of constitutional Y chromosome AZF deletions in patients with testicular germ cell tumors
BASIC SCIENCE
ABSENCE OF CONSTITUTIONAL Y CHROMOSOME AZF DELETIONS IN PATIENTS WITH TESTICULAR GERM CELL TUMORS M. F. LUTKE HOLZIK, K. STORM, R. H. SIJMONS, M. D’HOLLANDER, E. G. J. M. ARTS, M. L. VERSTRAATEN, D. T. SLEIJFER, AND H. J. HOEKSTRA
ABSTRACT Objectives. To investigate the frequency of azoospermia factor (AZF) deletions in Dutch patients with testicular germ cell tumors (TGCTs). Reduced fertility is associated with TGCTs and reduced fertility and TGCTs might share genetic risk factors according to the testicular dysgenesis hypothesis. Up to 8% of infertility and reduced fertility 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 AZF region. Methods. In 112 patients with TGCT, screening for constitutional deletions in the AZF region was performed by multiplex polymerase chain reaction analysis in DNA extracted from peripheral blood lymphocytes. A set of 24 primer pairs, of which 20 primer pairs are homologous to previously identified and mapped sequenced tag sites within the AZF region were used. Results. No deletions in the Yq11 region were detected in any of the 112 patients. Conclusions. Large Y chromosome microdeletions in the AZF region are not a major contributor to the development of TGCT and TGCT-associated reduced fertility. UROLOGY 65: 196–201, 2005. © 2005 Elsevier Inc.
A
lthough testicular germ cell tumors (TGCTs) constitute the most common malignancy in men aged 15 to 40 years, their etiology is still poorly understood.1 In the past few years, a decrease in fertility and an increase in TGCT has been reported.2 This could suggest that fertility and TGCT share a common etiologic factor. A typical illustration is the EastWest 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, This work was supported by a grant from the “Jan Kornelis de Cock” Foundation. From the Departments of Surgical Oncology, Clinical Genetics, Obstetrics and Gynecology, and Medical Oncology, Groningen University Medical Center, Groningen, The Netherlands; Department of Medical Genetics, University of Antwerp, Antwerp, Belgium Reprint requests: H. J. Hoekstra, M.D., Ph.D., Groningen University Medical Center, Department of Surgical Oncology, PO Box 30.001, 9700 RB, Groningen, The Netherlands. E-mail: [email protected] Submitted: May 24, 2004, accepted (with revisions): September 15, 2004 © 2005 ELSEVIER INC. 196
ALL RIGHTS RESERVED
which is inversely related to the lower sperm counts observed in Danish and Norwegian men compared with men from Finland and Estonia.3 A retrospective cohort study of more 30,000 men from infertile couples found an association between infertility and a subsequent risk of TGCT.2 Men with infertility were 1.6 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 1.6. This is a relatively constant risk for TGCT, and impaired spermatogenesis may, therefore, have been present many years before TGCT was diagnosed.2 These results are in line with our previous results showing that patients with TGCT have already impaired spermatogenesis before orchiectomy was performed.4 In the model postulated by Skakkebaek et al.,5 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 predis0090-4295/05/$30.00 doi:10.1016/j.urology.2004.09.022
posing gene mutations may result in TDS. Past research has traditionally focused more 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,1 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).6 – 8 AZFc deletions are the most commonly found (60%). 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.7 Foresta et al.9 recently observed a particularly high percentage of 27.5% AZF (a-c) deletions in patients with low sperm counts, as well as unilateral cryptorchism. Cryptorchism is an acknowledged risk factor for TGCT and is one of the postulated other possible manifestations of the TDS.5,10 Taken together, these observations suggest that, at least from a theoretical point of view, constitutional AZF deletions might be one of the genetic contributors to the 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 the precursor carcinoma in situ, which suggests that loss of Y chromosome material may indeed play a role in TGCT development.11 Bianchi et al.12 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, including 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. MATERIAL AND METHODS PATIENT SELECTION A total of 112 patients with TGCT treated at the Groningen University Medical Center (GUMC) in The NetherUROLOGY 65 (1), 2005
TABLE I. Patient characteristics (n ⴝ 112) Characteristic
Patients (%)
Neoplasia type Nonseminoma Pure seminoma Bilateral TGCT Cryptorchism Familial TGCT
94/112 (84) 18/112 (16) 4/112 (3.5) 21/112 (18.8) 10/112 (9)
KEY: TGCT ⫽ testicular germ cell tumor.
