Molecular Human Reproduction, Vol. 8, No. 3, 286-298,
March 2002
© 2002 European Society of Human Reproduction and Embryology
Reproductive genetics |
High frequency of DAZ1/DAZ2 gene deletions in patients with severe oligozoospermia
1 Reproduction Genetics, Institute of Human Genetics, University of Heidelberg, Heidelberg, Germany, 2 Department of Human Genetics, Faculty of Medicine, University of Porto, Porto, 3 National Institute of Health Dr. Ricardo Jorge, Human Genetics Center, Lisboa, Portugal, 4 Department of Growth and Reproduction, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark, 5 Department of Andrology, University of Marburg, Marburg, Germany and 6 Institute of Biomedical Sciences Abel Salazar, University of Porto, Porto, Portugal
Abstract
Deletions of the DAZ gene family in distal Yq11 are always associated with deletions of the azoospermia factor c (AZFc) region, which we now estimate extends to 4.94 Mb. Because more Y gene families are located in this chromosomal region, and are expressed like the DAZ gene family only in the male germ line, the testicular pathology associated with complete AZFc deletions cannot predict the functional contribution of the DAZ gene family to human spermatogenesis. We therefore established a DAZ gene copy specific deletion analysis based on the DAZ-BAC sequences in GenBank. It includes the deletion analysis of eight DAZ-DNA PCR markers [six DAZ-single nucleotide varients (SNVs) and two DAZ-sequence tag sites (STS)] selected from the 5' to the 3'end of each DAZ gene and a deletion analysis of the gene copy specific EcoRV and TaqI restriction fragments identified in the internal repetitive DAZ gene regions (DYS1 locus). With these diagnostic tools, 63 DNA samples from men with idiopathic oligozoospermia and 107 DNA samples from men with proven fertility were analysed for the presence of the complete DAZ gene locus, encompassing the four DAZ gene copies. In five oligozoospermic patients, we found a DAZ-SNV/STS and DYS1/EcoRV and TaqI fragment deletion pattern indicative for deletion of the DAZ1 and DAZ2 gene copies; one of these deletions could be identified as a `de-novo' deletion because it was absent in the DAZ locus of the patient's father. The same DAZ deletions were not found in any of the 107 fertile control samples. We therefore conclude that the deletion of the DAZ1/DAZ2 gene doublet in five out of our 63 oligozoospermic patients (8%) is responsible for the patients' reduced sperm numbers. It is most likely caused by intrachromosomal recombination events between two long repetitive sequence blocks (AZFc-Rep1) flanking the DAZ gene structures.
AZFc/DAZ deletions/DAZ-SNVs/male infertility/oligoazoospermia
Introduction
The Deleted in AZoospermia (DAZ) gene family is located in the distal euchromatic part of the long arm of the human Y chromosome (Yq11.23) in the so-called azoospermia factor c (AZFc) region (Vogt, 1998
; Saxena et al., 2000). It has been transposed to the Y chromosome during primate evolution after the divergence of the New World and Old World monkeys, i.e. 35x106 years ago (Shan et al., 1996; Seboun et al., 1997). The ancestor gene DAZ-Like 1 (DAZL1) is located on the short arm of chromosome 3 (3p25). DAZ and DAZL1 are transcribed exclusively in the germ line and encode RNA binding proteins of the highly conserved RNA-recognition motif (RRM) class (Burd et al., 1996).
The DAZL1 gene is an essential master gene for the pre-meiotic development of male and female germ cells (Ruggiu et al., 1997; Houston et al., 1998). The function of the DAZ genes in germ cells is not yet known. Human DAZ proteins have been found in late spermatids and in sperm tails (Habermann et al., 1998), and in spermatogonia and spermatocytes (Reijo et al., 2000), and a DAZ transgene can partially rescue the pathological phenotype of a DAZL1 knock-out mutant in the mouse (Slee et al., 1999). This suggests that the DAZ genes are functional at different phases of human spermatogenesis and might have evolved to improve or extend specifically the male germ line function of DAZL1 in Old World monkeys. However, deletion of the complete DAZ gene family in humans is compatible with the formation of mature spermatozoa, although only in small numbers; severe oligozoospermia is the primary phenotype (Vogt, 1998) and rare cases exist where complete DAZ deletions have been transmitted from father to son (Vogt et al., 1996; Kleiman et al., 1999). Comparative nucleotide mutation rates in DAZ exon and intron sequences have also led to the assumption that the Y-located DAZ gene family plays only a limited role in human spermatogenesis (Agulnik et al., 1998).
An association between specific DAZ gene mutations and a particular testicular pathology is still lacking, because all AZFc deletions described so far remove not only all of the DAZ genes, but also all other Y gene families which have been mapped to the AZFc interval (Lahn and Page, 1997; Vogt, 1998). Like the DAZ gene family, all these genes are expressed in testicular tissue only and therefore are also AZFc candidate genes.
The number and structure of the DAZ gene copies on the human Y chromosome is assumed to be variable in different men, ranging between three and seven gene copies (Yen et al., 1997; Gläser et al., 1998; Saxena et al., 2000) and the structures of the DAZ genes in the GenBank BAC sequences ( Tilford et al., 2001) have been proposed to be similar to those present in the DAZ-BACs isolated by Saxena et al. (Saxena et al., 2000). However, their detailed structures are not yet described. We assumed that these structures may be different because the BACs from GenBank were cloned from a different male donor (RPCI-11-donor) (Osoegawa et al., 1998) than the one used to clone the BACs isolated by Saxena et al. (CTA/CTB-donor) (Saxena et al., 2000; Shizuya et al., 1992). We therefore assembled a contig of 44 RPCI-11-BAC sequences from the GenBank data base, covering the complete AZFc region (4.94Mb, AZFc-BAC contig) and analysed the structures of the DAZ genes in this contig in detail. Only the DAZ1 gene structure was found to be identical to that proposed by Saxena et al. in the CTA/CTB BAC sequences (Saxena et al., 2000). Substantial differences were found in the repetitive exon regions of the DAZ2, DAZ3 and DAZ4 genes structures. In the AZFc RPCI-11-BAC contig, the molecular distance between the two identified DAZ gene doublets was estimated to be 1.47 Mb.
