Human Molecular Genetics

This Article Abstract FREE Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (34) Request Permissions Google Scholar Articles by Bosch, E. Articles by Jobling, M. A. Search for Related Content PubMed PubMed Citation Articles by Bosch, E. Articles by Jobling, M. A. Social Bookmarking          What's this?

Human Molecular Genetics, 2003, Vol. 12, No. 3 341-347
© 2003 Oxford University Press

Duplications of the AZFa region of the human Y chromosome are mediated by homologous recombination between HERVs and are compatible with male fertility

Elena Bosch and Mark A. Jobling^*

Department of Genetics, University of Leicester, University Road, Leicester LE1 7RH, UK

Received October 11, 2002; Accepted December 3, 2002

DDBJ/EMBL/GenBank accession nos AJ511660–AJ511667

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Deletions of the AZFa region on the long arm of the human Y chromosome cause male infertility. Previous work has shown that this is an example of a genomic disorder, since most deletions are caused by non-allelic homologous recombination between endogenous retroviral elements (HERVs) flanking the 780 kb region. The reciprocal products of these deletion events, AZFa duplications, have not been reported to date. Here we show that duplication chromosomes exist in population samples by detecting Y-chromosomal short tandem repeat (YSTR) allele duplications within the AZFa region, and by showing that two chromosomes carrying these duplicated alleles contain a third junction-specific HERV sequence. Sequence analysis of these cases, which most likely represent independent duplication events, shows that breakpoints lie in the same region of inter-HERV sequence identity as do deletion breakpoints, and thus that the mechanism of duplication is indeed the reciprocal of deletion. Consideration of the accumulated Y-STR allele diversity between duplicated copies of the AZFa region indicates that one of the duplication chromosomes has been in the population for at least 17 generations, and therefore must be compatible with male fertility.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Microdeletions of the long arm of the human Y chromosome represent an important cause of male infertility. Molecular analysis has defined three non-overlapping deletion intervals, named AZF (azoospermia factor) a, b and c (1). The most proximal and most rarely deleted of these intervals is AZFa, deletion of which leads most commonly to absence of germ-cells (Sertoli-cell only syndrome, SCOS). Two genes lie within the interval, namely DBY and DFFRY (also known as USP9Y), and observation of de novo deletions specifically affecting either DFFRY (2,3) or DBY (4) in milder oligozoospermic phenotypes suggests that both genes are critical and that there may be a synergistic effect in the more severe SCOS phenotype found in most AZFa-deleted individuals (5).

Previously, we (6) and others (7–9) have shown that all (10/10) known AZFa microdeletions arise through non-allelic homologous recombination between two 10 kb human endogenous retrovirus (HERV) elements flanking the 780 kb AZFa region (Fig. 1A and B). This form of male infertility can thus be classed as a ‘genomic disorder’, in which the lesion arises because of structural features of the genome (10).

View larger version (26K): [in this window] [in a new window]   Figure 1. Structure of HERVs, mechanisms of rearrangement and detection of duplication using STRs. (A) Structures of the proximal and distal AZFa HERVs, showing blocks of identity larger than 200 bp (hatched boxes), flanking specific primers, and the L1 fragment insertion in the distal HERV. Identity blocks are named A–C (6), with the name D being assigned to a fourth block between A and B. Note that block C extends beyond the distal end of each HERV. AZFa STR loci and the positions of proximal- or distal-specific primers between blocks of identity are also shown. (B) Schematic representation of a recombination event giving rise to reciprocal deletion and duplication products. In the case of a duplication, the junction HERV can in principle be amplified by the PCR primers 146R and HPR. (C) Schematic representation of electropherograms arising from two STRs, one within and one outside the AZFa region. (D) Electropherograms showing duplicated, and duplicated+mutated YSTR alleles found in this study. In the upper panels peak height for DYS389I (within AZFa) relative to DYS461 (outside AZFa, and here annotated as YA72) is elevated in case 2 compared with a control. DYS389II is duplicated and mutated in case 2 (two peaks). In the lower panels all loci are within AZFa, so duplication+mutation of DYS439 (case 2) gives two ‘half-height’ peaks. Size of each peak in base-pairs is indicated.

 
Apart from 1.5 kb of L1 material inserted in the distal HERV copy (Fig. 1A), the two directly oriented HERV elements share 94% sequence identity. However, the average degree of similarity varies markedly along the alignment of the two HERV elements (6). We define four blocks with a length of identity greater than 200 bp, which are called here A, D, B and C (see Fig. 1A). In mammalian cells, 200 bp is thought to be the minimum length of uninterrupted identity required for initiating homologous recombination in meiosis (also known as the minimal efficient processing segment, or MEPS) (11). Although AZFa deletion breakpoints are not identical in all patients, they all cluster within blocks of complete identity (9), which is the expected scenario if those identity blocks were providing the substrate for intrachromosomal recombination.

