Reduced copy number of DAZ genes in subfertile and infertile men

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1 MALE FACTOR FERTILITY AND STERILITY VOL. 77, NO. 1, JANUARY 2002 Copyright 2002 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Reduced copy number of DAZ genes in subfertile and infertile men Jan W. A. de Vries, M.Sc., a Mariëtte J. V. Hoffer, Ph.D., a,b Sjoerd Repping, M.Sc., a Jan M. N. Hoovers, Ph.D., b Nico J. Leschot, M.D., Ph.D., b and Fulco van der Veen, M.D., Ph.D. a Center for Reproductive Medicine, Academic Medical Center, Amsterdam, The Netherlands Received March 25, 2001; revised and accepted August 20, Presented at the 57th Annual Meeting of the American Society for Reproductive Medicine, October 20 24, 2001, Orlando, Florida. Reprint requests: Jan W. A. de Vries, M.Sc., Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Academic Medical Center, P.O. Box 22660, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands (FAX: ; J.W.devries@amc.uva.nl). a Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Academic Medical Center. b Department of Clinical Genetics, Academic Medical Center /02/$22.00 PII S (01) Objective(s): To determine the copy number and identity of the DAZ genes on the Y chromosomes of infertile patients. Design: Prospective study. Setting: University medical center. Patient(s): One hundred and thirty-nine patients with male factor infertility. Intervention(s): The separate genes were detected by polymerase chain reaction (PCR) digestion assays of sequence family variants in leukocyte DNA and by fluorescence in situ hybridization of interphase nuclei and chromatin fibers. Main Outcome Measure(s): Number of DAZ genes present. Result(s): One hundred twenty-nine patients had four genes, 6 patients had two genes, and 4 patients had none. Three patients had a deletion of the two proximal DAZ genes, and three were missing both distal genes. Semen analysis showed a less severe phenotype in patients with only two DAZ genes compared with patients missing all four genes. Conclusion(s): In six patients, two different partial deletions were found that were not detected by PCR with conventional markers. One patient with an AZFb deletion appeared to also have a partial AZFc deletion that was not detected by routine PCR. Phenotypic differences between patients with different deletions suggest a dose effect of the DAZ genes. (Fertil Steril 2002;77: by American Society for Reproductive Medicine.) Key Words: DAZ genes, partial deletions, AZFc, Y chromosome, male subfertility. To date, 10 gene families located on the nonrecombining region of the Y chromosome that are specifically expressed in the testis have been described (1, 2). The number of copies per gene family varies. RBMY, for instance, has many copies that are dispersed over the entire length of the Y chromosome, of which only one to four seem to be needed for spermatogenesis (3 5). The DAZ gene family consists of four copies located relatively close together in deletion interval 6 on chromosome band Yq11, designated as AZFc. As we demonstrated using fluorescence in situ hybridization (FISH), the four copies are located in two clusters, each comprising an inverted pair of DAZ genes (6). At least three different DAZ complementary DNAs have been identified, indicating that at least three DAZ genes are transcribed in the testis during spermatogenesis (6, 7). Because DAZ genes have multiple copies, sequence tagged site markers located in the DAZ genes tend to also have multiple copies. Polymerase chain reaction (PCR) analysis with conventional markers, the method used in all published deletion studies thus far, does not detect individual gene deletions within the DAZ gene family. Therefore, subfertile or infertile men may carry a deletion of fewer than four of the DAZ genes that goes undetected by conventional PCR techniques. The number of DAZ genes present can be determined by using FISH and gene-specific PCR digestion assays. Fluorescence in situ hybridization on interphase nuclei allows detection of the separate gene clusters, and FISH on extended DNA fibers (also called fiber FISH ) can be used to detect the separate genes within the clusters. A gene-specific PCR digestion 68

