Mol. Hum. Reprod. Advance Access originally published online on March 7, 2008
Molecular Human Reproduction 2008 14(4):251-258; doi:10.1093/molehr/gan014
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Identification of new breakpoints in AZFb and AZFc
1Department of Genetics, Faculty of Medicine, University of Porto, Alameda Prof. Hernâni Monteiro, 4200-319 Porto, Portugal 2Laboratory of Human Genetics, University of Madeira, Funchal, Madeira, Portugal 3Laboratory of Cell Biology, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, 4099-003 Porto, Portugal 4 Centre for Reproductive Genetics A Barros, 4100-009 Porto, Portugal
5 Correspondence address. Tel: +351-22-551-36-47; Fax: +351-22-551-36-48; E-mail: sf{at}med.up.pt
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Abstract |
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Microdeletions in AZFa, AZFb and AZFc regions lead to different patterns of male infertility, from severe oligozoospermia to non-obstructive azoospermia. Intrachromosomal homologous recombination mechanisms were already identified in patients with simultaneous microdeletions in the AZFb and AZFc regions. Ten patients with atypical AZFb and AZFc deletion patterns were studied. The definition of those microdeletions and the fine characterization of the respective breakpoints were performed using sequence tagged sites/single nucleotide variants-PCR and DNA sequencing. Y-chromosome haplogroups were determined to establish a putative association with the patterns obtained. Seven deletion patterns were identified, P5/terminal (30%; 3/10), P5/P1 distal (20%; 2/10), IR4/distal-P2, IR2/proximal-P1, IR4/distal-P1, P4/terminal and complete AZFb/c deletion (10%; 1/10). Breakpoint sequence analysis suggests that only in one patient the P5/P1 distal deletion pattern was due to a homologous recombination mechanism. Sequence alignment of the other deletion patterns suggest that they have resulted from non-homologous recombination mechanisms.
Key words: non-obstructive azoospermia/AZFb deletions/AZFb+c deletions/Y chromosome
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary materialFundingAcknowledgementsReferences |
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The Y chromosome is the shortest chromosome of the human genome. About 95% of its content consists of a non-recombining region (NRY; Page et al., 1982; Lahn and Page, 1997), recently renamed as male-specific region (MSY; Skaletsky et al., 2003). About one-third of the euchromatic MSY region is composed of large repetitive units (amplicons), of different composition, size, number of copies and orientation, occurring as tandem repeats, inverted repeats or dispersed throughout the short and long arms of the Y chromosome (Tilford et al., 2001). The highly repetitive nature of MSY sequence may favour the recombination between direct repeats leading to the occurrence of deletions, during meiosis (Vollrath et al., 1992; Edwards and Bishop, 1997).
The locus for azoospermia factor (AZF) in Yq11.2 (Tiepolo and Zuffardi, 1976) is subdivided into AZFa, AZFb and AZFc regions (Vogt et al., 1996), although there is recent evidence suggesting an overlap of 1.5 Mb between distal AZFb and proximal AZFc (Fig. 1A) (Vogt et al., 1996; Repping et al., 2002).
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fine mapping, (D) Genetic constitution of the AZFb and AZFc regions, (E) Various deletion patterns identified. Black with + symbol, presence; grey with – symbol, absence; grey without any symbol, sequence between the positive and negative STSs/SNVs, where the proximal and distal breakpoints are located. STSs, sequence tagged sites; SNVs, single nucleotide variants.Different pathologies have been associated with deletions in AZF regions, with Sertoli-cell-only syndrome (SCOS) being associated with a complete AZFa deletion (Vogt et al., 1996; Kamp et al., 2001; Dada et al., 2004) and pre-meiotic arrest (MA) to a complete AZFb deletion (Vogt et al., 1996; Dada et al., 2004). On the contrary, AZFc deletions are associated to different testicular pathologies, ranging from SCOS to MA, both with or without residual spermiogenesis, or hypospermatogenesis (HP), suggesting that AZFc genes might play a role in the maturation process of post-meiotic germ cells (Vogt et al., 1996; Silber et al., 1998; Ferlin et al., 1999; Foresta et al., 2001a; Vogt and Fernandes, 2003; Dada et al., 2004; Ferrás et al., 2004).