As well as their involvement in deletions, conversion-like events between the two HERVs have occurred, increasing the degree of identity between them (6), and in principle increasing the likelihood of HERV-sponsored rearrangements. The existence of a unique phylogeny of Y-chromosomal lineages (haplogroups) defined by binary, unique-event polymorphisms (12), provides an evolutionary context to studies of such rearrangements, and allows the number of independent conversions (in this case, two) to be determined.

The reciprocal product of the commonest class of AZFa deletion is expected to be a HERV-mediated duplication (Fig. 1B); such duplications have not yet been described. Their discovery would be of interest for three reasons: (i) it would confirm the expectation of the model for deletions; (ii) duplication chromosomes could represent intermediates for the generation of new HERV structures, for example by redeletion; and (iii) since males carrying duplications would possess extra copies of the genes within AZFa, they might have specific phenotypes in spermatogenesis.

The existence within AZFa of nine Y-chromosome-specific short tandem repeat loci (YSTRs), forming part of a polymorphic set used in population genetic studies (13) permits surveillance of population samples for duplications: after duplication, each AZFa YSTR will exist in two copies, while YSTRs outside the region remain as single copies, and detection of this difference in dosage when typing the STRs could signal the existence of the duplication. If a duplication persists through several generations, mutations are expected to accumulate in some AZFa YSTRs, differentiating the allele in one copy of the region from that in the other, and manifested as ‘double alleles’ at these particular loci (Fig. 1C).

Here we describe a survey of 1200 Y chromosomes from populations of the Iberian Peninsula, and the finding of AZFa duplication chromosomes. In two cases, the amplification of a junction fragment has been possible as well as the definition of the breakpoints involved in the rearrangement between proximal and distal HERV sequences.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Evidence of duplication from Y-STR haplotypes
Duplications of the AZFa region were sought by looking for duplicated AZFa YSTR loci (Fig. 1) during a population study of 1200 males from the Iberian Peninsula (13, and further typings). Three chromosomes presenting possible duplicated and mutated alleles (i.e. double peaks) for DYS389II and/or DYS439 were identified (see Fig. 1D for an example). Finding of mutations in these loci, but not others in the same interval, is not unexpected, as their relative diversities imply considerable mutation rate heterogeneity: DYS439 has the highest diversity of the AZFa YSTRs, DYS389II, DYS437 and DYS438 have intermediate diversity, while DYS434–436 have very low diversity (13). As an alternative to AZFa duplication, somatic STR mutation could also explain the finding of double peaks for one or more loci. However, the fact that double peaks are found in these cases only for YSTRs that lie within the AZFa region led us to further investigate the structure of these three chromosomes and try to confirm their potential AZFa duplications.

Nine commonly typed Y-STRs are known to lie in the AZFa region; six of these (DYS434–439) are coamplified in one multiplex, without any loci lying outside the region. AZFa duplication chromosomes in which STR mutations have not occurred cannot therefore be diagnosed using this multiplex. However, three AZFa YSTRs (DYS388, DYS389I and II) are coamplified with loci outside the AZFa region, and are therefore expected to show increased relative peak height in duplication chromosomes (see Fig. 1D for an example). Because the STR haplotyping method is not truly quantitative, identification of increased relative peak height is in practice more difficult than that of double peaks. Two further chromosomes were identified which showed reproducible evidence of AZFa duplication on these grounds, and these were also investigated further, along with the three chromosomes identified earlier.

Table 1 shows the complete 19-locus YSTR haplotypes of all five candidate duplication chromosomes, which originate from widely dispersed locations in the Iberian Peninsula.

View this table: [in this window] [in a new window]   Table 1. Haplotypes of potential AZFa duplication chromosomes

 
Junction HERV PCR confirms the presence of duplications
If AZFa duplicated chromosomes exist and represent the reciprocal product of HERV-mediated AZFa deletions, we expect them to contain three HERVs: the proximal and distal copies found in normal chromosomes, plus an additional junction HERV which will be a composite of the two original sequences (Fig. 1B).