2 assay that identifies the four different DAZ genes by their sequence family variants was recently developed (6). It makes use of single nucleotide variants in three different sequence tagged sites in the DAZ genes. These variants can be identified by using PCR digestion assays, producing a specific band pattern for each DAZ gene (the so-called DAZ gene signature) that identifies the four different DAZ genes (6). Deletions of all four DAZ genes have been described and are associated with azoospermia or severe oligozoospermia (8, 9). A partial deletion of the DAZ genes was recently described in a subfertile patient (10). It is not known how frequently this partial deletion is detected in subfertile or infertile men. We therefore examined the number of DAZ genes in patients with variable semen characteristics, all of whom had diagnosed male factor subfertility or infertility, by using gene-specific sequence family variant PCR and FISH. To determine whether different phenotypes in these patients are associated with the number of DAZ genes present, we related the number of copies found to semen characteristics. MATERIALS AND METHODS Patients and Semen Donors From October 1997 to December 1998, we enrolled 139 consecutive patients attending our clinic in whom male factor subfertility or infertility had been diagnosed. Semen analysis was performed according to World Health Organization criteria (11). To be included, patients had to have no history of alcohol abuse, orchitis, surgery on the vasa deferentia, bilateral orchidectomy, chemotherapy, radiotherapy, or obstructive azoospermia (confirmed by testis biopsy). An andrologic history was taken, testicular volume was measured, and blood was collected for karyotyping and DNA isolation. Blood from semen donors with proven fertility attending our semen bank was used as an internal control. The study was approved by the institutional review board, and all patients and semen donors gave informed consent before participating. Conventional PCR Assays Isolated DNA from leukocytes obtained by venipuncture was examined by using routine PCR amplification to detect deletions of the following loci: sy81, sy84, sy182, sy94, sy102, sy117, sy143, sy147 sy152, sy153, and sy157. Sequence Family Variants Sequence family variants that distinguish among DAZ genes were detected by PCR amplification of the three sequence tagged site markers sy581 (intron 3), sy586 (intron 6), and sy587 (intron 10). This was followed by enzyme digestion, as described elsewhere (6). Figure 1 shows the nucleotide variation between these markers and their association with the different DAZ genes. In brief, digestion of sy587 with Dra1 produces four fragments of 122 base pairs, 73 base pairs, 49 base pairs, and 26 base pairs in DAZ1 and DAZ2, which both contain the T variant of this sequence tagged site. In DAZ3 and DAZ4, which contain the C variant of sy587, three fragments of 195 base pairs, 49 base pairs, and 26 base pairs are detected. Similarly, digestion of sy581 with Sau3A produces a 189 base pair fragment and a 63 base pair fragment in DAZ1 and DAZ4 and three fragments of 130 base pairs, 59 base pairs, and 63 base pairs in DAZ2 and DAZ3. Finally, digestion of sy586 with Taq1 produces two fragments of 184 base pairs and 117 base pairs in DAZ1, DAZ3, and DAZ4, whereas Y586 of DAZ2 remains undigested, resulting in one 301 base pair fragment. Sequence family variant PCR in sy587 distinguished cluster 1 and cluster 2, and sequence family variant PCR in sy581 distinguished between left or right genes in both clusters. Fragment sizes of the PCR products after digestion with restriction enzymes are shown in Table 1. If incomplete DAZ signatures were detected, tests were repeated. FISH We used three sequenced DAZ cosmids as probes for FISH (6). Cosmid 18E8 (Genbank AC010089) encompasses the 5 end portions of two neighboring DAZ genes (Fig. 1). Cosmid 63C9 (Genbank AC000021) contains exons 2 through 11 almost an entire DAZ gene. Cosmid 46A6 (Genbank AC000022) derives from the 3 end portion of DAZ; it contains exons 8 through 11 and 35 kb downstream of the gene. Single-color FISH was performed on interphase nuclei from lymphocytes according to standard procedures (12). Cosmid 18E8 was used to detect the presence of the two DAZ clusters. Because the DAZ genes are located in two clusters, each containing two DAZ genes in a head-to-head orientation, cosmid 18E8 shows predominantly one signal per cluster. Because superimposition of the two signals can lead to misinterpretation, at least 200 interphase nuclei were examined before the presence of one or two signals was accepted. An average of 20 patients was tested in every FISH procedure. Lymphocytes from a