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Ethnic variation in the prevalence of AZF deletions in testicular cancer
Mutation Research 554 (2004) 45–51
Ethnic variation in the prevalence of AZF deletions in testicular cancer S.M. Richard a , N.O. Bianchi a,∗ , M.S. Bianchi a , P. Peltomäki b , R.A. Lothe c , W. Pavicic a a
c
Instituto Multidisciplinario de Biolog´ıa Celular (IMBICE), La Plata, Argentina b Department of Medical Genetics, University of Helsinki, Helsinki, Finland Department of Genetics, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo, Norway Received 17 December 2003; received in revised form 16 February 2004; accepted 1 March 2004 Available online 27 April 2004
Abstract Seventy-six percent of testicular cancers of origin in Finns have been reported to exhibit AZF deletions. We analyze here 40 testicular tumor cases from Norway and Argentina and we found that AZF deletions occur also in non-Finnish cases but at significantly lower frequency (25%) than in Finland testicular tumor cases. This frequency difference can be attributed to the condition of genetic isolate of the Finnish population and the subsequent prevalence in this ethnic group of genetic factors involved in the origin of AZF deletions associated with malignancies. The finding of a correlation between AZF deletions and a given Y haplogroup would indicate the existence of Y lineages carrying AZF deletion-enhancing gene or genes. This possibility was explored using a set of Y-DNA-markers allowing the identification of Y lineages occurring at high frequency in Finns. We characterized the Y haplogroups in 32 normal Finn males (control group) and 17 cases of testicular cancer in Finns with and without AZF deletions. We found no association between Y lineages and AZF microdeletions, nor between AZF microdeletions and a Y microdeletion (DYS7C) exhibiting high prevalence (>50%) in Finns. The lack of correlation between AZF deletions and Y haplogroups indicates that the origin of these deletions is independent from the Y chromosome genetic background. No AZF deletions were found in familial cases of testicular tumors; hence, hereditary factors inducing the appearance of testicular malignancies in certain genealogies apparently do not increase the susceptibility to AZF deficiencies. AZF deletions are de novo events occurring in prezygotic or in post-zygotic stages. We propose that most AZF deletions associated with testicular tumors are due to post-zygotic Y microdeletions, while most cases of deletions associated with infertility are due to deletions occurring in the germ cell line of proband fathers. © 2004 Elsevier B.V. All rights reserved. Keywords: Testicular tumors; AZF deletions; MGCT; AZF deletions and testicular tumors
1. Introduction
∗ Corresponding author. Fax: +54-221-425-3320. E-mail addresses: [email protected], [email protected] (N.O. Bianchi).
Several publications indicate that during the last 30–50 years there has been a progressive decrease in sperm concentration and quality of ejaculates from normal males, an increase in the incidence of male
0027-5107/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2004.03.001
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infertility, and a higher prevalence of testicular cancers in the general male population [1–4]. Moreover, the association between decreased sperm concentration and malignant diseases is further stressed by the finding that infertility may precede the appearance of lymphomas, testicular tumors, and several forms of endocrine cancers [5]. Since approximately 15–25% of idiopathic male infertility cases [6–9] and 0.87–2% of normal fertile males [7,10] have been reported to exhibit AZF deletions, we tested this gene family in a series of male tumors (MGCT) in order to determine whether AZF instability was an event also associated with testicular tumors. Thus, we screened 17 loci corresponding to AZF (a–d) subintervals, as defined by Kent-First et al. [7] in normal and tumor tissues of 17 Finn males with testicular malignancies. We found that 13 testicular cancer cases (76%) had AZF deletions [11]. Since 2 of the cases with Y microdeletions were testicular forms of non-Hodgkin lymphoma (the other 11 cases having Y instability were MGCTs), we extended the deletion testing to a series of 27 cases of Finn males with non-Hodgkin lymphoproliferative disorders: AZF deletions were detected in 66.6% (18/27) of patients [12]. Furthermore, no AZF deletions were detected in the fathers of testicular tumor cases or in the Y chromosomes from 32 normal Finn males or 48 normal males of European ancestry [11]. Thus far, all testicular tumor cases tested for AZF deletions were Finnish patients. In this report, we shall analyze whether the association of AZF deletions with testicular tumors is restricted to Finns or is a more general phenomenon taking place in other geographic populations as well. Moreover, we shall test whether the AZF deficiencies found in malignancies preferentially occur in a given Y lineage or in association with DYS7C deletions, a type of deficiency occurring at high frequency in normal Finn Y chromosomes [13].