In order to verify AZFa duplication we first attempted to amplify the entire junction HERV using primers 146R and HPR (Fig. 1B); this was possible only in case 2. Failure to amplify this large (10 kb) PCR product could reflect DNA quality, so we then attempted to amplify smaller predicted junction products using different combinations of primers specific for the proximal and distal HERV sequences (see Fig. 1). This yielded one further junction product (with primers Pp and Ld), in case 1. This therefore confirms the duplication for these two cases, and shows that the mechanism of duplication is HERV-mediated. The two confirmed cases belong to two related but distinct Y-chromosomal haplogroups, differentiated by the binary marker M153 (Table 1).

In the three remaining chromosomes, junction fragments could not be amplified. Without further evidence, it remains possible that these chromosomes do not carry AZFa duplications, and that their unusual YSTR patterns require other explanations. If they indeed do carry duplications of the AZFa region, their mechanisms may not be HERV-mediated.

Sequence structure of junction fragments
Sequence analysis was undertaken in order to illuminate the molecular mechanisms of duplication. To better define the breakpoint regions prior to sequencing, HERV junction structures in case 1 and 2 were determined by means of nested PCR using different combinations of proximal- and distal-specific primers (see Fig. 1A). In both junction HERVs breakpoints were located between the proximal boundary of identity block A, and the distal boundary of block D. This region is flanked by PCR primers P and L, each of which exists in a proximal- (Pp, Lp) and distal-specific (Pd, Ld) version. Sequence analysis of the PL region was then performed for the proximal, distal and junction HERVs in the two confirmed AZFa duplicated chromosomes (Fig. 2). The HERV sequences in the genome database derive from a Y chromosome (or Y chromosomes) about which we know very little. To provide a well-defined control with which to compare the duplicated chromosomes, we also determined proximal and distal PL sequences in an unduplicated chromosome, YCC26 (12); this chromosome was chosen because it belongs to the same sub-clade within the Y phylogeny as the two duplication cases, and is therefore more likely to reflect the sequence background on which the duplication(s) occurred than a chromosome chosen at random.

View larger version (23K): [in this window] [in a new window]   Figure 2. Structure and sequence of junction HERVs in AZFa duplication chromosomes. The upper part shows schematic representations of the proximal, distal and junction HERVs, indicating blocks of identity and proximal- and distal-specific primers used for amplification of junction fragments. The lower part of the figure shows the nucleotide present at each of the 41 positions of paralogous sequence variants within the first 2452 bp of the sequenced part of the PL PCR product for each HERV (the remaining 20 positions within the PL region were identical to the relevant reference sequence, and have been omitted). Nucleotides given in white on black are distal-specific, and those in black on white are proximal-specific, as defined by the reference sequence (‘Ref’). Numbers above the sequences give coordinates within the sequence: the nucleotide labelled ‘9’ is equivalent to nucleotide no. 10316 on the plus strand of AC002992 for the proximal HERV, and to nucleotide no. 145759 on the minus strand of AC005820 for the distal HERV.

 
In the reference sequence, the 3360 bp PL region contains 61 paralogous sequence variants (PSVs), nucleotide positions that vary between the HERVs. Our sequencing analysis reveals no additional PSVs between the HERVs. Figure 2 shows each nucleotide present at the 41 most proximal of these positions.

Sequences from the proximal HERVs in cases 1 and 2 and in the haplogroup R1b control were identical to each other, but different from the reference sequence. Four positions (from nucleotide 1460 to 1496) exist not in the proximal-specific forms found in the reference, but in the distal form; this represents a short tract of conversion of the proximal sequence by the distal sequence, of between 36 and 75 bp in length. This tract does not increase the length of identity block A (two proximal-specific positions separate the two), but begins 6–30 bp distal to its distal boundary. The finding of this conversion in the unduplicated R1b control sequence suggests that it arose before duplication, and is not one of its consequences. This was confirmed by screening a set of phylogenetically diverse chromosomes including the YCC collection (12) for the presence of the PpQd PCR product, which is diagnostic for the conversion (data not shown): it is found in all chromosomes belonging to clade R1b, and nowhere else in the phylogeny (i.e. is monophyletic).

In the distal HERV sequences we found a difference between case 1 and all other sequences, which again represents an apparent conversion, this time of the distal sequence by the proximal (Fig. 2). This conversion involves the first two non-identical positions distal to identity block A; conversion could therefore have involved block A as well as the two diagnostic bases, so the length of the conversion tract is between 5 and 1289 bp.