TABLE II. Distribution of semen concentration in 25 patients after orchiectomy Sperm Concentration Normozoospermia (⬎20 ⫻ 106/mL) Moderate oligozoospermia (5–20 ⫻ 106/mL) Severe oligozoospermia (⬍5 ⫻ 106/mL) Azoospermia
Patients (n) 13* 5 2 5*
* Normozoospermia and azoospermia groups both included 1 patient with cryptorchism.
lands were randomly selected for initial analysis. Patient characteristics are listed in Table I. Familial TGCT was defined as more than 1 case in the family. Histologic diagnosis was established in all patients by the Department of Pathology of the GUMC. 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 the semen concentration (Table II). All participants gave their written informed consent, and the ethical committee of the GUMC approved the study.
GENOTYPING High-molecular-weight genomic DNA was extracted from peripheral blood lymphocytes according to standard protocols.13 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.14 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 2.0.15 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 III). All the loci analyzed in this study have been recommended by the European Quality Monitoring Network Group for detection of Yq11 deletions associated with male infertility.15 Primers were combined into five primer sets to use in five parallel PCR amplifications (multiplex PCR A through E; Table III). The slight modifications to the protocol15 provided by the manufacturer were 500 ng DNA in a final volume of 25 L multiplex Master Mix, amplification in 35 197
FIGURE 1. Diagram of Y chromosome with AZF regions, previously cloned genes and pseudogenes, and STSs. Reprinted from Technical Manual No.248.15 Used with permission of Promega Corporation.
TABLE III. Overview of 24 STSs amplified in five multiplex PCR amplifications (A–E)* STS sY81 sY86 sY84 sY182 sY121 SYPR3 sY124 sY127 sY128 sY130 sY133 sY134 sY145 sY152 sY153 sY242 sY239 sY208 sY254 sY255 sY157 sY14
Locus
PCR Fragment (bp)
Multiplex PCR
Position
DYS271 DYS148 DYS273 KAL-Y DYS212 SMCY DYS215 DYS218 DYS219 DYS221 DYS223 DYS224 DYF51S1 DYS236 DYS237 DAZ DAZ DAZ DAZ DAZ DYS240 SRY SMCX ZFX/ZFY
209 232 177 125 190 350 109 274 228 173 177 303 142 285 139 233 200 140 370 126 285 400 83 496
A E E A C B D B C A D E C D D B B B A C A E A–D E
Distal to AZFa AZFa AZFa Proximal to AZFa AZFb AZFb AZFb AZFb AZFb AZFb AZFb AZFb Proximal to AZFc ⫽ AZFd Proximal to AZFc ⫽ AZFd AZFd (nonpathogenic) AZFc AZFc AZFc AZFc AZFc Distal to AZFc SRY gene X chromosome (control) X chromosome (control)
KEY: STSs ⫽ sequenced tag sites; PCR ⫽ polymerase chain reaction; AZF ⫽ azoospermia factor. * 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).
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 198
gels (Cambrex Bio Science), stained with Ethidium Bromide (Fig. 2). The multiplex primer sets A through D contained a control primer pair that amplified 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 amUROLOGY 65 (1), 2005
FIGURE 2. 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. plification 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 system15 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 among infertile and subfertile men who did not have a history of TGCT (data not shown). UROLOGY 65 (1), 2005
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 were detected in the blank (no DNA) control. As expected, the positive male control showed the ap199
propriate 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.
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.
COMMENT 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 I), and patients with proven normal or low sperm counts (Table II). No AZF deletions were observed in any of the patients. The distribution of nonseminomatous TGCTs and seminomatous TGCTs (Table I) is related to the referral pattern of TGCT’s to the GUMC. Our data confirmed and extended the findings of another study recently published while our study was in progress. Frydelund-Larsen et al.16 screened 160 Danish patients with TGCT 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 16 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.,9 AZF deletions were only found in a group of patients with a history 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 II) 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.16 presented data on fertility for 70 of 160 of their patients before TGCT treatment. A total of 37 of these patients (23% of the total group) had severe (n ⫽ 17; less than 5 ⫻ 106/mL) or very severe (n ⫽ 8; less than 0.2 ⫻ 106/mL) oligozoospermia or azoospermia (n ⫽ 200