Based on the DAZ gene sequences of the RPCI-11-DAZ-BACs, we established a gene copy specific marker analysis with six DAZ-single nucleotide variants (SNVs) and two DAZ-sequence tagged sites (STSs). Presence or absence of a specific DAZ gene copy could be correlated with the presence or absence of a specific set of SNV alleles and/or STS markers (DAZ deletion haplotype). This SNV/STS deletion analysis was confirmed by a deletion analysis of DAZ gene copy specific EcoRV and TaqI restriction fragments of the DYS1 locus (Ngo et al., 1986) in DNA blot experiments. The DYS1 DNA locus includes the repetitive DAZ exon 7 regions (Vogt et al., 1997).
We used this analysis to investigate the presence of putative DAZ gene deletions in the Y chromosomes of 63 infertile men with idiopathic oligozoospermia and of 107 men with normal fertility. In five oligozoospermic patients, we found DAZ-SNV/STS and DYS1-EcoRV/TaqI fragment deletion patterns indicative for deletion of the DAZ1 and DAZ2 gene copies, one of which could be identified as a `de-novo' deletion event because it was absent in the DAZ locus of the patient's father. Since the same DAZ deletion haplotype was not found in any of the 107 fertile control samples, we assume that the deletions of the DAZ1/DAZ2 gene doublet in five of our 63 oligozoospermic patients (8%) is the causative agent for the patients' reduced sperm numbers.
Materials and methods
Selection of oligozoospermic patients and the fertile men group
We first selected 77 infertile patients with a clinical diagnosis of idiopathic oligozoospermia, i.e. gonadal epididymal and prostatic abnormalities and testis tumours were excluded in all patients. Their sperm numbers ranged between 0.1 and 10x106 per ml of seminal fluid. Sperm motility and morphology were found to be below the World Health Organization reference values (World Health Organization, 1999) for spermatozoa with normal fertilization capacity. All patients were also analysed for the presence of a normal karyotype, 46XY, and for the absence of Yq11 microdeletions using the STS marker set which we established previously for the detection of complete AZFa, AZFb and AZFc deletions (Vogt et al., 1996). Using gene-specific STSs we also analysed each patient for a possible deletion of other AZFc candidate genes. According to these criteria, 63 of the 77 patients were found to be `normal'. Only these individuals were included in our DAZ gene copy specific deletion analysis (OZ samples).
The fertility status of 107 fertile men (designated as `FM samples') was proven by the fact that they had fathered one or more children. In addition, the normal reproductive status of 51 Danish FM controls (italic numbers in Table III) was confirmed by a normal sperm count (>20x106 per ml semen fluid), normal motility and morphology and normal levels of inhibin B, testosterone, LH and FSH according to the guidelines of the World Health Organization.
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SNVs/STSs PCR assays for partial DAZ deletions
Of the six DAZ-SNVs and two DAZ-STS listed in Table I
, three SNVs (I, IV, VI) and the two STSs specific for the detection of the DAZ1 and DAZ4 genes (DAZ-RRM3) and the DAZ3 gene (Y-DAZ3) were novel. Their primer sequences and respective PCR conditions were deposited in Genbank (accession numbers: Table I). The PCR reactions were performed in a 25 µl reaction volume with 1xPCR buffer (Gibco-BRL, Life Technologies GmbH, Karlsruhe, Germany), 2.5 mmol/l MgCl2 (SNV III, IV, Y-DAZ 3) or 2.0 mmol/l MgCl2 (SNV I, II, V, VI, DAZ-RRM3), 0.2 mmol/l dNTPs, 1 IU Taq DNA polymerase (Gibco) and 100 ng of genomic DNA. For the STS analyses, we developed multiplex PCR assays, containing two internal positive controls, one for the SRY locus (sY14) and one to confirm the proposed DAZ1/DAZ4 deletions (sY152), analysed also by DAZ-RRM3 STS. Genomic DNA samples from a fertile male and a female were used as positive and negative controls respectively in each PCR experiment. The DNA sample from the fertile man controls the sensitivity and specificity of each performed PCR assay, whilst the female DNA sample controls the Y specificity of the used primer pairs. In addition, a water sample containing all components except genomic DNA was run with each set of primers in order to detect any possible reagent contamination.
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The PCR reaction profile (for Biometra T-Gradient Personal Cycler) was as follows: 35 PCR cycles with a pre-soak for 3 min at 94°C, denaturation for 1 min at 94°C, annealing for 1 min at the annealing temperature specific for each primer pair, with a subsequent polymerization step of 1 min at 72°C and, at the end, a final extension step of 3 min at 72°C. The PCR products (5 µl aliquots for SNVs and 15 µl aliquots for STSs) were analysed on agarose gels stained with ethidium bromide. GeneRulerTM 100 bp DNA ladder-plus (Gibco) was used as molecular size marker. A positive result for STS markers was indicated by the presence of the respective fragment on the gel. For the subsequent SNVs analyses, the restriction digestions with the enzymes listed on Table I
were performed in a 25 µl reaction volume with the appropriate 1x buffer (MBI-Fermentas GmbH, St Leon-Rot, Germany for SNV II, III, IV, V and New England Biolabs GmbH, Frankfurt, Germany for SNV I and VI), 10 units of each enzyme and 20 µl of the PCR solution. Incubations were for 2 h at 37°C, except for TaqI digestion which was performed at 65°C. The digests (25 µl aliquots) were analysed on agarose gels (2 or 3% depending on the fragment size obtained for each SNV; Table I) and stained with ethidium bromide (see Figure 2).
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Southern blot analysis
A total of 8 µg of purified genomic DNA were digested with 2.5 IU/µg of the restriction enzyme EcoRV or TaqI (MBI). The fragments were separated by electrophoresis on 0.8% agarose gels (LE-SeaKem; FMC® Bioproducts, Rockland, Maine, USA) and transferred to nylon membranes (Hybond-N+; Amersham Pharmacia, Freiburg, Germany) by vacuum or capillary blot. Filters were UV-cross-linked (1200 J) and baked at 80°C for 2 h. These were pre-hybridized for 2 h and hybridized overnight at 65°C in Church buffer [7% sodium dodecyl suphate (SDS), 0.5 mol/l NaHPO4, 1 mmol/l EDTA, pH7.2] in roller bottles. The probe used was the 2.8 kb EcoRI fragment of plasmid p49f, gel-purified and labelled with 32P dCTP by random priming. After overnight hybridization, filters were washed for 30 min at 65°C in 2xstandard saline citrate/0.1% SDS and exposed for 1–6 days at –70°C with Kodak XAR films and intensifying screens (REGO) for visualization of the 49f cross-hybridizing genomic DNA fragments by autoradiography.