2. Material and methods 2.1. AZF testing Four non-commercial multiplex PCR reactions were used to detect the presence or absence of fragments corresponding to 17 AZF loci comprising the four subintervals (a–d), as defined by Kent-First et al. [7];
details on loci analyzed, the subinterval represented by each locus, and loci tested with each multiplex reaction are given in Table 1. Fragments lacking in multiplex PCR reactions were further evaluated in duplex PCR assays performed with two pairs of primers, one specific for the putative deleted fragment and the other specific for an AZF fragment that tested positive in the multiplex reaction and that was included in the duplex assay as an internal positive control. A fragment was considered deleted when it was absent in two independent multiplex reactions and in one or two duplex PCR assays. All fragments considered deleted in primary PCR reactions were retested in a secondary PCR assay using as target 0.5 l of the primary PCR amplified mixture, in a total volume of 15 l. Multiplex PCR reactions showing all the expected fragments were repeated once in order to identify false positives; no false positive fragments were detected. Primary and secondary PCR amplifications were carried out with the usual precautions used for ancient DNA testing. Sample handling and laboratory procedures were performed by female personnel in order to minimize chances of male DNA contamination in PCR reactions. PCR conditions employed are detailed in Bianchi et al. [11]. Interpretation of electrophoresis fragment patterns was performed at blind by two different investigators; in all cases there was agreement in the independent diagnosis of fragment deletions. AZF1 replaced AZF in recent nomenclature systems. In this report, however, we shall use the AZF acronym in order to maintain the same nomenclature employed in our previous publications [11,12]. 2.2. Y-specific markers Additional markers used were: YAP+ [14] or M1 according to Underhill et al. [15,16] nomenclature system (Y-specific Alu insertion); TAT-T →C transition [17] or M46, according to Underhill et al. [15,16]; M9 [15,16], and the DYS7C deletion (50f/2C locus, [13]). PCR conditions, restriction enzymes used, and electrophoresis identification patterns for each of the above markers are given in the aforementioned bibliographic references. DNA markers other than AZF deletions were analyzed in all samples from our previously published series of Finn testicular tumor cases Tt1–Tt17 (previously 1–17, [11]) and in 32 male DNA samples
+ + + +
+: no deletion; a: −/− deletion in normal and tumor samples; b: +/− deletion in tumor sample; c: −/+ deletion in normal sample. Tt 52 and Tt52* identify primary and contraleteral or secondary tumor samples, respectively. In case 51, the Tm sample corresponds to a pulmonary metastasis. Superscript letters (A–D) indicate the AZF fragments included in 1–4 multiplex PCR reactions. In cases with bilateral tumors asterisks indicate primary malignancies. a The first term corresponds to deletion patterns with primary PCRs; the second term after the bar indicates deletion patterns after PCR reamplification.
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + c/c + c/c + + b/b b/b + + c/+ + + c/c + + + + b/b b/b + + a/a b/b + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + b/+ + + + + + c/c b/+ + + + + b/b + + + + + + + + c/+ + + + + + + + c/c + c/c + + + + b/b + b/b c/+ + + c/c + b/b + b/b b/b b/b + + c/+ + c/c + b/b + + + + b/b + b/b c/+ + + + + + + + +
Tt26 Tt28 Tt32 Tt35 Tt36 Tt41 Tt51 Tm51 Tt52 Tt52* Tt53 Tt54
(SY81)
+ + + + c/+ + + b/b + + + +
+ c/c + + + + + b/+ + b/b + +
DAZ B (SY242) DYS223 D (SY133) DYS215 D (SY124) DYS212 C (SY121)
AZF b
KALY A (SY182) DYS271A
AZF a Cases
Table 1 AZF deletions in testicular tumor (Tt)a
DYS218 B (SY127)
DYS219 C (SY128)
DYS221 A (SY130)
AZF d
DYF51S1 C (SY145)
DYS237 D (SY153)
DYS236 D (SY152)
AZF c
DAZ B (SY239)
DAZ B (SY208)
DAZ A (SY254)
DAZ C (SY255)
DYS240 A (SY157)
S.M. Richard et al. / Mutation Research 554 (2004) 45–51
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randomly selected from a geographically weighted sampling of the entire Finnish population. 2.3. Samples analyzed Histological classification of testicular tumors was performed according to WHO recommendations. The main histological subtypes are seminomas and non-seminomas of which the latter may contain different histological elements including embryonal carcinoma, choriocarcinoma, yolk sac tumor, and teratoma. Testicular tumor samples were arbitrarily identified with the acronym Tt and correlatively numbered, the first number in this series being the next one (Tt18) to that in the series of testicular malignancies previously reported [11]. Tt18–52 are testicular tumor cases from Norway, and Tt53–57 are samples from La Plata city, Argentina. Tt19, 39, 40, 42, 48, 49 are six cases from five different families showing testicular malignancies in relatives of the proband. All other cases were sporadic forms of MGCT. Tt38, 41, 42, 43–48 and Tt52 had history of bilateral cancer. Both tumors from case 52 were included in the present study. Moreover, the primary tumor and a pulmonary metastasis were analyzed from case 51. In all cases reported here, AZF deletion testing was performed in DNA samples from non-malignant (blood) and malignant tissues. Histological diagnoses of the frozen testicular tumor samples in this series are: seminomas Tt19, 21, 24, 25, 27–29, 31–34, 38, 45–49, 53, 54. 56, 57; embryonal carcinoma components Tt20, 22, 23, 26, 30, 35, 40; yolk sac tumor component Tt37; nonseminoma (more than one component in the frozen tissue biopsy) Tt36, 39, 41, 42, 55; immature teratoma Tt51; combination of seminoma and non-seminoma or mixed germ cell tumors (S/NS) Tt18, 44, 50, 52. Concentration of tumor cells in tumor samples was in average 60%. The use of tumor and non-tumor samples for the research purposes reported here were approved by the Ethics Committees of Hospitals in which the patients were admitted. 3. Results 3.1. AZF deletions Over a total of 40 MGCTs analyzed, 10 (25%) showed AZF deletions involving 1–9 loci per case
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(Table 1). When the patterns of AZF deletions in non-tumor versus tumor tissues from the same donor were compared, we observed the pattern “a” (deletion in non-tumor and tumor tissues or: −/−) in one locus (3.1% of the 32 AZF deleted loci), the pattern “b” (deletion in tumor tissues only or: +/−) in 16 loci (50%), and the pattern “c” (deletion only in non-tumor tissues or: −/+) in 15 loci (46.9%) (Table 1). In three loci with deletion pattern “b” (+/−) and in six loci with deletion pattern “c” (−/+) the fragment lacking in primary PCRs appeared in PCR reamplifications; these loci are identified as b/+ and c/+, respectively, in Table 1. The appearance in PCR reamplifications of fragments that were lacking in primary PCRs is expected to occur in mosaicisms in which there is a marked prevalence of deleted over non-deleted Y chromosomes [11,12]. In order to test the ratio of non-deleted versus deleted Ys producing deletion in primary with presence of the fragment in secondary PCR and deletion in both PCR reactions, we used as PCR target a mixture of 50 ng of a DNA sample with deletions in one or more AZF loci plus increasing dilutions of a DNA sample without AZF deletions (the final volume of PCR mixtures was 15 l). We found that, when a non-deleted fragment reached a dilution of 1/300 in regard to the homologous deleted locus, the fragment did not show in primary PCRs but tested positive in PCR reamplifications; at dilutions higher than 1/1000 the fragment failed to appear in primary and secondary PCRs. One Norwegian MGCT case (Tt52) treated with orchidectomy developed a secondary tumor in the remaining testis. This case had no deletions in one of the tumors and three pattern “b” deletions in the contralateral one (Table 1). Differences in the deletion pattern of primary and secondary MTGCTs provide additional support to the hypothesis of an AZF mosaicism occurring in early embryogenesis. If non-tumor cells are a mosaic with two or more cell lineages having AZF deletions in different loci, and if the primary and secondary tumors are independent events involving transformation in distinct cell lineages, then AZF deletion patterns detected for each tumor are expected to differ. Moreover, the finding of a disparate deletion pattern in a testicular tumor and its pulmonary metastasis (Tt51/Tm51, Table 1) is also compatible with the assumption of a different origin and evolution of the primary and secondary malignant events.