The sequences of junction fragments in cases 1 and 2 are the same as each other. At the proximal end the sequence is identical to that of the distal HERV, and distal to identity block A it is identical to that of the proximal HERV, including the tract of conversion found in all R1b chromosomes. Thus, in each case, the breakpoint lies within identity block A.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
The Y chromosome is haploid, and escapes for most of its length the reshuffling effects of allelic recombination. This greatly facilitates the study of genomic rearrangements such as duplications and deletions of large genomic regions and intrachromosomal gene conversion between paralogous repeats. In addition, the availability of a detailed and robust Y chromosome phylogeny provides an evolutionary context to these rearrangements, allowing the independence or non-independence of such events to be recognized. There is no reason to suppose that similar events are not occurring throughout the genome, although they are likely to be disguised by diploidy, recombination and interallelic diversity.

The availability of YSTRs and their inclusion in population studies provides a convenient way of detecting deletions and duplications; this approach has also been applied to the detection of autosomal aberrations (14), using STR markers developed for whole genome scans. Currently used YSTRs are highly non-randomly distributed on the chromosome (13), but as the number of available YSTRs increases, further deletions and duplications will be detected, and a less biased understanding of the organization and dynamics of this most flexible of human chromosomes will emerge.

We have demonstrated for the first time duplications of the AZFa region in two chromosomes. The sequences of the breakpoints show that both duplications were mediated by non-allelic homologous recombination between the HERVs flanking the region, and that exchange has occurred within the largest uninterrupted block of identity between proximal and distal HERVs (block A). The majority (6/10) of breakpoints in HERV-mediated AZFa deletions also lie in this block (6–9), and this resemblance between deletion and duplication breakpoints strongly suggests that duplications are indeed generated as the reciprocal products of deletions.

Deletion of the entire AZFa region causes male infertility, manifested as absence of germ cells (Sertoli-cell only syndrome). The two candidate genes with the region both have highly similar X-linked homologues, and encode ubiquitously expressed proteins (although one of the DBY transcripts has been reported to be expressed specifically in testis) (4). The spermatogenesis-specific phenotype of AZFa deletion may therefore be dosage-based, and this raises the question of the phenotype of AZFa duplication, in which males have three, rather than two, doses of these XY-homologous genes.

Although we have confirmed the existence of duplications of the same region, we have no direct information on the phenotypes of carriers of the duplications, because samples had been anonymized and individuals were unavailable for further study. However, we can make some indirect inferences about phenotype by considering how long the duplications have been resident in the population: if they have existed for a number of generations, then the duplications cannot, at least, be associated with complete infertility. The accumulation of STR mutations between a pair of chromosomes originating from the same duplication event, or between copies of the AZFa region in a single chromosome, provides an indication of the age of a duplication event.

One of the confirmed duplication cases carries the M153 mutation (i.e. belongs to clade R1b6), and the other does not. The two duplications could share a common origin, in which case the M153 mutation occurred on a duplication chromosome background, and all R1b6 chromosomes should carry duplications. Although we do not have direct evidence for whether other R1b6 chromosomes are duplicated, >8% of an Iberian Peninsula sample in a previous study belonged to R1b6 (15); in our sample of 1200 chromosomes we would therefore expect to find 100 duplication chromosomes—a far greater number than we have observed. It is possible that the duplications within R1b6 have reverted, but this explanation is less parsimonious than the hypothesis that the two duplications have occurred independently.

Since the duplications in these two chromosomes are probably independent, comparing their haplotypes gives no information about the age of the duplications; however, the existence of STR mutations between the two copies of AZFa in case 2 suggests that this duplication has persisted for several generations. The age of the duplication was estimated by calculating the mean number of mutational steps between each of the AZFa duplicated haplotypes and a hypothetical ancestral haplotype (this is equivalent to the rho statistic calculated from a median joining network (16); see Materials and Methods). This figure, 1.5 mutations (with a standard deviation of 0.87), can be converted into a time in generations by dividing by the mean YSTR mutation rate (2.80x10^-3 per locus per generation) (17), yielding an age of 60 generations ±34 generations. If we factor in uncertainty over mutation rate (95% confidence interval limits, 1.72–4.27x10^-3) (16), the plausible range extends to 17–153 generations. While dating methods such as this entail many assumptions and uncertainties, observations of YSTR haplotype transmissions in deep-rooting pedigrees provide some empirical support for the ranges that we estimate: the only verified STR mutations among nine loci typed in contemporary members of 2–10-generation pedigrees are single-step changes at single loci (18,19). We therefore think it likely that this duplicated chromosome has existed for at least 17 generations, and this is strong evidence that duplications of AZFa are compatible with reproductive ability.