Sequence analysis
For sequence homology analysis in the National Center for Biotechnology Information (NCBI) and GenBank databases, restriction mapping and multiple alignment analysis, we used the HUSAR software package of the German Cancer Research Centre, Heidelberg (http://genome.dkfz-heidelberg.de/biounit). Database searches with the BLAST programs of the NCBI (http://www.ncbi.nlm.nih.gov) for the identification of the exon–intron structures of each DAZ gene copy were performed with a complete cDNA sequence of the DAZ2 gene (CT351Y, Genbank AF414184) using the universal splicing rules. The AZFc BAC contig was established by identifying first the BAC clones containing the AZFc border STSs: sY142 (BAC400I17) and sY167 (BAC557B9). Then inspection of the overlap data provided by GenBank were used to collect the internal 41 AZFc RPCI-11 BACs and assemble them in the unique order shown in Figure 1A. Only BACs with certified sequences (i.e. all regions were covered by sequences from more than one subclone and the assembly was confirmed by restriction digest) were selected. The sequence assembly of the RPCI-11-DAZ BAC contigs shown in Figure 1B was used to construct the restriction maps of EcoRV, TaqI and MluI in the genomic DAZ gene structures.
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Results
Mapping and structure of four DAZ gene copies in the RPCI-11-AZFc BAC contig
For the development of a DAZ gene copy specific deletion analysis, the knowledge of the detailed structure of each DAZ gene copy in the RPCI-11-BAC sequences was an essential prerequisite. We therefore screened the GenBank database with the complete DAZ cDNA sequence CT351Y (GenBank accession no. AF414184) to identify all DAZ-RPCI-11-BAC clones. Seven DAZ-BACs were identified: 26D12 (163948 bp; GenBank accession no. AC006982); 70G12 (180345 bp; GenBank accession no. AC006983); 140H23 (103432 bp; GenBank accession no. AC053490), 263A15 (168639 bp; GenBank accession no. AC007039); 289L7 (199458 bp; GenBank accession no. AC010088); 290O3 (103356 bp; GenBank accession no. AC010089) and 539D10 (186838 bp; GenBank accession no. AC006338). They were assembled in two small contigs of the large AZFc contig which we constructed by a successive analysis of the GenBank sequence overlap data of 44 RPCI-11 BAC sequences (i.e. 4.94 Mb; Figure 1A). The AZFc contig starts with the BAC400I17 sequence, containing the STSs sY142 (DYS230, GenBank accession no. G38345; sequence position: 3068–3268) and sY143 (DYS231; GenBank accession no. G38347; sequence position: 3074–3506). Both STSs have been identified as markers of the proximal AZFc border line (Simoni et al., 1999). The contig ends with the BAC557B9 sequence, which contains STS sY167 (DYS248; GenBank accession no. G12008; sequence position: 98016–98132). This STS is present in the Y chromosome of all our patients with an AZFc deletion (43 individuals) and therefore was designated as a marker for the distal AZFc border line (S.Fernandes and K.Huellen, unpublished results). Within this AZFc sequence, 1.39 Mb distal to sY142, the DAZ-contig containing BACs 289L7, 140H23, 263A15 and 70G12 was mapped proximal to the DAZ-contig including BACs 290O3, 26D12, 539D10 with a molecular distance of ~1.47 Mb between them (Figure 1A).
The proximal DAZ-BAC contig contains two DAZ gene copies with a 3'–5':: 5'–3' head-to-head orientation, a similar structure as found earlier in one of the DAZ-BAC contigs extracted from the CTA/CTB BAC libraries (Saxena et al., 2000). We therefore used the same nomenclature and designated them as DAZ1 starting with its 3' end in BAC289L7 and DAZ2 ending with its 3' end in BAC70G12 (Figure 1B). DAZ1 contains three tandem repeats of 10.8 kb, including exons 2–6 and a tandem repetitive structure of nine exon 7 copies. Nine sequence variants of exon 7 have been found in testicular DAZ cDNAs (Vogt et al., 1997; Yen et al., 1997). According to the nomenclature of Yen et al. we named them A, B, C, D, E, F, X, Y, Z with the Y variant as the most prominent consensus sequence (Yen et al., 1997). Eight variants are found in DAZ1 and organised in the order: 5'-B-C-D-E-F-E-X-Y-Z-3' (Figure 1B). Since identical repetitive exon 7 structures linked to a triplicated DAZ exon 2–6 domain have been found in testicular DAZ cDNAs (Saxena et al., 2000), we can assume that the DAZ1 gene is transcribed in human testis. Two additional exon 7 sequence variants with normal splicing signals were found in intron 6 of the first and second 10.8 kb repeat (exon 7G variant; in DAZ1: BAC140H23 sequence position: 39936–40007 and 50781–50852) and one in the intron 8 sequence (exon 7H variant; in DAZ1: BAC140H23 sequence position: 2563–2634) of the genomic DAZ gene structures (Figure 1B). However, no DAZ cDNA sequence from human testis tissue has yet been found to include one or both of these DAZ exon 7 variants. DAZ1 transcripts spliced with these variants must therefore be rare or not found in testis tissue.