The AZF region is deleted in 
13% of individuals with non-obstructive azoospermia and in 7–10% of patients with oligozoospermia (Reijo et al., 1995; Girardi et al., 1997). In infertile patients with Y chromosome microdeletions, the frequency of deletions in AZFa (5%) and AZFb (10–16%) is lower compared with the frequency of AZFc deletions (60%) (Vogt, 1998; Yen, 1998; Foresta et al., 2001b; Hopps et al., 2003). Deletions bearing two or the three AZF regions are detected in a frequency of 14% (Foresta et al., 2001b).
The highly repetitive structure of the Y chromosome and the presence of large repetitive blocks, mainly in AZFb and AZFc regions, favour deletions occurring as a result of intrachromosomal recombination mechanisms between homologous sequences (Vollrath et al., 1992; Kuroda-Kawaguchi et al., 2001; Repping et al., 2002). AZFa deletions seem to frequently arise as a consequence of homologous recombination between two human endogenous retrovirus (HERV15) sequence blocks, separated by 800 kb (Blanco et al., 2000; Kamp et al., 2000; Sun et al., 2000). Deletions removing simultaneously part of the AZFb and AZFc regions result from homologous recombination, in which the proximal breakpoints are located in the P5 palindrome and the distal breakpoints mapped in either proximal P1 or distal P1 (Repping et al., 2002). However, a few deletions based on mechanisms of non-homologous recombination were also identified. Krausz et al. (2006) described the occurrence of two AZFa deletions involving the USP9Y gene, one removing the sequence between intron 33 and the sequence downstream exon 46, and the second excising the upstream sequence of exon 1 up to intron 28. Two AZFb/c deletions were described as having resulted from non-homologous recombination, with the proximal breakpoints located on either P5 or P4 palindromes and the distal breakpoints mapped on distal P1 (Repping et al., 2002).
In this work, patients with AZFb/c microdeletions were studied to identify the breakpoints and the recombination mechanisms underlying these microdeletions. Additionally, Y-chromosome haplogroups were determined to study any possible association with the patterns obtained.
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Materials and Methods |
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Patients
Under informed consent, 10 patients were studied, one with severe oligozoospermia (<1 x 106 spermatozoa/ml—P1743) and nine with non-obstructive azoospermia (P36, P783, P1061, P1201, P1313, P1543, P1955, P2006 and P2115) (Table I). All cases presented a normal karyotype (46X,Y) and microdeletions in AZFb and AZFc regions, previously detected through routine molecular diagnosis of the Y chromosome. The molecular markers performed in this diagnosis are shown in Table II.
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Fine mapping of the breakpoints
DNA was extracted from patients' peripheral blood through salting out/clorophorm procedure (Müllenbach et al., 1989). For the fine mapping of the breakpoints, additional sequence tagged sites (STSs) and single nucleotide variants (SNVs) were used. Some were already published (Supplementary material, Tables SI and II) and others were newly designed (Supplementary material, Table SIII). The primer design was based on each patient microdeletion pattern, using the BACs sequence of the contigs NT_011875 [GenBank] and NT_011903 [GenBank] , from the RPCI-11 genomic library, deposited in the NCBI database (Fig. 1B and C) (Kuroda-Kawaguchi et al., 2001).
For each DNA sample, the presence/absence of STSs and SNVs, located close to the breakpoints, was analysed by successive PCR reactions containing 1x PCR buffer, 1–2.5 mM MgCl2, 0.03 mM of each dNTP (Invitrogen, Barcelona, Spain), 0.5 mM of each primer (Thermo Electron, Ulm, Germany; Supplementary material, Tables SI–III), 1 U Taq DNA polymerase (MBI Fermentas, St Leon-Rot, Germany) and 1 µl of DNA in a final volume of 25 µl.
PCR conditions consisted of an initial denaturation at 95°C for 5 min, followed by 35 amplification cycles: denaturation at 95°C for 1 min, annealing for 1 min at variable temperatures (Supplementary material, Tables SI–III) according to the STS or SNV, strand elongation at 72°C for 1 min and 30 s and a final extension at 72°C for 10 min. DNA from a fertile male and a female were used as positive and negative controls, respectively, to test the specificity of the primers. Negative controls, containing all the components except the DNA, were always used to exclude the hypothesis of PCR contaminations. SNV products were digested with specific restriction enzymes (Supplementary material, Table SIV). Enzymes were from Bioron, Ludwigshafen, Germany; MBI Fermentas; New England Biolabs (NEB), Frankfurt am Main, Germany.
Bridging—Long-Range PCR
The bridging (amplification of the sequence containing the breakpoint junction) was performed for those cases presenting a relatively small length between positive and negative STSs/SNVs at breakpoints, using the Expand High Fidelity PCR System Kit (Roche Diagnostics, Mannheim, Germany), according to manufacturer instructions—Long-Range PCR. Thermal profile consisted of an initial denaturation at 94°C for 5 min followed by two phases of cycles with distinct profiles. The first phase consisted of 10 cycles of denaturation at 94°C for 15 s, followed by annealing of primers (Supplementary material, Table SV) for 30 s, and elongation at 68°C for 8 min. The second phase was composed by 20 additional cycles of denaturation at 94°C for 15 s, primer annealing for 30 s, and elongation at 68°C for 8 min in first cycle, with successive increments of 5 s in each subsequent cycle. Finally, a temperature increase to 72°C for 7 min allowed the final extension of the already synthesized strands. All products were visualized on agarose gels stained with 0.5 µg/ml ethidium bromide. Long-Range PCR products were purified using GFX PCR DNA and Gel Band Purification Kit (Amersham Biosciences; Buckinghamshire, UK), according to manufacturer instructions and sequenced using inner primers—primer walking, with the BigDye Terminator Cycle Sequencing v1.1 Ready Reaction kit (Applied Biosystems, Foster City, CA, USA). Products were then precipitated, sequenced using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems) device and data analysed with appropriate software (DNA Sequencing Analysis; Applied Biosystems).
Y-chromosome haplogroups
The STSs containing Y-SNPs were assayed as described (Underhill et al., 2000, 2001). Genotyping was performed using both native and engineered restriction fragment length polymorphism methods. Haplogroup nomenclature and phylogeny was in accordance with the Y Chromosome Consortium (2002). Currently, 153 haplogroups are established, presenting a different distribution throughout the World. The haplogroup tree is composed of 18 main branches designated by the capital letters A to R. Each of these branches is further divided into internal branches defined by the letter A–R and an additional number. Lately, these branches may be further subdivided into minor branches whose designation, besides the letter A–R and the corresponding number, is followed by a lower case letter. The Y chromosome lineages that belong to a specific main branch, but not to its respective internal branches, since it is not based on derived characters, are defined as paragroups and distinguished from the haplogroups by the addiction of an asterisk (*) symbol to the corresponding branch (YCC, 2002).
Statistical analysis
Haplogroup frequency differences were calculated according to Fisher's exact test using the Arlequin software (Schneider et al., 2002), for 8 of the men with Y chromosome deletions and for 101 fertile men from Northern Portugal (Gonçalves et al., 2005). P-values <0.05 were considered statistically significant.
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Results |
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In the 10 patients studied, seven different deletion patterns were identified (Fig. 1E). The most detected pattern was P5/terminal, found in Patients P36, P1061 e P2115 (30%; 3/10), followed by P5/distal-P1 in Patients P1313 and P2006 (20%; 2/10). All other patterns, the complete deletion of AZFb and AZFc regions, IR4/distal-P2, IR2/proximal-P1, IR4/distal-P1 and P4/terminal, were identified each in only one patient (10%), P783, P1743, P1543, P1955 and P1201, respectively. In 50% (5/10) of the cases, terminal deletions were detected, involving the removal of the whole sequences, from the breakpoint to the heterochromatin. Although in some deletion patterns the proximal and/or distal breakpoints were mapped in the same amplicon, the majority of the breakpoints were different in each patient.
In spite of several attempts to precisely locate the breakpoints in those cases with a relatively small length between the positive and negative markers at the breakpoints, bridging was only achieved in Patient P2006. A fragment with a molecular weight between 7126 and 8144 bp was obtained after Long-Range PCR (Fig. 2) and further sequenced with inner primers, to finely map the deletion breakpoint. Sequence alignment with BAC RP11-509B6, where the proximal breakpoint was located, revealed the existence of mismatches after nucleotide 89 208 (Fig. 3A). On the other hand, most of this sequenced product also evidenced homology with the BAC RP11-497C14 sequence, where the distal breakpoint occurred, with existence of minor mismatches until nucleotide 40 843. Thus, the deletion breakpoints in Patient P2006 occurred somewhere in a sequence containing 22 bp (Fig. 3B).
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The alignment of the sequence breakpoints identified in all other patients suggests that their deletion patterns resulted from non-homologous recombination mechanisms (Waldman and Liskay, 1988), since they did not share the proposed 134–232 bp sequence of complete homology required for initiation of the process. The patterns identified were characterized by distinct deletion lengths, with longer deletions being verified in patients with the most severe phenotype (Table III).
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Y-chromosome haplogroups were assessed in 8 of the 10 patients, P783, P1201, P1313, P1543, P1743, P1955, P2006 and P2115. Patients were distributed into two of the main branches of the Y-haplogroup tree, the E and R branches. The most frequent haplogroup was E3b2 (5/8, 62.5%), identified in Patients P1201 (P4/terminal), P1743 (IR4/distal-P2), P2115 (P5/terminal), P1313 and P2006 (P5/distal-P1). The other three patients were ascribed to the R branch (3/8; 37.5%), Patients P783 (complete AZFb/c deletion) and P1955 (IR4/distal-P1) were included in haplogroup R1b (25%; 2/8) and P1543 (IR2/proximal-P1) in paragroup R1* (12.5%; 1/8). In this group of patients, haplogroup E3b2 was found to be associated with different deletion patterns, with some similarities in breakpoints location. With exception of the IR4/distal-P2 pattern, whose proximal and distal breakpoints differ from the other patterns identified within this haplogroup, in P5/distal-P1 and P5/terminal patterns the proximal breakpoints occurred in the same palindrome. The distal breakpoint in P5/distal-P1 pattern was located in distal AZFc, with deletion of a large portion of the AZFc sequence. The patterns involving terminal deletions (P5/terminal and P4/terminal) were also found only in patients with Y chromosomes belonging to the E3b2 haplogroup. To the haplogroup R were associated deletion patterns bearing distinct features on their proximal and distal breakpoints, being the complete AZFb/c deletion the most distinct one.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary materialFundingAcknowledgementsReferences |
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After Y-chromosome screening analysis of 1176 male infertile patients with severe oligozoospermia and non-obstructive azoospermia, 10 patients (0.8%) were found with a deletion encompassing the AZFb and AZFc regions. From the 10 patients studied, seven different deletion patterns were identified, three of which are first described here, the IR4/distal-P2, IR2/proximal-P1 and IR4/distal-P1 patterns. In 50% (5/10) of the patients, terminal patterns were detected, involving the deletion of the whole sequence downstream the breakpoint, located in palindromes P4 and P5, including the heterochromatin. The most frequently identified pattern was P5/terminal found in 30% (3/10) of the patients. The second most frequent pattern, with its proximal and distal breakpoints located in palindromes P5 and distal-P1 respectively, was detected in 20% (2/10) of the patients. The IR4/distal-P2, IR2/proximal-P1, IR4/distal-P1 and P4/terminal patterns were detected with a frequency of 10% (1/10) each. The pattern with the longest deletion length corresponded to the complete AZFb/c deletion (Hopps et al., 2003) and was identified in only one patient (10%).
Although some of these deletion patterns shared a common location at the proximal or distal breakpoints, the precise mapping of the breakpoints was different in all cases. This difference was even observed in patients sharing the same pattern (P5/distal-P1: P1313, P2006). The location of the proximal breakpoint in the P5 palindrome was found to bear two different patterns, identified in five patients (50%), whereas the distal breakpoint in the P1 palindrome was found in three patterns, identified in four patients (40%). These findings thus confirm the existence of hotspots within those palindromes (Repping et al., 2002).
Although AZF deletions are considered a consequence of the repetitive structure of the Y chromosome, which favours intrachromossomal recombination events between homologous sequences sharing the same polarity (Kuroda-Kawaguchi et al., 2001), not all deletions follow this mechanism. These atypical deletions resulting from non-homologous recombination mechanisms (Repping et al., 2002) are here confirmed. Exhaustive analysis of sequence alignments, where the proximal and distal breakpoints were located, revealed a total absence or reduced homology between the sequences involved in the breakpoints of the majority of patients, suggesting that the different deletions may have occurred as a consequence of non-homologous recombination mechanisms (Roth et al., 1985; Roth and Wilson, 1986; Thode et al., 1990). In the present series, only pattern P5/distal-P1 (P2006) occurred as a result of an intrachromosomal recombination between homologous sequences with the same orientation, from the BAC RP11-509B6 in yel3-yel4 amplicons and BAC RP11-497C14 in P1.1 amplicon, thus causing deletion of the whole internal sequence (Fig. 4). Only in this case and through DNA sequencing it was possible to finely map the proximal and distal breakpoints in a 22 bp sequence.
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Regardless the mechanism involved in each deletion, the identified patterns were characterized by different deletion lengths, which lead to the deletion of distinct groups and number of genes. In the complete AZFb/c deletion pattern, the
9.28 Mb deletion was associated with removal of all 51 genes and transcription units. The P5/terminal and P5/distal-P1 patterns were responsible for the deletion of a similar number of genes and transcription units (44–49). In the P4/terminal pattern, the number of genes and transcription units excised was lower as the breakpoint was located in a downstream sequence where the genetic content of the AZFc terminal sequence is less abundant. Patterns with a less deletion length (IR4/distal-P2, IR2/proximal-P1 and IR4/distal-P1) showed a corresponding number of genes and transcription units removed with the length of the deletion (Table III; Fig. 1D). The most frequently deleted gene copies belonged to the gene families RBMY1, PRY, BPY2 and DAZ, all of them already considered AZF candidates. Concerning the transcription units, the highest deleted frequency was observed for transcripts TTTY13, TTTY6, TTTY5, TTTY17 and TTTY4. Complete AZFa deletions were demonstrated to cause SCOS (Kamp et al., 2001), whereas complete AZFb (from P4 with or without AZFc deletion) deletions were suggested to cause pre-MA (Vogt et al., 1996). However, the latter were more recently shown to affect post-meiotic differentiation and cause an evolutive syndrome, meaning that during a patient's life it would evolve from oligozoospermia to azoospermia, then from HP to maturation arrest (MA), and, finally, to Sertoli only (Fernandes et al., 2002; Ferrás et al., 2004, Ferlin et al., 2007). Although FSH is supposed to rise as spermatogenesis becomes impaired, some patients do not seem to conform to this generalization most probably because of individual characteristics, independent of the AZF deletion patterns. Taking into account the histological diagnosis, the sites of the breakpoints and the type and number of the deleted genes in each deletion pattern (Table III), it is thus possible to suggest a putative critical region within AZFb that might be responsible for the initiation of spermatogenesis in humans, and located in yel4/b6, which includes genes XKRY2, CDY2 and HSFY1, as well as the LOC401630 and TTTY9 transcription units (Table III; Fig. 1D). Y-chromosome haplogroups could be studied in eight patients. The seven different AZFb/c deletion patterns were found to belong to two of the main branches of the haplogroup tree. Haplogroup E3b2, frequently found in the African population (Cruciani et al., 2002; Semino et al., 2002) but only with a frequency of 5.9% in North Portugal (Gonçalves et al., 2005), was identified in 62.5% (5/8) of the patients (P = 0.00). This included patterns IR4/distal-P2, P5/distal-P1, P4/terminal and P5/terminal with the last three presenting some homogeneity in breakpoints location, length of the deleted sequence and genes and transcript units removed, pointing for a structure that favours the same type of deletions.
In spite of haplogroup R being the most frequent in Western Europe since Palaeolithic times (Semino et al., 2000; Jobling and Tyler-Smith, 2003), it was only identified in three patients, each with distinct deletion patterns. This seems to strengthen the theory that some haplogroups, like the E3b, are higher associated to the same non-random deletions in Y-chromosome. In spite of different deletion patterns being ascribed to the same haplogroup, it was not possible to establish an association between these deletion patterns and Y haplogroups, although it remains possible to predict the occurrence of deletions in individuals belonging to a specific haplogroup such as E3b (Fernandes et al., 2006).
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Supplementary material |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary materialFundingAcknowledgementsReferences |
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Supplementary material is available at MOLEHR Journal online.
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Funding |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary materialFundingAcknowledgementsReferences |
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This study was partially supported by Foundation for Science and Technology (FCT-SFRH/BD/6664/01, POCI/SAU-MMO/60555/04, 59997/04; UMIB).
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Acknowledgements |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary materialFundingAcknowledgementsReferences |
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The authors wish to thank Cristiano Oliveira (Gynaecology, Centre for Reproductive Genetics), Luís Ferrás (Department Urology, Vila Nova de Gaia Hospital) for clinical expertise and Joaquina Silva and Ana Gonçalves (Centre for Reproductive Genetics) for technical assistance.
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Submitted on December 20, 2007; resubmitted on February 28, 2008; accepted on March 3, 2008.
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