A generous estimate of the incidence of AZFa deletions among men is 0.04%, since roughly 7.5% of men are infertile (20), up to 25% of these are infertile due to deletion in the Y chromosome (21) and AZFa deletions are estimated to account for, at most, 2% (5) of these. In contrast, we have shown that the incidence of confirmed AZFa duplication is at least 0.17% in the Iberian Peninsula; that is, at least four times more frequent than AZFa deletions. These duplications can act as intermediates for redeleted structures which may resemble the products of gene conversion or double crossover between HERVs, such as the chromosomes described previously (6), and our findings show that such complexity needs to be taken into account when modelling the evolution of paralogous direct repeats. Provided that the deletion and duplication mutations are equally likely to occur during meiosis, the relatively high frequency of AZFa duplications also supports the idea that men carrying them can father children.

The question of whether the fertility of duplication carriers is completely normal remains open, and careful genotype–phenotype correlations, including semen analysis and testicular biopsy, are clearly desirable to investigate this. It is possible that duplication chromosomes are targets for positive, or negative, selection. In principle, direct assays for duplication, using PCR primers designed to amplify only from the junction-specific HERV, could be used to screen both population samples and samples of men with unusual semen parameters. However, the evidence we have found for patchy gene conversion between HERVs means that apparent duplication chromosomes would need careful verification.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
DNA samples
Iberian DNA samples were from collections of the authors and are largely included in Bosch et al. (13) (see sources of potential duplication cases below); other samples of known binary haplotype were from the Y Chromosome Consortium (YCC) collection (12).

STR haplotyping
Typing of 19 YSTRs was carried out within three multiplexes (MS1, DYS19, DYS388, DYS390, DYS391, DYS392 and DYS393; CTS, DYS434, DYS435, DYS436, DYS437, DYS438 and DYS439; and EBF, DYS385, DYS389, DYS460, DYS461, DYS462 and amelogenin) on an ABI377 sequencer as described (13).

Haplotyping of binary markers
A number of binary markers were typed in the putative duplication chromosomes, but not all markers could be typed in all chromosomes, because amounts of DNA were limiting (see Table 1). 92R7 was typed according to Hurles et al. (22), SRY-2627 according to Veitia et al. (23), and PN25 by an allele-specific assay to be described elsewhere. M153 was typed by sequencing using the primers (5'–3') ACC CCG AAA GTT TTA TTT TA and GAC ACC AAT GGT CCT ATC TT.

Amplification of HERV sequences
PCR reactions were performed in a 10 µl final volume containing PCR buffer (24) supplemented with 12.5 mM Tris HCl, 1 µM each primer (HPF and HPR for proximal, 146R and 12f2B for distal and 146R and HPR for junction HERVs; sequences given below), 0.07 U Taq polymerase (Advanced Biotechnologies), 0.007 U Pfu DNA polymerase (Stratagene) and 10–50 ng of genomic DNA. Primer sequences (5'–3') were as follows: HPF, ATC ACA GGA ACA TCT CCA TGA; HPR, ACC TCA GAA GCA CCC TCC ATG G; 12f2B, GAA TTC CTA GAC TCA TGC AAT CTA C; 146R (also known as 494–146kP2) (6), ATG GCT TCA TCC CAA CTG AG.

PCR cycling conditions were as follows: 95°C for 5 min; 10 cycles of 94°C for 15 s, 60°C for 30 s, 66°C for 10 min; 25 cycles of 94°C for 15 s, 60°C for 30 s , 66°C for 10 min (+20 s/cycle); final extension at 66°C for 15 min. PCR products were isolated from a 0.6% (w/v) agarose gel as a plug and soaked for 2 h at 37°C in 200 µl of water containing 5 ng/µl of herring sperm DNA (Sigma). Two microlitres of eluate were used as template for subsequent HERV-specific amplifications.

HERV structure
The gross structure of each HERV was determined by means of nested PCR using different combinations of proximal-specific (Pre, Lre, Q2p, sY83bf, sY83br, R and O) and distal-specific (Pd, Qd, Q2d, Ld, G, D and S) primers. PCR reactions were performed using the previously isolated HERVs as a template in the same reaction mix as described above except for the addition of herring sperm DNA (0.1 ng/µl).

PCR cycling conditions were as follows: 95°C for 5 min; 10 cycles of 94°C for 20 s, 57.5°C for 30 s, 68°C for 3 min; 15 cycles of 94°C for 20 s, 57.5°C for 30 s, 68°C for 3 min (+20 s/cycle). Primer sequences (5'–3') were as follows: Pp, CAG CTG AAA CTA CTT CTT CAG TT; Lp (also known as 12f2L) (6): TTA ATT CAG CCC TCT GAG CG; sY83bF, CCA TCC TAC AAA AGA TCT AC; sY83bR, TGT CTA TCA ACT CAT AAT GC; R, GTG CTT TCC AAG CAT TTG GAA; O, GGA GAT CTG TGC AGG GTG AG; Pd, CAC CTG AAA CCA TTC TTC AGG C; Qd, CTC TTT TCT TTG GCC TCT GTG; Q2d, CTT CTT TTC CCC CAG TAA TGT A; Q2p, CTT CTT ATC CCT CAA TAA CGT G; Ld, TTA ATT CAG CCT TCG GAG CA; G (also known as 12f2G) (25), GGA TCC CTT CCT TAC ACC TTA TAC; D (also known as 12f2D) (6), CTG ACT GAT CAA AAT GCT TAC AGA TC; S, CTC TTG GCA TCA GCT AAA GAC.