CONCLUSIONS 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.17 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.18 ACKNOWLEDGMENT. To Jan Osinga for technical support in the AZF analysis. REFERENCES 1. Lutke Holzik MF, Rapley EA, Hoekstra HJ, et al: Genetic predisposition to testicular germ-cell tumours. Lancet Oncol 5: 363–371, 2004. 2. Jacobsen R, Bostofte E, Engholm G, et al: Risk of testicular cancer in men with abnormal semen characteristics: cohort study. BMJ 321: 789 –792, 2000. 3. Jorgensen N, Carlsen E, Nermoen I, et al: 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 17: 2199 –2208, 2002. 4. Nijman JM, Schraffordt Koops H, Kremer J, et al: Fertility and hormonal function in patients with a nonseminomatous tumor of the testis. Arch Androl 14: 239 –246, 1985. 5. Skakkebaek NE, Rajpert-De Meyts E, and Main KM: Testicular dysgenesis syndrome: an increasingly common deUROLOGY 65 (1), 2005
velopmental disorder with environmental aspects. Hum Reprod 16: 972–978, 2001. 6. Kent-First M, Muallem A, Shultz J, et al: Defining regions of the Y-chromosome responsible for male infertility and identification of a fourth AZF region (AZFd) by Y-chromosome microdeletion detection. Mol Reprod Dev 53: 27– 41, 1999. 7. Layman LC: Human gene mutations causing infertility. J Med Genet 39: 153–161, 2002. 8. Vogt PH, Edelmann A, Kirsch S, et al: Human Y chromosome azoospermia factors (AZF) mapped to different subregions in Yq11. Hum Mol Genet 5: 933–943, 1996. 9. Foresta C, Moro E, Garolla A, et al: Y chromosome microdeletions in cryptorchidism and idiopathic infertility. J Clin Endocrinol Metab 84: 3660 –3665, 1999. 10. Herrinton LJ, Zhao W, and Husson G: Management of cryptorchism and risk of testicular cancer. Am J Epidemiol 157: 602– 605, 2003. 11. van Echten J, and de Jong B: Cytogenetics of testicular germ cell tumors of adults. Cancer J 11: 242–246, 1998. 12. Bianchi NO, Richard SM, Peltomaki P, et al: Mosaic AZF deletions and susceptibility to testicular tumors. Mutat Res 503: 51– 62, 2002.
UROLOGY 65 (1), 2005
13. Miller SA, Dykes DD, and Polesky HF: A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16: 1215–1216, 1988. 14. Aknin-Seifer IE, Touraine RL, Lejeune H, et al: A simple, low cost and non-invasive method for screening Ychromosome microdeletions in infertile men. Hum Reprod 18: 257–261, 2003. 15. Y chromosome deletion detection system, version 2.0. Technical Manual No. 248. USA, Promega Corporation, 2003. 16. Frydelund-Larsen L, Vogt PH, Leffers H, et al: No AZF deletion in 160 patients with testicular germ cell neoplasia. Mol Hum Reprod 9: 517–521, 2003. 17. Nathanson K, Kanetsky P, Hawes R, et al: The Y deletion gr/gr confers susceptibility to testicular germ cell cancer. Abstract presented at the 2004 annual meeting of the American Society of Human Genetics (ASHG), Toronto, Canada. Session 59. Program Number 237. (http://genetics.faseb.org/ genetics/ashg/annmeet/2004/sess-59.shtml). 18. Repping S, Skaletsky H, Brown L, et al: Polymorphism for a 1.6-Mb deletion of the human Y chromosome persists through balance between recurrent mutation and haploid selection. Nat Genet 35: 247–251, 2003.
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ABSENCE OF CONSTITUTIONAL Y CHROMOSOME AZF DELETIONS IN PATIENTS WITH TESTICULAR GERM CELL TUMORS M. F. LUTKE HOLZIK, K. STORM, R. H. SIJMONS, M. D’HOLLANDER, E. G. J. M. ARTS, M. L. VERSTRAATEN, D. T. SLEIJFER, AND H. J. HOEKSTRA
ABSTRACT Objectives. To investigate the frequency of azoospermia factor (AZF) deletions in Dutch patients with testicular germ cell tumors (TGCTs). Reduced fertility is associated with TGCTs and reduced fertility and TGCTs might share genetic risk factors according to the testicular dysgenesis hypothesis. Up to 8% of infertility and reduced fertility 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 AZF region. Methods. In 112 patients with TGCT, screening for constitutional deletions in the AZF region was performed by multiplex polymerase chain reaction analysis in DNA extracted from peripheral blood lymphocytes. A set of 24 primer pairs, of which 20 primer pairs are homologous to previously identified and mapped sequenced tag sites within the AZF region were used. Results. No deletions in the Yq11 region were detected in any of the 112 patients. Conclusions. Large Y chromosome microdeletions in the AZF region are not a major contributor to the development of TGCT and TGCT-associated reduced fertility. UROLOGY 65: 196–201, 2005. © 2005 Elsevier Inc.