The DAZ2 gene structure maps to three overlapping BAC sequences. Typical for DAZ2 is the presence of the A variant and absence of the B variant in the repetitive exon 7 structure next to six tandem Y variants (Figure 1B). DAZ2 transcripts with this exon 7 variant structure have been found in testicular DAZ cDNA sequences (Yen et al., 1997; Saxena et al., 2000), our cDNA screening probe CT351Y being one example (GenBank accession no. AF414184). This indicates that the DAZ2 gene is also transcribed in human testis tissue. However, there have been no reports of DAZ2 testis transcripts containing a duplicated E–F variant motif and an exon 7D variant flanked proximal by exon 7X or distal by exon 7E, as would be predicted by the genomic DAZ2 sequence in BAC263A15 (Figure 1B). Such DAZ2 transcripts might therefore be rare or do not exist. Alternatively, an artifact of the sequence assembly programmes used for the construction of the genomic BAC263A15 sequence might have produced this exon 7 variant composition. The structure of the DAZ2-BAC sequence from the CTB library (BAC352E14) contains an A and seven tandem Y variants in the repetitive exon 7 structure, but the duplicated E–F motif found in the RPCI-11 BAC263A15 sequence was absent (Saxena et al., 2000).
In the distal region of the AZFc contig (Figure 1A), 1.47 Mb distal to the 3'end of DAZ2, the sequence of BAC290O3 contains the third DAZ gene copy, followed by the overlapping BACs 26D12 and 539D10 with the fourth DAZ gene copy (Figure 1B). Both have a similiar 3'–5':: 5'–3' head-to-head orientation as found for the DAZ3 and DAZ4 gene structures extracted from the CTB-BAC library (Saxena et al., 2000). However, substantial differences were found in their repetitive exon structures.
Typical for DAZ3 in the CTB-BAC132B16 sequence was the absence of the Y variants and a triplication of the E–X motif in the repetitive exon 7 structure (Saxena et al., 2000). This was also found in the DAZ3 gene of the RPCI-11-BAC290O3 sequence (Figure 1B). However, two additional exon 7 variants (E and F) in the CTB-DAZ3 gene were not present in DAZ3 of the RPCI-11-BAC. Since testicular DAZ cDNA sequences with both exon 7 variant compositions have been found (Yen et al., 1997), we can assume that the repetitive sequence region of the DAZ3 gene structure can be variable on different Y chromosomes, as marked here by the presence or absence of the exon 7 E–F motif.
The DAZ4 gene structure in the RPCI-11 BAC539D10 sequence is substantially different from that of DAZ4 in the CTB-BAC546E5 sequence. Both DAZ4 genes were recognised by their 10.8 kb repeat unit; however, three copies of this large repeat unit were found in the RPCI-11-DAZ4 gene and only two 10.8 kb repeats were found in the CTB-DAZ4 gene (Saxena et al., 2000). The number and composition of the exon 7 variants in both DAZ4 genes was also different. We found 15 exon 7 copies with the composition 5'-B-C-D-E-F-E-X-E-X-Y-Y-Y-Y-Y-Z-3' in the RPCI-11-DAZ4 gene, whereas only nine exon 7 repeats with the composition 5'-B-C-D-E-F-E-X-Y-Z-3' were identified in the DAZ4 gene of the CTB-BACs (Saxena et al., 2000). Testicular DAZ cDNAs with an exon 7 variant structure typical for the RPCI-11-DAZ4 structure (i.e. B variant linked to five tandem Y variants) have been described by Yen et al. (Yen et al., 1997), although complete DAZ4-cDNAs with 15 exon 7 copies, linked to a triplicated 10.8 repeat, have not yet been isolated.
Establishment of a DAZ gene copy specific deletion analysis
DAZ gene copy specific sequence variants (SNVs) and STSs
DAZ gene locus restriction maps and sequence analyses have shown that the genomic structures of the four DAZ genes present in the AZFc region are more than 99% homologous to each other (Saxena et al., 1996, 2000; J.Zeisler and S.Fernandes, unpublished results). For the identification of DAZ gene copy specific deletion events, our search for DAZ copy specific markers along the complete gene sequences therefore first focused on a systematic identification of small sequence variants, including specific restriction sites (DAZ-SNVs) and copy-specific DAZ-STSs in the RPCI-11-DAZ-BAC sequences. We established six DAZ-SNVs (I–VI) spanning the 5' to the 3' end of these DAZ gene sequences and two DAZ gene copy-specific STSs (DAZ-RRM3, Y-DAZ3; Table I). We would like to acknowledge the work of our colleagues who first identified the SNV in intron 3 of the DAZ1 gene (Agulnik et al., 1998) and in intron 7A of the DAZ2 gene (sY586) (Saxena et al., 2000). Because both were also found in the RPCI-11-DAZ-BAC sequences, we included them in our SNV marker set as DAZ-SNV II and DAZ-SNV III respectively. We also included the sY587 marker of Saxena et al. as DAZ-SNV V for analysis of the DraI restriction site in the DAZ1 and DAZ2 genes (Saxena et al., 2000).
Additionally, we established three novel DAZ-SNVs (SNV I, SNV IV, SNV VI). SNV I was identified 90 nucleotides upstream of the putative transcription initiation site of the DAZ genes with a polymorphic FspI restriction site only present in DAZ4. SNV IV was found 1159 nucleotides downstream of DAZ exon 7H with a polymorphic AluI restriction site absent in DAZ2. SNV VI was found 480 nucleotides downstream of DAZ exon 10, where due to a 4 bp deletion (CCTG) at the 3'end of DAZ4, an AflIII site was created. An extension of this deletion to eight nucleotides (TCCTGTAT) was identified at the 3' end of the DAZ3 gene and used for the development of a DAZ3 specific STS (Y-DAZ3). A second STS (DAZ-RRM3) was established in the repeated intron 1–intron 6 structure specific for DAZ1 and DAZ4. This marker is in the same intronic region as sY152 (Figure 1B) declared earlier to be a marker for the DAZ1 and DAZ4 genes (Saxena et al., 2000). However, we found the same sY152 primer pair sequences in the RPCI-11 DAZ2 and DAZ3 gene sequences (3' to exon 7A and 7B respectively) with only three internal mismatches in the forward primer. Therefore, we often obtained false positive amplification products in our PCR experiments and developed the DAZ-RRM3 STS as a more stringent alternative for the identification of the DAZ1 and DAZ4 gene copies using our PCR multiplex conditions. All novel DAZ-SNVs and STSs including the optimal PCR conditions were deposited in GenBank (accession numbers in Table I).