Lately, Frydelund-Larsen et al. [18] found no AZF deletions in blood DNA from a series of 160 Danish cases of MGCT. The deletion screening performed by these researchers comprised AZF a–c subintervals that were represented by 9 loci in 103 cases and by 17 loci in the remaining 57 cases. A decrease in the frequency of AZF deletions in testicular cancer cases of Danish origin in regard to deletion frequencies observed in other geographic populations should be borne in mind in order to explain the discrepancy between Frydelund-Larsen and our results. Yet, some other factors may also give rise to the above interlaboratory differences. All loci showing the AZF deletion pattern b (+/−) will be labeled as non-deleted if the deletion screening is restricted to non-tumor samples. Table 1 of this report shows that the pattern b represents 61.4% of the total deletion events and that almost half of our cases with deletions would have been considered non-deleted if tumor tissues would have been excluded from the deletion screening. Moreover, in our experience, the frequency of AZF deletions in MGCT cases varies depending on the locus tested. Only two of the loci tested by Frydelund-Larsen et al. [18] (DAZ-SY254) and DYS237) were also used by us in this report. No DAZ (SY254) or DYS237 fragment deletion occurred in the cases reported here (Table 1) and only one DAZ-SY254 deletion in primary but not in secondary PCR was detected in tumor tissues in one case from our Finnish series [11]. Accordingly, two aspects to tackle in the future would be: (i) to detect the AZF loci most frequently deleted in MGCTs; and (ii) to determine the best number of AZF loci to test for an efficient identification of deletions in testicular tumors. 3.2. Y-chromosome lineages, DYS7C deletions One of the aims of this investigation was to establish whether AZF deletions are associated with a given Y haplogroup or whether there is a correlation between AZF and DYS7C deletions in testicular tumors. Finn Y haplogroups have been well characterized by Raitio et al. [19]. Moreover, the highest incidence of AZF deletions in malignancies and the highest incidence of DYS7C deletions in normal males are found in Finnish populations [11–13]. Accordingly, the use of YAP (M1), M46, and M9 markers to characterize Y lineages and the search for a correlation between
S.M. Richard et al. / Mutation Research 554 (2004) 45–51
AZF and DYS7C deletions were restricted to the series of testicular tumors in Finns reported in Bianchi et al. [11]. We detected three haplogroups: H2 (YAP-/M9C/ M46T), H3+ (YAP-/M9G/M46T), and H16 (YAP-/ M9G/M46C) corresponding to the most frequent Finn Y lineages reported by Raitio et al. [19]. H2 and H3+ had both the same distribution: they were detected in seven controls and in three and one MGCTs with and without deletions respectively. H16, on the other hand, occurred in 18 controls and in seven and two MGCTs with and without deletions respectively. Differences in the distribution of haplogroups in control, malignant deleted, and malignant non-deleted cases were not significant indicating lack of association between AZF deletions and Y parental lineages. The frequency of DYS7C deleted Y chromosomes was equal (63.6%) in normal Finn males and in Finn cases with malignancies (21/33 and 28/44, respectively). This percentage is in good agreement with the 55% of DYS7C deletions reported by Jobling et al. [13] for normal Finns. Hence, no correlation between AZF and DYS7C deletions was found.