PCR products generated using Pp/d and Lp/d primers were purified from agarose gels using the Qiaquick extraction kit (Qiagen) and used as templates for sequencing.

Sequencing analysis
Sequencing reactions were performed using the ABI PRISM Big Dye terminator chemistry (version 2) according to the manufacturer's recommendations and analysed on a ABI3100 sequencer (Applied Biosystems). Additional primer sequences (5'–3') were as follows: PQ forw, GGA AGG CAT TGA ATA TCC ATA AC; PQ rev, GTT ATG GAT ATT CAA TGC CTT CC; QL forw, CTA TTC GCT GCC CCC AGG A; QL rev, TCC TGG GGG CAG CGA ATA G; UNIV, TTG TGG AGC CTA TGG TCT CTA T; PPQfor, TAA ATT GGA AAC AGC ATG CCC; PPQrev, GGG CAT GCT GTT TCC AAT TTA; PQQfor, ACC CTG GAC TTG TTA GAG GGT G; PQQrev, CAC CCT CTA ACA AGT CCA GGG T; QQL1rev, GTC CTC AGC CCA TAC ACC TG; QQL2for, GTG CAC AAG CCA AAT GGT G; QLLfor, GAC TTG GTG GCC TTA GGT TG; QLLrev, CAA CCT AAG GCC ACC AAG TC. Sequence traces are available from the authors on request.

Calculation of duplication age
The rho statistic (the mean number of mutations to the root of a network), including a standard deviation, was calculated using the program program Network 3.1.1.1 (16). From this value an age in generations was calculated using the formula t=/µ, and a mutation rate of 2.80x10^-3 (95% confidence interval limits: 1.72–4.27x10^-3) per locus per generation (17)—see the text.

	ACKNOWLEDGEMENTS

 
We thank the donors of DNA samples; Matt Hurles for primer sequences, advice and comments on the manuscript; and Turi King for developing the PN25 assay. E.B. was supported by the Wellcome Trust, and M.A.J. by a Wellcome Trust Senior Fellowship in Basic Biomedical Science (grant no. 057559).

	FOOTNOTES

 
^* To whom correspondence should be addressed. Tel: +44 1162523377; Fax: +44 1162523378; Email: maj4{at}leicester.ac.uk

	REFERENCES

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 

1. Vogt, P.H., Edelmann, A., Kirsch, S., Henegariu, O., Hirschmann, P., Kiesewetter, F., Köhn, F.M., Schill, W.B., Farah, S., Ramos, C. et al. (1996) Human Y chromosome azoospermia factors (AZF) mapped to different subregions in Yq11. Hum. Mol. Genet., 5, 933–943.[Abstract/Free Full Text]

2. Sun, C., Skaletsky, H., Birren, B., Devon, K., Tang, Z., Silber, S., Oates, R. and Page, D.C. (1999) An azoospermic man with a de novo point mutation in the Y-chromosomal gene USP9Y. Nat. Genet., 23, 429–432.[CrossRef][Web of Science][Medline]

3. Sargent, C.A., Boucher, C.A., Kirsch, S., Brown, G., Weiss, B., Trundley, A., Burgoyne, P., Saut, N., Durand, C., Levy, N. et al. (1999) The critical region of overlap defining the AZFa male infertility interval of proximal Yq contains three transcribed sequences. J. Med. Genet., 36, 670–677.[Abstract/Free Full Text]

4. Foresta, C., Ferlin, A. and Moro, E. (2000) Deletion and expression analysis of AZFa genes on the human Y chromosome revealed a major role for DBY in male infertility. Hum. Mol. Genet., 9, 1161–1169.[Abstract/Free Full Text]

5. Affara, N.A. (2001) The role of the Y chromosome in male infertility. Exp. Rev. Mol. Med., 3 January, www-ermm.cbcu.cam.ac.uk/01002319h.htm.