On the Y chromosome of men with normal fertility, we expect the presence of four DAZ gene copies to be indicated by the presence of A and B alleles for each of the six DAZ-SNVs as well as the presence of the STSs DAZ-RRM3 and Y-DAZ3, as shown in Figure 1B. Absence of all DAZ markers would indicate the absence of the complete DAZ gene family on the Y chromosome. The deletion of a specific DAZ gene copy would then be recognized by the absence of specific SNV A or B alleles associated with a particular DAZ gene copy and the deletion pattern of the DAZ-RRM3 and Y-DAZ3 STSs (Figure 1B), i.e. by a specific DAZ deletion haplotype. In this way, we propose 13 specific DAZ deletion haplotypes (DAZ-del-haps), each one indicative for the deletion of a specific set of DAZ gene copies (Table II).
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DAZ gene deletions in men with severe oligozoospermia and in men with proven fertility
We analysed the presence of each of the 13 DAZ-del-haps in the genomic DNA samples of 63 men with severe oligozoospermia (OZ samples) and of 107 men with proven fertility (FM samples). All OZ samples had a normal karyotype, 46XY, and Yq11 microdeletions analysed with the STS marker set established previously for the detection of complete AZFa, AZFb and AZFc deletions (Vogt et al., 1996
) were not detectable. Four DAZ-del-haps (#2, 4, 5, 9) could be identified. Surprisingly, most of them were found in both groups (i.e. fertile and infertile men) with similar frequencies (Tables III and IV). DAZ-del-hap 2, indicative for a deletion of DAZ2, was found in 26 FM samples (Table III) and in eight OZ samples (Table IV) and is associated with the absence of the B allele for SNV VI, which would suggest an additional deletion in the distal part of DAZ4 including the SNV VI AflIII restriction site (Figure 1B). Derivatives of DAZ-del hap 2, i.e. absence of only the A allele of SNV III, or only the A allele of SNV IV (with or without absence of the B allele of SNV VI and absence of the Y-DAZ3 STS marker), were also identified in both groups of men (Tables III and IV). A similar pattern of SNV and STS deletion events was also observed in both sample groups for DAZ-del hap 4 (indicative for deletion of DAZ4) and DAZ-del-hap 9 (indicative for deletion of DAZ2 and DAZ4) (Tables III and IV). It strongly suggests that these DAZ deletions are not associated with the occurrence of the oligozoospermic phenotype in the OZ individuals, but are most likely familial variants, inherited from father to son. We were able to confirm this by also finding DAZ-del-hap 2 in the fathers of FM 46 and FM 53 and DAZ-del-hap 4 in the father of FM 39.
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Contrasting with these results, we found a deletion of the DAZ1 gene linked to a deletion of the DAZ2 gene (i.e. DAZ-del-hap 5; Table II
) in only five DNA samples of the oligozoospermic men (OZ 5, 20, 35, 65 and 73; Table IV; Figure 2). Two of them (OZ 20, 35) additionally displayed absence of the A allele for SNV I and of the B allele for SNV VI (Figure 2). This would indicate additionally partial deletions in the 5' region of the DAZ3 gene and in the 3' region of the DAZ4 gene. In OZ 5, we also found a deletion of the DAZ-RRM3 marker (Table IV), which would suggest that the internal part of the repetitive DAZ4 RRM region (location of the DAZ-RRM3 marker; Figure 1B) is absent in this patient in addition to the deletion of the DAZ1 and DAZ2 genes (Table IV). DAZ-del-hap 5 occurred in five of our 63 OZ samples (8%). It could be shown as a `de-novo' mutation event in the family of OZ 5 as the father of OZ 5 displayed the same complete DAZ-SNV pattern as our fertile male control (Figure 2). Our request for a genomic DNA sample from the fathers of OZ 20, 35, 65 and 73 was unsuccessful because they were deceased or did not want to collaborate. The alternative use of DNA samples from fertile brothers was not possible either, because no brothers were present in these families.
Specific DYS1-EcoRV and -TaqI DNA blot patterns in men with DAZ gene deletions
We found that the deletion of single DAZ gene copies could also be identified by DYS1-DNA blot experiments after restriction digest of genomic DNA samples with EcoRV and TaqI. A detailed DAZ gene copy specific restriction analysis on the sequences of the RPCI-11-DAZ BAC contigs revealed that EcoRV restriction fragments specific for DAZ1 and DAZ4 and TaqI restriction fragments specific for DAZ2 and DAZ3 were present in the repetitive DAZ gene regions (Figure 1B). They are part of the well-known DYS1 locus (Ngo et al., 1986) because they cross-hybridize with the DYS1 cognate fragment 49f (subclone from cosmid 49) (Bishop et al., 1984). Our sequence analysis identified 49f as an EcoRI fragment of the DAZ1 gene, starting 5' in intron 3 of the third DAZ1 RRM domain (BAC140H23 sequence position: 34027) and ending 3' in DAZ1 intron 7B (BAC140H23 sequence position: 31181; Figure 1B; GenBank accession no. AF414183).
From these findings, we expect a deletion of the DYS1-EcoRV fragments 10845 and 10843 bp to be associated with a deletion of the DAZ1 gene, and a deletion of the 7327 bp DYS1-EcoRV fragments to be associated with a deletion of the DAZ4 gene (Figure 1B). Similarly, a deletion of the DYS1-TaqI fragment of 3112 bp should be associated with a deletion of the DAZ2 gene, and a deletion of the DYS1-TaqI fragment of 19612 bp should be associated with a deletion of the DAZ3 gene. The MluI restriction patterns identified as specific for each DAZ gene copy by Saxena et al. (Saxena et al., 2000) could not be used for genomic DAZ gene deletion analysis, because the methylation of most MluI sites in the genomic DAZ DNA inhibited restriction (S.Fernandes, unpublished results).