A
lthough testicular germ cell tumors (TGCTs) constitute the most common malignancy in men aged 15 to 40 years, their etiology is still poorly understood.1 In the past few years, a decrease in fertility and an increase in TGCT has been reported.2 This could suggest that fertility and TGCT share a common etiologic factor. A typical illustration is the EastWest 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, This work was supported by a grant from the “Jan Kornelis de Cock” Foundation. From the Departments of Surgical Oncology, Clinical Genetics, Obstetrics and Gynecology, and Medical Oncology, Groningen University Medical Center, Groningen, The Netherlands; Department of Medical Genetics, University of Antwerp, Antwerp, Belgium Reprint requests: H. J. Hoekstra, M.D., Ph.D., Groningen University Medical Center, Department of Surgical Oncology, PO Box 30.001, 9700 RB, Groningen, The Netherlands. E-mail: [email protected] Submitted: May 24, 2004, accepted (with revisions): September 15, 2004 © 2005 ELSEVIER INC. 196
ALL RIGHTS RESERVED
which is inversely related to the lower sperm counts observed in Danish and Norwegian men compared with men from Finland and Estonia.3 A retrospective cohort study of more 30,000 men from infertile couples found an association between infertility and a subsequent risk of TGCT.2 Men with infertility were 1.6 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 1.6. This is a relatively constant risk for TGCT, and impaired spermatogenesis may, therefore, have been present many years before TGCT was diagnosed.2 These results are in line with our previous results showing that patients with TGCT have already impaired spermatogenesis before orchiectomy was performed.4 In the model postulated by Skakkebaek et al.,5 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 predis0090-4295/05/$30.00 doi:10.1016/j.urology.2004.09.022
posing gene mutations may result in TDS. Past research has traditionally focused more 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,1 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).6 – 8 AZFc deletions are the most commonly found (60%). 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.7 Foresta et al.9 recently observed a particularly high percentage of 27.5% AZF (a-c) deletions in patients with low sperm counts, as well as unilateral cryptorchism. Cryptorchism is an acknowledged risk factor for TGCT and is one of the postulated other possible manifestations of the TDS.5,10 Taken together, these observations suggest that, at least from a theoretical point of view, constitutional AZF deletions might be one of the genetic contributors to the 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 the precursor carcinoma in situ, which suggests that loss of Y chromosome material may indeed play a role in TGCT development.11 Bianchi et al.12 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, including 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. MATERIAL AND METHODS PATIENT SELECTION A total of 112 patients with TGCT treated at the Groningen University Medical Center (GUMC) in The NetherUROLOGY 65 (1), 2005
TABLE I. Patient characteristics (n ⴝ 112) Characteristic
Patients (%)
Neoplasia type Nonseminoma Pure seminoma Bilateral TGCT Cryptorchism Familial TGCT
94/112 (84) 18/112 (16) 4/112 (3.5) 21/112 (18.8) 10/112 (9)
KEY: TGCT ⫽ testicular germ cell tumor.
TABLE II. Distribution of semen concentration in 25 patients after orchiectomy Sperm Concentration Normozoospermia (⬎20 ⫻ 106/mL) Moderate oligozoospermia (5–20 ⫻ 106/mL) Severe oligozoospermia (⬍5 ⫻ 106/mL) Azoospermia
Patients (n) 13* 5 2 5*
* Normozoospermia and azoospermia groups both included 1 patient with cryptorchism.
lands were randomly selected for initial analysis. Patient characteristics are listed in Table I. Familial TGCT was defined as more than 1 case in the family. Histologic diagnosis was established in all patients by the Department of Pathology of the GUMC. 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 the semen concentration (Table II). All participants gave their written informed consent, and the ethical committee of the GUMC approved the study.
GENOTYPING High-molecular-weight genomic DNA was extracted from peripheral blood lymphocytes according to standard protocols.13 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.14 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 2.0.15 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 III). All the loci analyzed in this study have been recommended by the European Quality Monitoring Network Group for detection of Yq11 deletions associated with male infertility.15 Primers were combined into five primer sets to use in five parallel PCR amplifications (multiplex PCR A through E; Table III). The slight modifications to the protocol15 provided by the manufacturer were 500 ng DNA in a final volume of 25 L multiplex Master Mix, amplification in 35 197
FIGURE 1. Diagram of Y chromosome with AZF regions, previously cloned genes and pseudogenes, and STSs. Reprinted from Technical Manual No.248.15 Used with permission of Promega Corporation.
TABLE III. Overview of 24 STSs amplified in five multiplex PCR amplifications (A–E)* STS sY81 sY86 sY84 sY182 sY121 SYPR3 sY124 sY127 sY128 sY130 sY133 sY134 sY145 sY152 sY153 sY242 sY239 sY208 sY254 sY255 sY157 sY14
Locus
PCR Fragment (bp)
Multiplex PCR
Position
DYS271 DYS148 DYS273 KAL-Y DYS212 SMCY DYS215 DYS218 DYS219 DYS221 DYS223 DYS224 DYF51S1 DYS236 DYS237 DAZ DAZ DAZ DAZ DAZ DYS240 SRY SMCX ZFX/ZFY
209 232 177 125 190 350 109 274 228 173 177 303 142 285 139 233 200 140 370 126 285 400 83 496
A E E A C B D B C A D E C D D B B B A C A E A–D E
Distal to AZFa AZFa AZFa Proximal to AZFa AZFb AZFb AZFb AZFb AZFb AZFb AZFb AZFb Proximal to AZFc ⫽ AZFd Proximal to AZFc ⫽ AZFd AZFd (nonpathogenic) AZFc AZFc AZFc AZFc AZFc Distal to AZFc SRY gene X chromosome (control) X chromosome (control)
KEY: STSs ⫽ sequenced tag sites; PCR ⫽ polymerase chain reaction; AZF ⫽ azoospermia factor. * 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).
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 198
gels (Cambrex Bio Science), stained with Ethidium Bromide (Fig. 2). The multiplex primer sets A through D contained a control primer pair that amplified 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 amUROLOGY 65 (1), 2005
FIGURE 2. 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. plification 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 system15 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 among infertile and subfertile men who did not have a history of TGCT (data not shown). UROLOGY 65 (1), 2005
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 were detected in the blank (no DNA) control. As expected, the positive male control showed the ap199
propriate 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.
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.
COMMENT 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 I), and patients with proven normal or low sperm counts (Table II). No AZF deletions were observed in any of the patients. The distribution of nonseminomatous TGCTs and seminomatous TGCTs (Table I) is related to the referral pattern of TGCT’s to the GUMC. Our data confirmed and extended the findings of another study recently published while our study was in progress. Frydelund-Larsen et al.16 screened 160 Danish patients with TGCT 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 16 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.,9 AZF deletions were only found in a group of patients with a history 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 II) 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.16 presented data on fertility for 70 of 160 of their patients before TGCT treatment. A total of 37 of these patients (23% of the total group) had severe (n ⫽ 17; less than 5 ⫻ 106/mL) or very severe (n ⫽ 8; less than 0.2 ⫻ 106/mL) oligozoospermia or azoospermia (n ⫽ 200
CONCLUSIONS 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.17 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.18 ACKNOWLEDGMENT. To Jan Osinga for technical support in the AZF analysis. REFERENCES 1. Lutke Holzik MF, Rapley EA, Hoekstra HJ, et al: Genetic predisposition to testicular germ-cell tumours. Lancet Oncol 5: 363–371, 2004. 2. Jacobsen R, Bostofte E, Engholm G, et al: Risk of testicular cancer in men with abnormal semen characteristics: cohort study. BMJ 321: 789 –792, 2000. 3. Jorgensen N, Carlsen E, Nermoen I, et al: 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 17: 2199 –2208, 2002. 4. Nijman JM, Schraffordt Koops H, Kremer J, et al: Fertility and hormonal function in patients with a nonseminomatous tumor of the testis. Arch Androl 14: 239 –246, 1985. 5. Skakkebaek NE, Rajpert-De Meyts E, and Main KM: Testicular dysgenesis syndrome: an increasingly common deUROLOGY 65 (1), 2005
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13. Miller SA, Dykes DD, and Polesky HF: A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16: 1215–1216, 1988. 14. Aknin-Seifer IE, Touraine RL, Lejeune H, et al: A simple, low cost and non-invasive method for screening Ychromosome microdeletions in infertile men. Hum Reprod 18: 257–261, 2003. 15. Y chromosome deletion detection system, version 2.0. Technical Manual No. 248. USA, Promega Corporation, 2003. 16. Frydelund-Larsen L, Vogt PH, Leffers H, et al: No AZF deletion in 160 patients with testicular germ cell neoplasia. Mol Hum Reprod 9: 517–521, 2003. 17. Nathanson K, Kanetsky P, Hawes R, et al: The Y deletion gr/gr confers susceptibility to testicular germ cell cancer. Abstract presented at the 2004 annual meeting of the American Society of Human Genetics (ASHG), Toronto, Canada. Session 59. Program Number 237. (http://genetics.faseb.org/ genetics/ashg/annmeet/2004/sess-59.shtml). 18. Repping S, Skaletsky H, Brown L, et al: Polymorphism for a 1.6-Mb deletion of the human Y chromosome persists through balance between recurrent mutation and haploid selection. Nat Genet 35: 247–251, 2003.
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