fertile donor were included in every FISH procedure as internal control on the validity of the FISH procedure. The technician performing the counting of the signals was not aware of the outcome of the PCR experiments. Extended chromatin fibers from lymphocytes were prepared by using SDS/ethylenediamine tetraacetic acid extraction (13). Fiber FISH was performed by using a combination of probes 18E8 with 46A6 to detect the 5 and 3 portions of the DAZ genes and a combination of probes 18E8 with 63C9 to detect the 5 portion and the body of the DAZ genes in single clusters. Probes were labeled with biotin or digoxigenin, hybridized to target DNA, and detected by avidin or antidigoxigenin antibodies conjugated to fluorochromes Cy3 (red) or fluorescein (green). FERTILITY & STERILITY 69

3 FIGURE 1 Schematic drawing of the two DAZ gene clusters. Sequence family variants (SFVs) between the four different genes. sy587 distinguishes between clusters 1 and 2, sy581 distinguishes between the genes within the clusters, and sy586 distinguishes between DAZ2 and the other three genes. The location of the three cosmids used for fluorescence in situ hybridization is also shown. Statistical Analysis The t-test was used for all statistical analyses. RESULTS Semen Characteristics Twenty-five patients had azoospermia, and 84 patients had oligozoospermia; of these 84 patients, 57 had severe oligozoospermia ( 5 million sperm cells/ml). Thirty patients had asthenozoospermia or teratozoospermia with normal sperm counts. Sperm count varied from 0 to cells/ml. A testicular volume less than 15 ml in both testes was detected in 71 patients, of whom 15 had a normal sperm count and asthenozoospermia or teratozoospermia and 45 had oligozoospermia (counts cells/ml); of these 45 azoospermic patients, 34 had a sperm count less than cells/ml and 11 had azoospermia. Karyotypes Six of the 139 patients had an aberrant karyotype. Three patients had a 47,XXY karyotype and azoospermia, one patient had a 47,XYY karyotype and oligozoospermia (3 TABLE 1 Fragment sizes after restriction digestion Sequence tagged site marker Enzyme DAZ1 DAZ2 DAZ3 DAZ4 SY587 intron 10 Dra1 122, 73, 49, , 73, 49, , 49, , 49, 26 SY581 intron 3 Sau3A 189, , 59, , 59, , 63 SY586 intron 6 Taq1 184, , , 117 Note: In cluster (DAZ1 and DAZ2), sy587 has the T variant, producing an extra restriction site for Dra1. In cluster 2 (DAZ3 and DAZ4), sy587 shows the C variant. In DAZ2 and DAZ3, sy581 has the T variant, producing an extra restriction site for Sau3A. In DAZ1 and DAZ4, sy581 shows the C variant. In DAZ2, sy586 is in the T variant, causing loss of a restriction site for Dra1. Fragment sizes are expressed in base pairs. 70 de Vries et al. Reduced DAZ gene copy number Vol. 77, No. 1, January 2002

4 TABLE 2 Microdeletions detected by routine polymerase chain reaction. AZFa AZFb AZFc Patient SY81 SY84 SY182 SY94 SY102 SY117 SY143 SY152 SY153 SY147 SY Note: Table shows presence ( ) or absence ( ) of sequence tagged sites detected in polymerase chain reaction in the three deletion intervals of the Y chromosome cells/ml), one patient had a translocation 46,XY, der (22)t(y;22)(q12;p13) and oligozoospermia ( cells/ ml), and one patient had 47,XY, mar ish der(15) and azoospermia. Microdeletions Using the standard panel of sequence tagged site markers for the Y chromosome, we found five patients with deletions (Table 2). Deletions of the AZFc region (sy157, sy152, sy153, and sy147) were detected in four patients. One patient retained the markers for the AZFc region but was missing sy117 and sy143, corresponding to an AZFb deletion. Detection of the Sequence Family Variants Table 3 shows data on the presence of the different DAZ genes, as determined by results of sequence family variant PCR for sy581 and sy587. In 126 patients, sequence family variants for all four DAZ genes were detected. In 13 patients, not all sequence family variants were detected, indicating the absence of one or more of the DAZ genes. In two patients, DAZ1 and DAZ2 were detected but DAZ3 and DAZ4 were absent. In two patients, only DAZ3 and DAZ4 were detected. In four patients, only DAZ3 was detected; one of these patients also had an AZFb deletion. In one patient, only DAZ1 was detected. In the four patients with AZFc deletions, all sequence family variants were absent, as expected. Although PCR for sy581 and sy587 showed a normal sequence family variant pattern in 126 patients, 32 of these patients did not have the 301 base