4. Discussion 4.1. Frequency of AZF deletions in testicular tumors from different geographic populations Our results indicate that AZF deletions in testicular malignancies are not restricted to the Finn population. Yet, the deletion frequency is three-fold higher in Finns than in males from other geographic populations: 76.4% in Finn [11] versus 25% for Norwegian/La Plata cases of testicular cancer (P < 0.01, Fisher exact test). Due to several factors, the population of Finland has the characteristics of a genetic isolate [20–22]. The prevalence in Finland of hereditary syndromes, rare in other geographic regions conform the so called Finnish Disease Heritage group of syndromes [23,24]. Moreover, the existence of Y chromosome lineages that are almost Finn-specific [19,25], and the high incidence in Finns of a recurrent Y chromosome deletion (DYS7C) at the 50f2/C locus [13] are peculiarities that can be grouped under the name of Finnish Y Heritage (FYH) and that probably result from the condition of genetic
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isolate of the Finnish population. Thus, the high incidence of AZF deletions found in Finn patients with testicular malignancies should be included within the FYH and might be due to the prevalence in Finn males of genetic factors favoring the appearance of Y deletions. The risk of developing a testicular tumor for the sibling of a tumor case is 10-fold higher than the lifetime risk for a random case from the general population [26]. Moreover, testicular tumor cases have a 27-fold risk for developing a secondary tumor in the remaining testis [27,28]. Accordingly, familial and bilateral testicular tumor cases form a group of malignancies in which the genetic causes inducing testicular carcinogenesis apparently play a more relevant role than in sporadic cases of testicular tumors. In the Norwegian series of testicular malignancies reported here, AZF deletions were found in sporadic but not in familial cases of MGCTs. Hence, we might assume that genetic factors favoring the development of familial testicular cancers are not related with the susceptibility to AZF deletions. A Y haplogroup can be defined as a monophyletic group of Y chromosomes sharing the same allelic states in the non-recombinant Y region. From this, we may infer that the finding of a preferential association of AZF deletions with a given haplogroup would indicate the existence of Y lineages carrying a deletion-enhancing gene or genes. At least two reports, Kuroki et al. [29] on Japanese and Krausz et al. [30] on Danish males claim to have detected an association between oligo/azoospermia and specific Y haplogroups. On the other hand, Carvalho et al. [31] were unable to establish a correlation between male infertility, AZF deletions, and Y haplogroups in Japanese males having reduced sperm counts. Our results for MGCTs are coincident with those from Carvalho et al. [31]. By using a set of three biallelic markers we were able to identify three haplogroups of wide distribution in Finns [19]. The frequency of these lineages in control and MGCT cases showed no difference indicating a lack of association between testicular malignancies and a given Y haplogroup. DYS7C deletions occur in the 50f2C locus that maps in 6C deletion interval, in relatively close proximity to the AZFd subinterval. DYS7C deficiencies are known to be associated with five to seven different Y haplogroups indicating a recurrent process involving five to seven indepen-
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dent DYS7C deletion events [13]. The lack of correlation between AZF and Y haplogroups and between AZF and DYS7C deficiencies further confirms that the AZF instability in testicular malignancies is independent from Y chromosome genetic background. 4.2. AZF deletions in infertility and malignancies Most AZF deficiencies are de novo events due to deletions occurring in germ cells or in post-zygotic stages. Germ cell deletions generate a mosaic sperm population carrying normal and AZF deleted Y chromosomes. If a viable product results from the fertilization of a normal oocyte by a Y-deleted sperm, the outcome will be a sexually mature individual having the same form of AZF deletion in all tissues and a spermatogenesis impairment ranging from severe to minor or even no impairment at all [7,32]. PCR deletion analyses of these cases usually show extensive AZF deficiencies involving several loci in the same or in adjacent AZF subintervals [7,10]. On the other hand, post-zygotic deletions take place during the early embryogenesis, giving rise to mosaic individuals exhibiting somatic and germ cell lineages with and without AZF deletions. Due to the mixture of Y chromosomes having deleted and non-deleted AZF loci, the testing of AZF deletions by PCR predominantly exhibits an interstitial or scattered pattern of Y microdeletions [11]. Since AZF deficiencies associated with malignant processes show a scattered form of deletions, it can be tentatively assumed that these deletions preferentially occur through a post-zygotic mechanism [11,12]. Conversely, although post-zygotic deletions occasionally occur in infertile individuals [33], most AZF deletions associated with infertility are due to Y microdeletions taking place in the germ cell line of the proband father. Post-zygotic AZF deletions might be assumed to be markers of susceptibility to testicular cancer on the basis that they occur during early embryogenesis and precede by many years the appearance of tumors [11]. In this regard, it is worth commenting here the findings of Kent-First et al. [7] and Pryor et al. [10]. The group of Pryor found four cases (2%) with AZF deletions in a series of 200 normal males, whereas Kent-First et al. [7] detected eight cases (0.87%) with AZF deleted Y chromosomes in a series of 920 fertile males. Deletions in these cases were interstitial and involved 1–4 loci out of a total of 36 [10] or 48 [7] AZF
loci tested; a pattern compatible with that assumed to occur in post-zygotic deletion mosaicisms. Whether or not these AZF deleted males were at high risk of developing MGCTs is still an open question. The screening for post-zygotic deletions in a general male population and the subsequent follow up of deleted cases for the appearance of testicular malignancies would be time consuming. Conversely, the search for AZF deletions in male relatives of familial and sporadic cases of testicular tumors would be useful to evaluate with less effort whether these deletions are a marker of susceptibility to testicular cancer.
Acknowledgements This work was supported by grants from CONICET, CICPBA, and ANPCyT of Argentina, and the Academy of Finland. DNA samples from normal Finn males were kindly provided by A. de la Chapelle and A.-E. Lehesjoki. References [1] E. Carlsen, A. Giwercman, N. Keiding, N.E. Skakkebaek, Evidence for decreasing quality of semen during the past 50 years, Br. Med. J. 305 (1992) 609–613. [2] J. Auger, J.M. Kunstmann, F. Czyglik, P. Jouannet, Decline in semen quality among fertile men in Paris during the past 20 years, N. Engl. J. Med. 332 (1995) 281–285. [3] R. Jacobsen, E. Bostofte, G. Engholm, J. Hansen, N.E. Skakkebaek, H. Moller, Fertility and offspring sex ratio of men who develop testicular cancer: a record linkage study, Hum. Reprod. 15 (2000) 1958–1961. [4] J. Schuz, D. Schon, W. Batzler, C. Baumgardt-Elms, B. Eisinger, M. Lehnert, C. Stegmaier, Cancer registration in Germany: current status, perspectives and trends in cancer incidence 1973–93, J. Epidemiol. Biostat. 5 (2000) 99–107. [5] D. Meirow, J.G. Schenker, Cancer and male infertility, Hum. Reprod. 10 (1995) 2017–2022. [6] L. Stuppia, V. Gatta, G. Calabrese, P. Guanciali Franchi, E. Morizio, C. Bombieri, R. Mingarelli, V. Sforza, G. Frajese, R. Tenaglia, G. Palka, A quarter of men with idiopathic oligoazoospermia display chromosomal abnormalities and microdeletions of different types in interval 6 of Yq11, Hum. Genet. 102 (1998) 566–570. [7] M. Kent-First, A. Muallem, J. Shultz, J. Pryor, K. Roberts, W. Nolten, L. Meisner, A. Chandley, G. Gouchy, L. Jorgensen, T. Havighurst, J. Grosch, 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 (1999) 27–41.
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Ethnic variation in the prevalence of AZF deletions in testicular cancer S.M. Richard a , N.O. Bianchi a,∗ , M.S. Bianchi a , P. Peltomäki b , R.A. Lothe c , W. Pavicic a a
c
Instituto Multidisciplinario de Biolog´ıa Celular (IMBICE), La Plata, Argentina b Department of Medical Genetics, University of Helsinki, Helsinki, Finland Department of Genetics, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo, Norway Received 17 December 2003; received in revised form 16 February 2004; accepted 1 March 2004 Available online 27 April 2004
Abstract Seventy-six percent of testicular cancers of origin in Finns have been reported to exhibit AZF deletions. We analyze here 40 testicular tumor cases from Norway and Argentina and we found that AZF deletions occur also in non-Finnish cases but at significantly lower frequency (25%) than in Finland testicular tumor cases. This frequency difference can be attributed to the condition of genetic isolate of the Finnish population and the subsequent prevalence in this ethnic group of genetic factors involved in the origin of AZF deletions associated with malignancies. The finding of a correlation between AZF deletions and a given Y haplogroup would indicate the existence of Y lineages carrying AZF deletion-enhancing gene or genes. This possibility was explored using a set of Y-DNA-markers allowing the identification of Y lineages occurring at high frequency in Finns. We characterized the Y haplogroups in 32 normal Finn males (control group) and 17 cases of testicular cancer in Finns with and without AZF deletions. We found no association between Y lineages and AZF microdeletions, nor between AZF microdeletions and a Y microdeletion (DYS7C) exhibiting high prevalence (>50%) in Finns. The lack of correlation between AZF deletions and Y haplogroups indicates that the origin of these deletions is independent from the Y chromosome genetic background. No AZF deletions were found in familial cases of testicular tumors; hence, hereditary factors inducing the appearance of testicular malignancies in certain genealogies apparently do not increase the susceptibility to AZF deficiencies. AZF deletions are de novo events occurring in prezygotic or in post-zygotic stages. We propose that most AZF deletions associated with testicular tumors are due to post-zygotic Y microdeletions, while most cases of deletions associated with infertility are due to deletions occurring in the germ cell line of proband fathers. © 2004 Elsevier B.V. All rights reserved. Keywords: Testicular tumors; AZF deletions; MGCT; AZF deletions and testicular tumors
1. Introduction
∗ Corresponding author. Fax: +54-221-425-3320. E-mail addresses: [email protected], [email protected] (N.O. Bianchi).
Several publications indicate that during the last 30–50 years there has been a progressive decrease in sperm concentration and quality of ejaculates from normal males, an increase in the incidence of male
0027-5107/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2004.03.001
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infertility, and a higher prevalence of testicular cancers in the general male population [1–4]. Moreover, the association between decreased sperm concentration and malignant diseases is further stressed by the finding that infertility may precede the appearance of lymphomas, testicular tumors, and several forms of endocrine cancers [5]. Since approximately 15–25% of idiopathic male infertility cases [6–9] and 0.87–2% of normal fertile males [7,10] have been reported to exhibit AZF deletions, we tested this gene family in a series of male tumors (MGCT) in order to determine whether AZF instability was an event also associated with testicular tumors. Thus, we screened 17 loci corresponding to AZF (a–d) subintervals, as defined by Kent-First et al. [7] in normal and tumor tissues of 17 Finn males with testicular malignancies. We found that 13 testicular cancer cases (76%) had AZF deletions [11]. Since 2 of the cases with Y microdeletions were testicular forms of non-Hodgkin lymphoma (the other 11 cases having Y instability were MGCTs), we extended the deletion testing to a series of 27 cases of Finn males with non-Hodgkin lymphoproliferative disorders: AZF deletions were detected in 66.6% (18/27) of patients [12]. Furthermore, no AZF deletions were detected in the fathers of testicular tumor cases or in the Y chromosomes from 32 normal Finn males or 48 normal males of European ancestry [11]. Thus far, all testicular tumor cases tested for AZF deletions were Finnish patients. In this report, we shall analyze whether the association of AZF deletions with testicular tumors is restricted to Finns or is a more general phenomenon taking place in other geographic populations as well. Moreover, we shall test whether the AZF deficiencies found in malignancies preferentially occur in a given Y lineage or in association with DYS7C deletions, a type of deficiency occurring at high frequency in normal Finn Y chromosomes [13].