Examples with relevant DNA blot experiments using genomic DNA samples of patients with DAZ-del-hap 5 are shown in Figure 3A. A deletion of the DAZ1 and DAZ2 genes was confirmed in all DAZ-del-hap 5 samples by absence of the DAZ1-specific 10.8 kb-EcoRV and the DAZ2-specific 3.1 kb-TaqI fragments of the DYS1 locus. Interestingly, both OZ 5 and his father displayed additionally a familial deletion of DAZ4, detected through the absence of the DAZ4-specific 7.3 kb EcoRV fragment in both DNA samples (Figure 3A). This familial DAZ4 deletion includes most likely the entire repetitive DAZ4 gene structure, as the signal intensities of the 5 and 2.4 kb EcoRV fragments are weak in comparison with the normal male control (Figure 3A). These two DYS1-EcoRV fragments are not gene copy-specific but span the repetitive DAZ exon 7 structure in each DAZ gene. The presence of the DAZ4-specific B alleles of SNV I and SNV VI in OZ 5 and his father (Figure 2), marking the 5' and 3' end of the DAZ4 gene, suggests that the DAZ4 deletion in this family is partial.
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-HindIII marker that was used are given on the left.We also performed a DYS1 blot analysis for some DNA samples of men with DAZ-del-hap 2 (FM 18, OZ 1 and 15; DYS1-TaqI) and DAZ-del-hap 9 (FM 16, OZ -60; DYS1-TaqI and EcoRV). We confirmed a deletion of DAZ2 through absence of the DAZ2-specific 3.1 kb TaqI fragment in all of them (Figure 3B
), but we could not identify a deletion of the DAZ4-specific 7.3 EcoRV fragment in FM 16 and OZ 60. Discussion
Thanks to the Human Genome Sequencing project, it was possible to cover the genomic Y sequence of the AZFc region with a contig of 44 BAC sequences and to estimate its molecular extension to be 4.94 Mb (Figure 1A). This starts at STS sY142 and ends at STS sY167 in distal Yq11.23. sY167 was mapped not >100 kb proximal to the DYZ1 border of the Yq12 heterochromatin, found in BAC428D10 (Figure 1A). A similar BAC contig of the AZFc region was recently published by Kuroda-Kawaguchi et al. (Kuroda-Kawaguchi et al., 2001). Their data completely confirm the correct assembly of our AZFc-BAC contig in Figure 1. They extended this analysis by additional iterative alignment processes and revealed a unique repetitive and palindromic AZFc sequence structure along 93% of its length.
AZFc deletions are the most common known genetic cause of human male infertility and are found in different human populations with a rate of ~10–4 (Vogt, 1998). In spite of this, it is not yet known whether the Y gene families mapped to this chromosomal region, and which are expressed exclusively in testicular tissue (Lahn and Page, 1997; Vogt, 1998; Kuroda-Kawaguchi et al., 2001) are indeed essential for human spermatogenesis. Gene-specific deletions or point mutations have not been reported in any of the proposed AZFc candidate genes. Therefore it has been proposed that the observed AZFc testicular pathology might only be caused by deletion of the entire AZFc gene content of the human Y chromosome (Vogt, 1998).
In this paper, we present strong experimental evidence that this might not be the case since we found that, in five of 63 OZ individuals, deletions of the DAZ1/DAZ2 gene doublet are most likely associated with an oligozoospermic phenotype similar to that observed after deletion of the entire AZFc region. Our results are a major step towards the solution of the frequently discussed enigma on the function of the different DAZ gene copies in human spermatogenesis.
Variable numbers of DAZ gene copies in the AZFc region of men with proven fertility?
The number and structure of the DAZ genes on the human Y chromosome has been assumed to be variable, in a range between three and seven gene copies (Yen et al., 1997; Gläser et al., 1998; Saxena et al., 2000). We did not find more than four DAZ gene copies in the RPCI-11 BAC library, nor the presence of truncated DAZ pseudogenes. The Y chromosome of the RPCI-11 BAC-library donor should therefore contain four DAZ gene copies. They were mapped as DAZ1/DAZ2 and DAZ3/DAZ4 doublets in the middle of the 4.94 Mb AZFc BAC contig with a distance of 1.47 Mb between them (Figure 1A). This genomic AZFc structure was first proposed in the AZFc duplication model by Kirsch et al. (Kirsch et al., 1996). The presence of four DAZ genes in AZFc with the same doublet structure was first found by Saxena et al., although with different repetitive exon 7 domains in DAZ2, DAZ3 and DAZ4 (Saxena et al., 2000). We also found the four copy gene structure found on the Y chromosome of the RPCI-11 donor in 50 of our 107 fertile control individuals and in 32 of our 63 OZ samples, i.e. in 48% of the samples analysed. That is indicated by the same DAZ-SNV and STS patterns (Tables III and IV). Therefore, we predict that the 4-DAZ-gene pattern found in the RPCI-11-DAZ-BACs represents the most common DAZ gene structure in Caucasian male populations.
In addition, our DAZ-deletion analyses suggest that at least in some individuals with DAZ-del-hap 2, two of our two sample groups (FM and OZ ), the DAZ copy number is reduced to three by deletion of the DAZ2 gene copy (Figure 3B). The presence of only three DAZ gene copies on the Y chromosome of some fertile men was first proposed by Yen et al. (Yen et al., 1997). We could not perform a TaqI-DYS1 blot experiment with all DNA samples carrying the DAZ-del-hap 2 listed in Tables III and IV, because the DNA amount was insufficient and a request for a second genomic DNA sample was not possible; for ethical reasons most fertile male samples had to be collected anonymously, since these individuals had no health problem. We were unable to confirm the additional deletion of DAZ4 in FM 16 and OZ -60 with DAZ-del-hap 9, since the DAZ4-specific 7.3 kb EcoRV fragment of the DYS1 locus was present in both DNA samples. DNA samples with DAZ-del-hap 4, indicative for deletion of only DAZ4, were found in the fertile men exclusively (Table III). For the practical reasons given above, DYS1 blot experiments were not possible for these individuals. However, the DAZ4 deletion in the father of OZ 5 (Figure 3A) and in the father of FM 39 strongly suggests that DAZ4 deletions are common variants in the normal fertile male population and are not associated with the occurrence of male infertility.
Our DAZ-SNV/STS analyses also suggest that the B alleles of SNV I and VI in DAZ4 are polymorphic. Although they are present in the DAZ4 gene of the RPCI-11-donor and of 88 individuals in our two sample groups (Tables III and IV), absence of the B allele of SNV VI was found, independently from absence of the B allele of SNV I, in all DNA samples with DAZ-del-hap 2 and in three FM -DNA samples with DAZ-del-hap 4 (FM 2, 15, 26). Absence of the B allele for SNV I was found, independently from absence of the B allele of SNV VI in six FM and two OZ samples of DAZ-del-hap 4 (Tables III and IV). Similar derivatives of DAZ-del-hap 2 (i.e. absence of the A allele of only SNV III, or of only SNV IV) were found in FM and OZ samples as well (Tables III and IV). We therefore conclude that in DNA samples where the deletion of DAZ2 or DAZ4 could not be confirmed by appropriate DYS1 blot experiments, most probably familial gene conversion events occurred between the highly homologous DAZ gene copies (<99%) in the DAZ locus of the Y chromosome in these families. Gene conversion events, especially within the DAZ2 and DAZ4 gene structures, would easily explain the complex pattern of derivatives which we observed for DAZ-del-hap 2, 4 and 9 in both groups of men. Similar conclusions were drawn recently for other human gene loci, based on an unexpected linkage disequilibrium between closely spaced SNP markers (Ardlie et al., 2001). Obviously, gene conversion events prevent the identification of a specific DAZ copy deletion by only SNV/STS analyses and the additional analysis by DYS1 blot experiments is required.
We do not think that our findings would be improved by additional fibre–fluorescence in-situ hybridization (FISH) experiments, because FISH probes including the repetitive DAZ gene regions can also result in an over-estimation of the number of DAZ gene copies (Saxena et al., 2000) and FISH probes specific for each DAZ gene copy cannot be created because of their high sequence homology (<99%). Moreover, irrespective of whether real DAZ deletions or simple SNV/STS polymorphisms due to gene conversion events had led to the derivatives of the DAZ-del-hap 2, 4, 9 classification in a portion of our FM and OZ samples, both molecular events would not interfere with the men's fertility, as they were found in both sample groups.
Only DAZ1/DAZ2 deletions were associated with severe oligozoospermia
A possible link of a specific DAZ-deletion haplotype specific to the occurrence of the patients' oligozoospermic phenotype was found only for the OZ samples with DAZ-del-hap 5, pointing to a deletion of the DAZ1 and DAZ2 gene copies in the Y chromosome of these men. These were confirmed by the DYS1-DNA blot analysis (Figure 3A). DAZ-del-hap 5 was not found in the DNA samples of 107 fertile men nor in the father of OZ 5 (Figure 2, 3A). We therefore assume that not only in OZ 5, but in all OZ samples with DAZ-del-hap 5, the DAZ1/DAZ2 deletions are most likely `de-novo' mutation events, i.e. restricted to the DAZ locus of the patient. A spermatogenic influence of the DAZ4 deletion in the OZ 5 family can be excluded because the father of OZ 5 was reported to have normal fertility (J.Goncalves, personal communication). No other AZFc STS deletions nor the deletion of any other AZFc candidate genes were identified in the Y chromosome of the DAZ-del-hap 5 patients, although small deletions or point mutations in the other AZFc candidate genes cannot yet be excluded.
Our extensive analyses of DAZ gene copy specific restriction fragments in the RPCI-11 DAZ-BAC contigs (Figure 1B) were only successful for the repetitive DYS1-DAZ gene regions. The exact molecular extensions of the indicated DAZ1/DAZ2 gene deletions could therefore not be estimated. However, absence of the DAZ1-specific DYS1-EcoRV fragments in all DAZ-del-hap 5 patients includes a deletion of at least two of the DAZ1 RNA binding domains (RRM) in exons 2–5 (Figure 1B). A distortion of the putative DAZ1 germ line-specific RNA binding function is therefore expected. The most probable explanation for the occurrence of the oligozoospermic phenotype in OZ 5, 20, 35, 65 and 73 is therefore the deletion of the entire DAZ1/DAZ2 gene doublet in all of them.
Putative molecular mechanisms for the DAZ gene deletions in the AZFc region
Our comparison of the DAZ gene structures of the RPCI-11 donor with that of the CTA/CTB donors described by Saxena et al. reflects a dynamic structure, especially of the DAZ2, DAZ3 and DAZ4 gene copies, but not of the DAZ1 gene copy (Saxena et al., 2000). We found that the DAZ gene doublets DAZ1/DAZ2 and DAZ3/DAZ4 are located in two palindromic sequence domains, separated by a distance of 1.47 Mb (Figure 1A). The observed dynamic rearrangements which distinguished the DAZ2, DAZ3 and DAZ4 gene copies of the CTA/CTB- and the RPCI-11 donors, as well as the proposed gene conversion events discussed above, might therefore be caused by the structural-specific organization in the genomic region of both DAZ gene doublets and/or of the entire AZFc sequence, now shown to be composed of massive palindromes in 93% of its length (Kuroda-Kawaguchi et al., 2001). Palindromes and inverted repeat structures in the genome are generally known to induce local instabilities which depend on their lengths and repetitive sequence contents. Local rearrangements increasing their asymmetry could stabilize them (Zhou et al., 2001). In the DAZ1/DAZ2 and DAZ3/DAZ4 palindromes, this could explain the amplification of RRM domains in only DAZ1 and DAZ4 and the variable structure of the repetitive exon 7 in DAZ2, DAZ3 and DAZ4.
Deletions of the DAZ1 gene alone were found neither in our fertile men nor in our infertile men, but only in combination with the DAZ2 gene. We therefore assume that molecular mechanisms inducing the observed DAZ1/DAZ2 gene deletion events are most likely based on the nature of their unique palindromic sequence structure or on the recombination of homologous repetitive sequence blocks flanking the DAZ1/DAZ2 gene doublet (Chen et al., 1997). Intrachromosomal recombination events of two repetitive 10 kb sequence blocks in proximal Yq11 were recently found to be causative agents for the occurrence of most AZFa deletions (Kamp et al., 2000; Sun et al., 2000). Long homologous repetitive sequence blocks were also found around the DAZ genes. Two long repetitive blocks (AZFc-Rep1, Figure 1) in the same polarity and with a surprisingly high sequence homology (<99%), starting in BAC5C5 and BAC160O2 (respectively positions 134890 and 50000) and extending to the 3'ends of the DAZ1 and DAZ3 gene respectively, were easily identified by BLAST alignment (Figure 1A). Their molecular lengths were estimated from the AZFc-BAC contig sequence to be 368 (DAZ1-AZFc-Rep1) and 320 kb (DAZ3-AZFc-Rep1). Different amplifications of the human ß-satellite 68 bp repeat unit (Waye et al., 1989) explain their length differences. Intrachromosomal recombination events between these two AZFc-Rep1 blocks would delete not only the DAZ1 gene, but also the entire DAZ2 gene, as demonstrated in our five OZ samples with DAZ-del-hap 5.
We also found a considerable extension of the palindromic inverted repeats wherein the DAZ1/DAZ2 and DAZ3/DAZ4 gene doublets were embedded. These structures would certainly also promote a deletion of the complete gene doublets (Zhou et al., 2001).
The presence of the long repetitive AZFc-Rep1 sequence block with the same polarity upstream of the DAZ1/DAZ2 and DAZ3/DAZ4 gene doublets respectively was confirmed by the work of Kuroda-Kawaguchi et al. (Kuroda-Kawaguchi et al., 2001). In their paper, the AZFc-Rep1 blocks are the green AZFc amplicons, g1 and g2. A third homologous amplicon, g3, but with an opposite polarity, was found 3' downstream of the DAZ3/DAZ4 gene doublet (Kuroda-Kawaguchi et al., 2001; Figure 1 in that paper). Based on this palindromic AZFc sequence structure, we indeed expect that a deletion of the complete DAZ1/DAZ2 gene doublet can be caused by intrachromosomal recombination events between the AZFc-Rep1 blocks as shown in Figure 1, or, as a second possibility, between the AZFc amplicons b1 and b3 (Kuroda-Kawaguchi et al., 2001). DAZ1/DAZ2 deletions caused by b1/b3 recombination events would be indicated by the absence of two AZFc-STSs: DYS227/sY139 (GenBank accession no. G12002) mapped to AZFc-u2 (S.Fernandes, unpublished results) and sY1192 (GenBank accession no. G67166) mapped to AZFc-u3 (Kuroda-Kawaguchi et al., 2001). DAZ1/DAZ2 deletions caused by AZFc-Rep1 (or g1/g2) recombination events would be indicated by absence of the P1.2 amplicon of the AZFc-yellow-palindrome (Kuroda-Kawaguchi et al., 2001). Therefore, for the analysis of AZFc-Rep1 recombination events we established a novel SNV in the AZFc-P1.1/P1.2 amplicons (AZFc-yellow-palindrome-SNV I; GenBank accession no. G73351). Only AZFc-P1.2 located between g1/g2 has the A allele and AZFc-P1.1 has the B allele (DdeI restriction site; S.Fernandes, unpublished results).
We exclude a b1/b3 recombination event as a causative agent for the proposed complete deletion of the DAZ1/DAZ2 gene doublet in the OZ samples with DAZ-del-hap 5 (OZ 5, 20, 35, 65 and 73) because STS sY139 and sY1192 were found to be present in all these DNA samples. However, absence of the A allele for SNV I of the AZFc-yellow-palindrome was noted in OZ 5 and OZ 65, indicating deletion of AZFc-P1.2. This confirms our assumption of a AZFc-Rep1 recombination event as the causative agent for a complete deletion of the DAZ1/DAZ2 gene doublet in these OZ samples. Interestingly, the three other OZ samples with DAZ-del-hap 5 (OZ 20, 35 and 73) displayed a deletion of the SNV I B allele of the AZFc-yellow-palindrome, suggesting a deletion of the P1.1 amplicon in these samples. Although not yet proven, we speculate that in the Y chromosome of these three OZ families both AZFc-yellow amplicons (P1.1 and P1.2) have an inverted orientation when compared with that in the AZFc sequence of the Y chromosome of the RPCI-11 donor (Kuroda-Kawaguchi et al., 2001). If this holds true, we can suggest that a general reference for the AZFc sequence as proposed by Kuroda-Kawaguchi et al. might not exist (Kuroda-Kawaguchi et al., 2001) and that in the OZ 20, 35 and 73 samples, a recombination event between the AZFc-Rep1 blocks has caused a complete deletion of the DAZ1/DAZ2 gene doublet, as indicated by absence of the B allele of the AZFc-yellow-palindrome SNV I.
Acknowledgements
We are grateful to Hartmut Voss for sequence analysis of the 49f fragment. Drs N.Joergensen, A.G.Andersen and J.Hinrichsen are thanked for their help in collecting and isolating DNA samples from fertile male controls (partially supported by the European Research Commission). J.Riekenberg and A.Barbosa are thanked for their help in collecting the samples from the oligozoospermic men. We also wish to thank Kartrin Christophers-Genzmer and Anne Jordan for their help in preparing the final version of this manuscript and especially for critically improving our English expression and grammar. This study was supported by grants to S.F. from the Fundacao para a Ciencia e Tecnologia (FCT) (SFRH/BD/811/2000), to J.G. from the project PRAXIS XXI/PSAU/97/96 of the FCT and to P.H.V. from the Deutsche Forschungsgemeinschaft (DFG: Vo403/10–2).
Notes
7 To whom correspondence should be addressed at: Im Neuenheimer Feld 328, D-69120 Heidelberg, FRG, Germany. E-mail: peter_vogt{at}med.uni-heidelberg.de 
Recently, a reduced number of DAZ gene copies in subfertile and infertile men has also been reported by de Vries et al. (2002, Fertil. Steril., 77, 68–75), using DAZ-SNV deletion and DAZ-Fibre-FISH experiments. Surprisingly, the Fibre-FISH experiments did not support their SNV deletion analyses and Fibre-FISH was also not able to distinguish between the deletion of one DAZ gene copy and the deletion of a complete DAZ gene cluster (DAZI/DAZ2 or DAZ3/DAZ4, respectively). To confirm partial DAZ gene deletions, the use of DYS1 blot experiments instead of Fibre-FISH, as proposed in this paper, is therefore strongly recommended.
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Submitted on November 2, 2001; accepted on January 15, 2002.
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