6. Blanco, P., Shlumukova, M., Sargent, C.A., Jobling, M.A., Affara, N. and Hurles, M.E. (2000) Divergent outcomes of intra-chromosomal recombination on the human Y chromosome: male infertility and recurrent polymorphism. J. Med. Genet., 37, 752–758.[Abstract/Free Full Text]

7. Sun, C., Skaletsky, H., Rezen, S., Gromoll, J., Nieschlag, E., Oates, R. and Page, D.C. (2000) Deletion of azoospermia factor a (AZFa) region of human Y chromosome caused by recombination between HERV15 proviruses. Hum. Mol. Genet., 9, 2291–2296.[Abstract/Free Full Text]

8. Kamp, C., Hirschmann, P., Voss, H., Huellen, K. and Vogt, P.H. (2000) Two long homologous retroviral sequence blocks in proximal Yq11 cause AZFa microdeletions as a result of intrachromosomal recombination events. Hum. Mol. Genet., 9, 2563–2572.[Abstract/Free Full Text]

9. Kamp, C., Huellen, K., Fernandes, S., Sousa, M., Schlegel, P.N., Mielnik, A., Kleiman, S., Yavetz, H., Krause, W., Kupker, W. et al. (2001) High deletion frequency of the complete AZFa sequence in men with Sertoli-cell-only syndrome. Mol. Hum. Reprod., 7, 987–994.[Abstract/Free Full Text]

10. Stankiewicz, P. and Lupski, J.R. (2002) Molecular-evolutionary mechanisms for genomic disorders. Curr. Opin. Genet. Devl., 12, 312–319.[CrossRef][Web of Science][Medline]

11. Lukacsovich, T. and Waldman, A.S. (1999) Suppression of intrachromosomal gene conversion in mammalian cells by small degrees of sequence divergence. Genetics, 151, 1559–1568.[Abstract/Free Full Text]

12. Y Chromosome Consortium (2002) A nomenclature system for the tree of human Y-chromosomal binary haplogroups. Genome Res., 12, 339–348.[Abstract/Free Full Text]

13. Bosch, E., Lee, A.C., Calafell, F., Arroyo, E., Henneman, P., de Knijff, P. and Jobling, M.A. (2002) High resolution Y chromosome typing: 19 STRs amplified in three multiplex reactions. Forens. Sci. Int., 125, 42–51.

14. Rosenberg, M.J., Vaske, D., Killoran, C.E., Ning, Y., Wargowski, D., Hudgins, L., Tifft, C.J., Meck, J., Blancato, J.K., Rosenbaum, K. et al. (2000) Detection of chromosomal aberrations by a whole-genome microsatellite screen. Am. J. Hum. Genet., 66, 419–427.[CrossRef][Web of Science][Medline]

15. Bosch, E., Calafell, F., Comas, D., Oefner, P.J., Underhill, P.A. and Bertranpetit, J. (2001) High-resolution analysis of human Y-chromosome variation shows a sharp discontinuity and limited gene flow between Northwestern Africa and the Iberian Peninsula. Am. J. Hum. Genet., 68, 1019–1029.[CrossRef][Web of Science][Medline]

16. Bandelt, H.-J., Forster, P. and Röhl, A. (1999) Median-joining networks for inferring intraspecific phylogenies. Mol. Biol. Evol., 16, 37–48.[Abstract]

17. Kayser, M., Roewer, L., Hedman, M., Henke, L., Henke, J., Brauer, S., Krüger, C., Krawczak, M., Nagy, M., Dobosz, T. et al. (2000) Characteristics and frequency of germline mutations at microsatellite loci from the human Y chromosome, as revealed by direct observation in father/son pairs. Am. J. Hum. Genet., 66, 1580–1588.[CrossRef][Web of Science][Medline]

18. Heyer, E., Puymirat, J., Dieltjes, P., Bakker, E. and de Knijff, P. (1997) Estimating Y chromosome specific microsatellite mutation frequencies using deep rooting pedigrees. Hum. Mol. Genet., 6, 799–803.[Abstract/Free Full Text]

19. Jobling, M.A., Heyer, E., Dieltjes, P. and de Knijff, P. (1999) Y-chromosome-specific microsatellite mutation rates re-examined using a minisatellite, MSY1. Hum. Mol. Genet., 8, 2117–2120.[Abstract/Free Full Text]

20. Skakkebaek, N.E., Giwercman, A. and de Kretser, D. (1994) Pathogenesis and management of male infertility. Lancet, 343, 1473–1479.[CrossRef][Web of Science][Medline]