2. Material and methods 2.1. AZF testing Four non-commercial multiplex PCR reactions were used to detect the presence or absence of fragments corresponding to 17 AZF loci comprising the four subintervals (a–d), as defined by Kent-First et al. [7];
details on loci analyzed, the subinterval represented by each locus, and loci tested with each multiplex reaction are given in Table 1. Fragments lacking in multiplex PCR reactions were further evaluated in duplex PCR assays performed with two pairs of primers, one specific for the putative deleted fragment and the other specific for an AZF fragment that tested positive in the multiplex reaction and that was included in the duplex assay as an internal positive control. A fragment was considered deleted when it was absent in two independent multiplex reactions and in one or two duplex PCR assays. All fragments considered deleted in primary PCR reactions were retested in a secondary PCR assay using as target 0.5 l of the primary PCR amplified mixture, in a total volume of 15 l. Multiplex PCR reactions showing all the expected fragments were repeated once in order to identify false positives; no false positive fragments were detected. Primary and secondary PCR amplifications were carried out with the usual precautions used for ancient DNA testing. Sample handling and laboratory procedures were performed by female personnel in order to minimize chances of male DNA contamination in PCR reactions. PCR conditions employed are detailed in Bianchi et al. [11]. Interpretation of electrophoresis fragment patterns was performed at blind by two different investigators; in all cases there was agreement in the independent diagnosis of fragment deletions. AZF1 replaced AZF in recent nomenclature systems. In this report, however, we shall use the AZF acronym in order to maintain the same nomenclature employed in our previous publications [11,12]. 2.2. Y-specific markers Additional markers used were: YAP+ [14] or M1 according to Underhill et al. [15,16] nomenclature system (Y-specific Alu insertion); TAT-T →C transition [17] or M46, according to Underhill et al. [15,16]; M9 [15,16], and the DYS7C deletion (50f/2C locus, [13]). PCR conditions, restriction enzymes used, and electrophoresis identification patterns for each of the above markers are given in the aforementioned bibliographic references. DNA markers other than AZF deletions were analyzed in all samples from our previously published series of Finn testicular tumor cases Tt1–Tt17 (previously 1–17, [11]) and in 32 male DNA samples
+ + + +
+: no deletion; a: −/− deletion in normal and tumor samples; b: +/− deletion in tumor sample; c: −/+ deletion in normal sample. Tt 52 and Tt52* identify primary and contraleteral or secondary tumor samples, respectively. In case 51, the Tm sample corresponds to a pulmonary metastasis. Superscript letters (A–D) indicate the AZF fragments included in 1–4 multiplex PCR reactions. In cases with bilateral tumors asterisks indicate primary malignancies. a The first term corresponds to deletion patterns with primary PCRs; the second term after the bar indicates deletion patterns after PCR reamplification.
+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + c/c + c/c + + b/b b/b + + c/+ + + c/c + + + + b/b b/b + + a/a b/b + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + b/+ + + + + + c/c b/+ + + + + b/b + + + + + + + + c/+ + + + + + + + c/c + c/c + + + + b/b + b/b c/+ + + c/c + b/b + b/b b/b b/b + + c/+ + c/c + b/b + + + + b/b + b/b c/+ + + + + + + + +
Tt26 Tt28 Tt32 Tt35 Tt36 Tt41 Tt51 Tm51 Tt52 Tt52* Tt53 Tt54
(SY81)
+ + + + c/+ + + b/b + + + +
+ c/c + + + + + b/+ + b/b + +
DAZ B (SY242) DYS223 D (SY133) DYS215 D (SY124) DYS212 C (SY121)
AZF b
KALY A (SY182) DYS271A
AZF a Cases
Table 1 AZF deletions in testicular tumor (Tt)a
DYS218 B (SY127)
DYS219 C (SY128)
DYS221 A (SY130)
AZF d
DYF51S1 C (SY145)
DYS237 D (SY153)
DYS236 D (SY152)
AZF c
DAZ B (SY239)
DAZ B (SY208)
DAZ A (SY254)
DAZ C (SY255)
DYS240 A (SY157)
S.M. Richard et al. / Mutation Research 554 (2004) 45–51
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randomly selected from a geographically weighted sampling of the entire Finnish population. 2.3. Samples analyzed Histological classification of testicular tumors was performed according to WHO recommendations. The main histological subtypes are seminomas and non-seminomas of which the latter may contain different histological elements including embryonal carcinoma, choriocarcinoma, yolk sac tumor, and teratoma. Testicular tumor samples were arbitrarily identified with the acronym Tt and correlatively numbered, the first number in this series being the next one (Tt18) to that in the series of testicular malignancies previously reported [11]. Tt18–52 are testicular tumor cases from Norway, and Tt53–57 are samples from La Plata city, Argentina. Tt19, 39, 40, 42, 48, 49 are six cases from five different families showing testicular malignancies in relatives of the proband. All other cases were sporadic forms of MGCT. Tt38, 41, 42, 43–48 and Tt52 had history of bilateral cancer. Both tumors from case 52 were included in the present study. Moreover, the primary tumor and a pulmonary metastasis were analyzed from case 51. In all cases reported here, AZF deletion testing was performed in DNA samples from non-malignant (blood) and malignant tissues. Histological diagnoses of the frozen testicular tumor samples in this series are: seminomas Tt19, 21, 24, 25, 27–29, 31–34, 38, 45–49, 53, 54. 56, 57; embryonal carcinoma components Tt20, 22, 23, 26, 30, 35, 40; yolk sac tumor component Tt37; nonseminoma (more than one component in the frozen tissue biopsy) Tt36, 39, 41, 42, 55; immature teratoma Tt51; combination of seminoma and non-seminoma or mixed germ cell tumors (S/NS) Tt18, 44, 50, 52. Concentration of tumor cells in tumor samples was in average 60%. The use of tumor and non-tumor samples for the research purposes reported here were approved by the Ethics Committees of Hospitals in which the patients were admitted. 3. Results 3.1. AZF deletions Over a total of 40 MGCTs analyzed, 10 (25%) showed AZF deletions involving 1–9 loci per case
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(Table 1). When the patterns of AZF deletions in non-tumor versus tumor tissues from the same donor were compared, we observed the pattern “a” (deletion in non-tumor and tumor tissues or: −/−) in one locus (3.1% of the 32 AZF deleted loci), the pattern “b” (deletion in tumor tissues only or: +/−) in 16 loci (50%), and the pattern “c” (deletion only in non-tumor tissues or: −/+) in 15 loci (46.9%) (Table 1). In three loci with deletion pattern “b” (+/−) and in six loci with deletion pattern “c” (−/+) the fragment lacking in primary PCRs appeared in PCR reamplifications; these loci are identified as b/+ and c/+, respectively, in Table 1. The appearance in PCR reamplifications of fragments that were lacking in primary PCRs is expected to occur in mosaicisms in which there is a marked prevalence of deleted over non-deleted Y chromosomes [11,12]. In order to test the ratio of non-deleted versus deleted Ys producing deletion in primary with presence of the fragment in secondary PCR and deletion in both PCR reactions, we used as PCR target a mixture of 50 ng of a DNA sample with deletions in one or more AZF loci plus increasing dilutions of a DNA sample without AZF deletions (the final volume of PCR mixtures was 15 l). We found that, when a non-deleted fragment reached a dilution of 1/300 in regard to the homologous deleted locus, the fragment did not show in primary PCRs but tested positive in PCR reamplifications; at dilutions higher than 1/1000 the fragment failed to appear in primary and secondary PCRs. One Norwegian MGCT case (Tt52) treated with orchidectomy developed a secondary tumor in the remaining testis. This case had no deletions in one of the tumors and three pattern “b” deletions in the contralateral one (Table 1). Differences in the deletion pattern of primary and secondary MTGCTs provide additional support to the hypothesis of an AZF mosaicism occurring in early embryogenesis. If non-tumor cells are a mosaic with two or more cell lineages having AZF deletions in different loci, and if the primary and secondary tumors are independent events involving transformation in distinct cell lineages, then AZF deletion patterns detected for each tumor are expected to differ. Moreover, the finding of a disparate deletion pattern in a testicular tumor and its pulmonary metastasis (Tt51/Tm51, Table 1) is also compatible with the assumption of a different origin and evolution of the primary and secondary malignant events.
Lately, Frydelund-Larsen et al. [18] found no AZF deletions in blood DNA from a series of 160 Danish cases of MGCT. The deletion screening performed by these researchers comprised AZF a–c subintervals that were represented by 9 loci in 103 cases and by 17 loci in the remaining 57 cases. A decrease in the frequency of AZF deletions in testicular cancer cases of Danish origin in regard to deletion frequencies observed in other geographic populations should be borne in mind in order to explain the discrepancy between Frydelund-Larsen and our results. Yet, some other factors may also give rise to the above interlaboratory differences. All loci showing the AZF deletion pattern b (+/−) will be labeled as non-deleted if the deletion screening is restricted to non-tumor samples. Table 1 of this report shows that the pattern b represents 61.4% of the total deletion events and that almost half of our cases with deletions would have been considered non-deleted if tumor tissues would have been excluded from the deletion screening. Moreover, in our experience, the frequency of AZF deletions in MGCT cases varies depending on the locus tested. Only two of the loci tested by Frydelund-Larsen et al. [18] (DAZ-SY254) and DYS237) were also used by us in this report. No DAZ (SY254) or DYS237 fragment deletion occurred in the cases reported here (Table 1) and only one DAZ-SY254 deletion in primary but not in secondary PCR was detected in tumor tissues in one case from our Finnish series [11]. Accordingly, two aspects to tackle in the future would be: (i) to detect the AZF loci most frequently deleted in MGCTs; and (ii) to determine the best number of AZF loci to test for an efficient identification of deletions in testicular tumors. 3.2. Y-chromosome lineages, DYS7C deletions One of the aims of this investigation was to establish whether AZF deletions are associated with a given Y haplogroup or whether there is a correlation between AZF and DYS7C deletions in testicular tumors. Finn Y haplogroups have been well characterized by Raitio et al. [19]. Moreover, the highest incidence of AZF deletions in malignancies and the highest incidence of DYS7C deletions in normal males are found in Finnish populations [11–13]. Accordingly, the use of YAP (M1), M46, and M9 markers to characterize Y lineages and the search for a correlation between
S.M. Richard et al. / Mutation Research 554 (2004) 45–51
AZF and DYS7C deletions were restricted to the series of testicular tumors in Finns reported in Bianchi et al. [11]. We detected three haplogroups: H2 (YAP-/M9C/ M46T), H3+ (YAP-/M9G/M46T), and H16 (YAP-/ M9G/M46C) corresponding to the most frequent Finn Y lineages reported by Raitio et al. [19]. H2 and H3+ had both the same distribution: they were detected in seven controls and in three and one MGCTs with and without deletions respectively. H16, on the other hand, occurred in 18 controls and in seven and two MGCTs with and without deletions respectively. Differences in the distribution of haplogroups in control, malignant deleted, and malignant non-deleted cases were not significant indicating lack of association between AZF deletions and Y parental lineages. The frequency of DYS7C deleted Y chromosomes was equal (63.6%) in normal Finn males and in Finn cases with malignancies (21/33 and 28/44, respectively). This percentage is in good agreement with the 55% of DYS7C deletions reported by Jobling et al. [13] for normal Finns. Hence, no correlation between AZF and DYS7C deletions was found.
4. Discussion 4.1. Frequency of AZF deletions in testicular tumors from different geographic populations Our results indicate that AZF deletions in testicular malignancies are not restricted to the Finn population. Yet, the deletion frequency is three-fold higher in Finns than in males from other geographic populations: 76.4% in Finn [11] versus 25% for Norwegian/La Plata cases of testicular cancer (P < 0.01, Fisher exact test). Due to several factors, the population of Finland has the characteristics of a genetic isolate [20–22]. The prevalence in Finland of hereditary syndromes, rare in other geographic regions conform the so called Finnish Disease Heritage group of syndromes [23,24]. Moreover, the existence of Y chromosome lineages that are almost Finn-specific [19,25], and the high incidence in Finns of a recurrent Y chromosome deletion (DYS7C) at the 50f2/C locus [13] are peculiarities that can be grouped under the name of Finnish Y Heritage (FYH) and that probably result from the condition of genetic
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isolate of the Finnish population. Thus, the high incidence of AZF deletions found in Finn patients with testicular malignancies should be included within the FYH and might be due to the prevalence in Finn males of genetic factors favoring the appearance of Y deletions. The risk of developing a testicular tumor for the sibling of a tumor case is 10-fold higher than the lifetime risk for a random case from the general population [26]. Moreover, testicular tumor cases have a 27-fold risk for developing a secondary tumor in the remaining testis [27,28]. Accordingly, familial and bilateral testicular tumor cases form a group of malignancies in which the genetic causes inducing testicular carcinogenesis apparently play a more relevant role than in sporadic cases of testicular tumors. In the Norwegian series of testicular malignancies reported here, AZF deletions were found in sporadic but not in familial cases of MGCTs. Hence, we might assume that genetic factors favoring the development of familial testicular cancers are not related with the susceptibility to AZF deletions. A Y haplogroup can be defined as a monophyletic group of Y chromosomes sharing the same allelic states in the non-recombinant Y region. From this, we may infer that the finding of a preferential association of AZF deletions with a given haplogroup would indicate the existence of Y lineages carrying a deletion-enhancing gene or genes. At least two reports, Kuroki et al. [29] on Japanese and Krausz et al. [30] on Danish males claim to have detected an association between oligo/azoospermia and specific Y haplogroups. On the other hand, Carvalho et al. [31] were unable to establish a correlation between male infertility, AZF deletions, and Y haplogroups in Japanese males having reduced sperm counts. Our results for MGCTs are coincident with those from Carvalho et al. [31]. By using a set of three biallelic markers we were able to identify three haplogroups of wide distribution in Finns [19]. The frequency of these lineages in control and MGCT cases showed no difference indicating a lack of association between testicular malignancies and a given Y haplogroup. DYS7C deletions occur in the 50f2C locus that maps in 6C deletion interval, in relatively close proximity to the AZFd subinterval. DYS7C deficiencies are known to be associated with five to seven different Y haplogroups indicating a recurrent process involving five to seven indepen-
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dent DYS7C deletion events [13]. The lack of correlation between AZF and Y haplogroups and between AZF and DYS7C deficiencies further confirms that the AZF instability in testicular malignancies is independent from Y chromosome genetic background. 4.2. AZF deletions in infertility and malignancies Most AZF deficiencies are de novo events due to deletions occurring in germ cells or in post-zygotic stages. Germ cell deletions generate a mosaic sperm population carrying normal and AZF deleted Y chromosomes. If a viable product results from the fertilization of a normal oocyte by a Y-deleted sperm, the outcome will be a sexually mature individual having the same form of AZF deletion in all tissues and a spermatogenesis impairment ranging from severe to minor or even no impairment at all [7,32]. PCR deletion analyses of these cases usually show extensive AZF deficiencies involving several loci in the same or in adjacent AZF subintervals [7,10]. On the other hand, post-zygotic deletions take place during the early embryogenesis, giving rise to mosaic individuals exhibiting somatic and germ cell lineages with and without AZF deletions. Due to the mixture of Y chromosomes having deleted and non-deleted AZF loci, the testing of AZF deletions by PCR predominantly exhibits an interstitial or scattered pattern of Y microdeletions [11]. Since AZF deficiencies associated with malignant processes show a scattered form of deletions, it can be tentatively assumed that these deletions preferentially occur through a post-zygotic mechanism [11,12]. Conversely, although post-zygotic deletions occasionally occur in infertile individuals [33], most AZF deletions associated with infertility are due to Y microdeletions taking place in the germ cell line of the proband father. Post-zygotic AZF deletions might be assumed to be markers of susceptibility to testicular cancer on the basis that they occur during early embryogenesis and precede by many years the appearance of tumors [11]. In this regard, it is worth commenting here the findings of Kent-First et al. [7] and Pryor et al. [10]. The group of Pryor found four cases (2%) with AZF deletions in a series of 200 normal males, whereas Kent-First et al. [7] detected eight cases (0.87%) with AZF deleted Y chromosomes in a series of 920 fertile males. Deletions in these cases were interstitial and involved 1–4 loci out of a total of 36 [10] or 48 [7] AZF
loci tested; a pattern compatible with that assumed to occur in post-zygotic deletion mosaicisms. Whether or not these AZF deleted males were at high risk of developing MGCTs is still an open question. The screening for post-zygotic deletions in a general male population and the subsequent follow up of deleted cases for the appearance of testicular malignancies would be time consuming. Conversely, the search for AZF deletions in male relatives of familial and sporadic cases of testicular tumors would be useful to evaluate with less effort whether these deletions are a marker of susceptibility to testicular cancer.
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