pair fragment for sy586, which is specific for DAZ2. This fragment was also missing TABLE 3 Combinations of DAZ signatures in 139 patients and the presence of one or two clusters on fluorescence in situ hybridization. Sequence family variant a DAZ2 detected by PCR of sy586 b FISH Fiber FISH No. of patient (%) DAZ1 DAZ2 DAZ3 DAZ4 Yes No 2 clusters 1 cluster Normal cluster 126 (90.6) c 126 Yes 2 (1.4) 1 1 c 2 Yes 2 (1.4) 1 c 1 2 Yes 4 (2.9) 4 1 d 3 Yes 1 (0.7) 1 1 Yes 4 (2.9) Not determined a Columns show presence ( ) or absence ( ) ofthedaz genes as detected by polymerase chain reaction digestion assay of sy581 and SY587. b Data are expressed as the number of patients in whom the 301 base pair band, typical for the DAZ2 gene, was found or was absent. c This result conflicts with that of the sy587 and sy581 assay. d Patient with a 47,XYY karyotype had two clusters per Y chromosome. FERTILITY & STERILITY 71

5 in one of the two patients who had both DAZ1 and DAZ2 according to PCR for sy581 and sy587. Conversely, the 301 base pair band was present in one of the two patients in whom only DAZ3 and DAZ4 were detected on PCR for sy581 and sy587 FISH Fluorescence in situ hybridization with probe 18E8 on interphase nuclei showed two clusters in 129 patients. In six patients, however, the mean number of signals per nucleus was significantly lower than in control samples from semen donors. The mean ( SD) number of signals in these six patients ranged from to , compared with to in the control samples (P.001). On fiber FISH, all six patients were shown to have retained one complete DAZ cluster with two genes. Of the 129 patients with two signals on FISH, four genes were also detected by sequence family variant analysis in 126, including the 32 patients who were missing the 301 base pair band representing DAZ2 (Table 3). Fiber FISH again showed only normal cluster images (comprising two genes each) in all these patients. Six of the nine patients with incomplete sequence family variants had predominantly one cluster per cell, and FISH clearly showed two clusters with two genes each in the remaining three patients. Interphase FISH and sequence family variant PCR were concordant in the 2 patients in whom only DAZ1 and DAZ2 were detected, in the one patient in whom only DAZ1 was detected, and in three of the four patients in whom only DAZ3 was detected. In contrast, both patients in whom only DAZ3 and DAZ4 were detected and one patient in whom only DAZ3 was detected clearly had two normal clusters with two genes each on FISH. The latter patient had a 47,XYY karyotype, and FISH showed two clusters per Y chromosome. Of note, one of the patients in whom only DAZ3 was detected on sequence family variant PCR and one cluster on FISH also had an AZFb deletion (Fig. 2). All patients with an aberrant number of DAZ genes had normal clusters comprising two genes on fiber FISH, even though some patients had sequence family variants concordant with only one gene. In the four patients with AZFc deletions, FISH confirmed absence of the DAZ gene clusters. Semen Characteristics in Relation to DAZ Gene Clusters The number of DAZ gene clusters was determined by FISH, and the cluster was identified by sequence family variant PCR (Table 4). For example, if one normal cluster was found on interphase and fiber FISH and sequence family variant PCR indicated the presence of only DAZ3, the cluster was identified as cluster 2. Table 4 shows the relation of DAZ gene clusters with semen characteristics. One of the three patients with a deletion of cluster 2 had severe oligozoospermia. The other two FIGURE 2 Results of fiber fluorescence in situ hybridization on DNA from patient 4937, who had an AZFb deletion and a normal DAZ gene cluster. Red signal, 18E8; green signal, 63C9. patients had a normal sperm count ( cells/ml) with asthenozoospermia (8% 22% motility) and teratozoospermia (12% 13% normoform). Of the three patients with deletion of cluster 1, two had azoospermia and one had oligozoospermia ( cells/ml). Of the four patients with deletion of both clusters, two had azoospermia and two had severe oligozoospermia ( cells/ml). DISCUSSION DAZ genes on the Y chromosome arose from transposition of the autosomal DAZL gene on chromosome 3. Intragenic amplification, exon pruning, and duplication produces four almost identical genes on the Y chromosome in two clusters, each consisting of two genes oriented in a head-tohead manner (6). These genes are located in the AZFc deletion interval (9). Yen (14) identified three of the four genes, but observed a gap in their YAC contig at the place where we suspect the fourth gene was. Our two-cluster concept is in good agreement with the findings of Glaser et al. (15). However, those investigators concluded that seven DAZ genes are present on the Y chromosome. We suspect that the probes used by that group detected the 10.8-kb segment containing exons 2 through 6 of the DAZ gene, in which the sy581 marker is located. This region represents the RNA recognition motif and is present seven times in the DAZ gene clusters: three times in DAZ1, twice in DAZ4, and 72 de Vries et al. Reduced DAZ gene copy number Vol. 77, No. 1, January 2002

6 TABLE 4 Semen characteristics relative to the presence of DAZ gene clusters. No. of patients DAZ gene Severe cluster deleted Azoospermia a oligozoospermia a,b Mild oligozoospermia a,b Asthenozoospermia or teratozoospermia a 129 None Cluster Cluster 1 2 c 1 4 both 2 2 Note: The DAZ gene cluster number was determined by FISH and the clusters were identified by SFVs. a Data are the number of patients. b Mild oligozoospermia is a sperm count of cells ml to cells ml; severe oligozoospermia is a sperm count less than cells/ml. c One of the two patients was the AZFb deleted patient with the AZFb deletion. once each in DAZ2 and DAZ3 (Fig. 1). Of the 139 patients in our study, 129 had four DAZ genes in two clusters. Deletion events on the Y chromosome and, especially, in the AZFc region are relatively frequent; rates range from 1% to 20% in men with severe oligozoospermia and azoospermia (9, 16 22). Thus far, all AZFc deletions described are deletions of all four DAZ genes, except in the patient described by Moro et al. (10). In these deletion studies, only complete deletions of the four DAZ genes could be detected because markers that are shared by the DAZ genes were used. Although the four DAZ genes are 99.9% identical in coding sequence, they can be distinguished by detection of single nucleotide variants in three different introns of the DAZ gene (6). This distinction is based on Bacterial Artificial Chromosome (BAC) clones that each contain no more than two of the four DAZ genes. In genomic DNA, however, four DAZ genes are present; because the specific band patterns of the four DAZ gene signatures overlap, a single gene deletion cannot be excluded, even if all band patterns for the sequence family variants are present. DAZ genes can be detected at the cluster level by using FISH on interphase nuclei. On fiber FISH, only one complete or incomplete cluster can be seen in a single microscopic image because of the distance between the clusters and breaking of the chromatin fibers. Using a combination of sequence family variant PCR and FISH techniques, we found that six patients had a deletion of one of the two DAZ gene clusters. At least three different breakpoints must be involved, because either cluster could be absent independently. These partial deletions of the DAZ genes would have been missed if we had used only conventional PCR markers. From these partial deletions, we obtained new information on the relative orientation of the DAZ gene clusters. Because the patient with the AZFb deletion was also missing cluster 1 and the father had no detectable deletion on the Y chromosome (data not shown), we conclude that cluster 1 is the proximal cluster and cluster 2 is the distal cluster. The orientation of the genes within the clusters, however, remains to be elucidated. Sequence family variant PCR is based on three silent nucleotide substitutions that were found in different BAC clones from one Y chromosome (6). On the basis of results of sequence family variant PCR and FISH on the Y chromosomes of 139 men, we conclude that polymorphism in these sequence family variants exists. In 32 of 126 patients in whom all genes were detected on sequence family variant PCR for sy581 and sy587 and FISH, the T substitution in sy586 in DAZ2 was absent, indicating polymorphism for sy586. In three patients in whom only DAZ3 was detected and one patient in whom only DAZ1 was detected on sequence family variant PCR for sy581, single but complete clusters were found on FISH. In two patients in whom only cluster 2 was detected on sequence family variant PCR for sy587, two normal clusters were found on FISH. These results indicate polymorphism for sy581 and sy587 as well, albeit to a lesser extent. Polymorphism of the restriction site in sy581 has been described in relation to ethnicity (23). However, neither the number of copies of the DAZ genes nor fertility status were evaluated. Therefore, it is unknown whether the men with only the T or C variant for sy581 in that study represent true polymorphisms or deletions of some of the DAZ genes. In our study, almost all patients with aberrant copy numbers were of European origin; one patient in whom only cluster 2 was detected came from West Africa (data not shown). Both AZFb and AZFc harbor more than one gene family (1). The AZFb candidate gene RBMY is dispersed over the entire Y chromosome, and fiber FISH has shown that it also resides in between the DAZ gene clusters (24). AZFb and AZFc deletions show different breakpoints in different patients (16, 17, 19, 25 27). In this study, we describe a patient with an AZFb deletion that extends into AZFc. If many breakpoints exist in this area and if the areas appear to overlap, it might be better to not distinguish between these FERTILITY & STERILITY 73

7 two intervals but rather to examine the entire contiguous interval by using gene-specific probes for all genes known to reside in this deletion interval. Multicopy genes can be an evolutionary advantage through dose repetition with identical gene products (28) or variant repetition with related but not identical gene products (29). Since at least three but probably all four genes are transcribed in the adult testis (6), a gene dose effect for DAZ can be expected. The predicted products of the four DAZ genes differ in the number of RNA recognition motifs; whether these products differ functionally is not yet known (6). We related the phenotype on the spermiogram to the presence or absence of the different clusters. The absence of both clusters does not always lead to complete absence of spermatogenesis (30). Indeed, two of the four patients with an AZFc deletion had some spermatozoa in their ejaculate. A less severe phenotype was found if only one cluster was absent, suggesting a gene dose effect of the DAZ genes. Furthermore, absence of cluster 1 seems to lead to a more severe phenotype than does absence of cluster 2, suggesting possible different functions of the various DAZ genes. To determine the clinical value of these partial deletions, a larger sample of affected patients and controls must be studied. It has been proposed that men with sperm concentrations above cells/ml need not be examined for Y chromosome deletions (31). However, in our study, three of the six patients with only one DAZ gene cluster had sperm concentrations greater than cells/ml. These deletions will be transmitted to male offspring, and we do not yet know the risk for widening of these deletions in future generations, as others have proposed (32). Acknowledgments: The authors thank Laura Brown and David Page, Ph.D., for providing probes 18E8, 63C9, and 46A6; they also thank Saskia van Daalen and Cindy Korver for technical support. References 1. Lahn BT, Page DC. Functional coherence of the human Y chromosome. Science 1997;278: Vogt PH. Human chromosome deletions in Yq11, AZF candidate genes and male infertility: history and update. Mol Hum Reprod 1998;4: Prosser J, Inglis JD, Condie A, Ma K, Kerr S, Thakrar R, et al. Degeneracy in human multicopy RBM (YRRM), a candidate spermatogenesis gene. Mamm Genome 1996;7: Elliott DJ, Millar MR, Oghene K, Ross A, Kiesewetter F, Pryor J, et al. Expression of RBM in the nuclei of human germ cells is dependent on a critical region of the Y chromosome long arm. Proc Natl Acad Sci U S A 1997;94: Chai NN, Zhou H, Hernandez J, Najmabadi H, Bhasin S, Yen PH. 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8 30. Mulhall JP, Reijo R, Alagappan R, Brown L, Page D, Carson R, et al. Azoospermic men with deletion of the DAZ gene cluster are capable of completing spermatogenesis: fertilization, normal embryonic development and pregnancy occur when retrieved testicular spermatozoa are used for intracytoplasmic sperm injection. Hum Reprod 1997;12: Calogero AE, Garofalo MR, D Agata R. Factors influencing the variable incidence of Y chromosome microdeletions in infertile patients. Hum Reprod 1999;14: Stuppia L, Calabrese G, Franchi PG, Mingarelli R, Gatta V, Palka G, et al. Widening of a Y-chromosome interval-6 deletion transmitted from a father to his infertile son accounts for an oligozoospermia critical region distal to the RBM1 and DAZ genes. Am J Hum Genet 1996;59: FERTILITY & STERILITY 75