21. Stuppia, L., Gatta, V., Calabrese, G., Franchi, P.G., Morizio, E., Bombieri, C., Mingarelli, R., Sforza, V., Frajese, G., Tenaglia, R. et al. (1998) A quarter of men with idiopathic oligo-azoospermia display chromosomal abnormalities and microdeletions of different types in interval 6 of Yq11. Hum. Genet., 102, 566–570.[CrossRef][Web of Science][Medline]

22. Hurles, M.E., Veitia, R., Arroyo, E., Armenteros, M., Bertranpetit, J., Pérez-Lezaun, A., Bosch, E., Shlumukova, M., Cambon-Thomsen, A., McElreavey, K. et al. (1999) Recent male-mediated gene flow over a linguistic barrier in Iberia, suggested by analysis of a Y-chromosomal DNA polymorphism. Am. J. Hum. Genet., 65, 1437–1448.[CrossRef][Web of Science][Medline]

23. Veitia, R., Ion, A., Barbaux, S., Jobling, M.A., Souleyreau, N., Ennis, K., Ostrer, H., Tosi, M., Meo, T., Chibani, J. et al. (1997) Mutations and sequence variants in the testis-determining region of the Y chromosome in individuals with a 46,XY female phenotype. Hum. Genet., 99, 648–652.[CrossRef][Web of Science][Medline]

24. Jeffreys, A.J., Neumann, R. and Wilson, V. (1990) Repeat unit sequence variation in minisatellites: a novel source of DNA polymorphism for studying variation and mutation by single molecule analysis. Cell, 60, 473–485.[CrossRef][Web of Science][Medline]

25. Rosser, Z.H., Zerjal, T., Hurles, M.E., Adojaan, M., Alavantic, D., Amorim, A., Amos, W., Armenteros, M., Arroyo, E., Barbujani, G. et al. (2000) Y-chromosomal diversity within Europe is clinal and influenced primarily by geography, rather than by language. Am. J. Hum. Genet., 67, 1526–1543.[CrossRef][Web of Science][Medline]

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				M. A. Jobling, I. C. C. Lo, D. J. Turner, G. R. Bowden, A. C. Lee, Y. Xue, D. Carvalho-Silva, M. E. Hurles, S. M. Adams, Y. M. Chang, et al. Structural variation on the short arm of the human Y chromosome: recurrent multigene deletions encompassing Amelogenin Y Hum. Mol. Genet., February 1, 2007; 16(3): 307 - 316. [Abstract] [Full Text] [PDF]

				K. Writzl, B. Zorn, and B. Peterlin Preliminary analysis of AZFb region duplication by quantitative real-time PCR Hum. Reprod., March 1, 2006; 21(3): 753 - 754. [Abstract] [Full Text] [PDF]

				P. H. Vogt AZF deletions and Y chromosomal haplogroups: history and update based on sequence Hum. Reprod. Update, July 1, 2005; 11(4): 319 - 336. [Abstract] [Full Text] [PDF]

				T E King, E Bosch, S M Adams, E J Parkin, Z H Rosser, and M A Jobling Inadvertent diagnosis of male infertility through genealogical DNA testing J. Med. Genet., April 1, 2005; 42(4): 366 - 368. [Full Text] [PDF]

				N. Bannert and R. Kurth Retroelements and the human genome: New perspectives on an old relation PNAS, October 5, 2004; 101(suppl_2): 14572 - 14579. [Abstract] [Full Text] [PDF]

				E. Bosch, M. E. Hurles, A. Navarro, and M. A. Jobling Dynamics of a Human Interparalog Gene Conversion Hotspot Genome Res., May 1, 2004; 14(5): 835 - 844. [Abstract] [Full Text] [PDF]

				C. J. Shaw and J. R. Lupski Implications of human genome architecture for rearrangement-based disorders: the genomic basis of disease Hum. Mol. Genet., April 1, 2004; 13(90001): R57 - 64. [Abstract] [Full Text] [PDF]

				M. Babcock, A. Pavlicek, E. Spiteri, C. D. Kashork, I. Ioshikhes, L. G. Shaffer, J. Jurka, and B. E. Morrow Shuffling of Genes Within Low-Copy Repeats on 22q11 (LCR22) by Alu-Mediated Recombination Events During Evolution Genome Res., December 1, 2003; 13(12): 2519 - 2532. [Abstract] [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (34) Request Permissions Google Scholar Articles by Bosch, E. Articles by Jobling, M. A. Search for Related Content PubMed PubMed Citation Articles by Bosch, E. Articles by Jobling, M. A. Social Bookmarking          What's this?

Online ISSN 1460-2083 - Print ISSN 0964-6906

Copyright © 2009 Oxford University Press

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics