Fine mapping of re-arranged Y chromosome in three infertile patients with non-obstructive azoospermia/cryptozoospermia
1 INSERM, U823, Grenoble F-38706, France 2 Université Joseph Fourier, Institut Albert Bonniot, Grenoble F-38706, France 3 Département de Génétique et Procréation, CHU de Grenoble BP 217, 38 043 Grenoble Cedex 09, France 4 Laboratoire de Biologie de la Reproduction et/ou service de Génétique Moléculaire, Hôpital Nord, 42 055 Saint Etienne, France 5 Clinilab, 38 400 Saint Martin d'Hères, France
6 Correspondence address. Tel: +33-4-76-54-95-12; Fax: +33-4-76–54-95-95; E-mail: sophie.rousseaux{at}ujf-grenoble.fr
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Abstract |
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BACKGROUND: Cytogenetically detectable aberrations of the Y chromosome, such as isodicentrics, rings or translocations are sometimes associated with male non-obstructive infertility. This report presents a detailed analysis of the clinical, cytogenetic and molecular data in three patients with a re-arranged Y chromosome.
METHODS: Patients A and B were azoospermic, whereas patient C was cryptozoospermic. All had a somatic mosaic karyotype including a population of 45,X cells and a cell line with a re-arranged Y chromosome. A molecular and FISH analysis of their re-arranged Y was undertaken, which specifically focussed on the presence of the AZFa, b and c regions.
RESULTS: The AZFa region was present in all the three patients. The AZFb and AZFc regions were absent in patients A and B, whereas, in patient C, the distal part of AZFb and the whole AZFc region were deleted. Moreover, in this patient, the AZF FISH analysis revealed a mosaicism for the size of the AZF deletion within the re-arranged Y, suggesting a progressive enlargement of the deletion during cell mitotic divisions.
CONCLUSIONS: This investigation allowed not only a more precise description of the abnormal Y, but also shed light on how this re-arrangement could be involved in the infertility phenotype.
Key words: azoospermia/Y deletion/sex chromosomes/chromosomal abnormalities
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Introduction |
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Y microdeletions, generally resulting from intrachromosomal recombination events between large homologous repetitive sequence blocks in Yq11, are the most frequent known genetic cause of non-obstructive severe oligozoospermia or azoospermia, with a frequency ranging from 10% to 15% (Krausz, 2005
; Kuroda-Kawaguchi, 2001; Noordam and Repping, 2006).
Among cytogenetically detectable aberrations of the Y chromosome, the isodicentric Y, idic(Y), is the most common. It results from a break occurring in the juxtacentromeric region, followed by the duplication of the centromere-containing fragment of the chromosome. They can sometimes be mistaken for a normal Y chromosome by the routine Giemsa staining procedure, because of their similarity in size compared to the normal Y chromosome (Siffroi et al., 2000). A ring chromosome, r(Y), can also be associated with male infertility. It results from the fusion of the two broken short and long arms of a Y chromosome, forming a circular configuration (Tharapel, 2005).
This report presents a detailed analysis of the clinical, cytogenetic and molecular data in three patients with a re-arranged Y chromosome.
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Materials and Methods |
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Clinical reports
Patients A and B were azoospermic, whereas patient C was cryptozoospermic. Their clinical and biological parameters are described in Table 1. They were all mosaics with two different cell lines. One of the cell populations is monosomic (45,X) whereas the second contains 46 chromosomes with a re-arranged Y.
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Karyotyping and SRY FISH analysis
Chromosome analysis was performed on peripheral blood metaphases using the standard techniques and R, G and, in some cases, C banding. FISH studies were performed to assess the presence or absence of the SRY gene (LSI SRY (Yp11.3)) (Vysis, IL, USA).
Molecular analysis of the AZF regions
STS analysis was performed on genomic DNA extracted from buccal cells using the International Recommendations (Fig. 1) (Simoni et al., 2004). For a first screening, eight STS were analysed in two multiplex PCRs: sY84 and sY86 for AZFa, sY127 and sY134 for AZFb, sY254 and sY255 for AZFc. SRY (sY14) and ZFY were included as internal positive controls. All deleted samples were subjected to a complementary screening using 18 STS in nine duplex experiments: sY82, sY83, sY87, sY88, sY95, sY117, sY114, sY1015, sY135, sY143, sY142, sY145, sY1197, sY1192, sY152, sY158, sY157, sY1125. Several controls were used for each PCR: a blank without DNA, a female DNA, a male DNA with a known AZFc deletion, as well as a fertile male DNA.
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AZF FISH experiments
FISH experiments were performed on metaphases using probes cloned in Bacterial artificial chromosome (BAC) vectors. Each probe was, respectively, specific for the three AZF a, b and c, regions on Yq. The BACs were chosen from the RP11 library according to the mapping of Tilford and collaborators (Tilford et al., 2001
), and provided by the Wellcome Trust Sanger Institute (Cambridge, UK) (http://www.sanger.ac.uk/). They were as follow: BAC clone RP11-492N16 for the AZFa region, BAC clone RP11-424G14 for the AZFb region, and BAC clone RP11-539D10 for the AZFc region (Fig. 1A). The DNA were extracted from the BACs, labelled and hybridized according to standard protocols.
The localization and identification of the Y chromosome was confirmed by co-hybridization of each AZF probe with a probe specific for the Yp11.1–q11.1 
-satellite region (CEP Y alpha (DYZ3) (named thereafter ‘centromeric probe’) (Vysis, IL, USA).
On metaphase chromosomes of control fertile patients, all three probes displayed a strong spot-like signal on each chromatid, which localized exclusively to the proximal part of the long arm of the Y chromosome, whereas, as expected, no signal was observed when they were hybridized on metaphases of infertile patients with a deletion of the AZFa, AZFb or AZFc region.
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Results |
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The results of these investigations are detailed in Table 1 and Fig. 1B and C. For both patients A and B, the initial somatic karyotype showed a mosaicism, including a 45,X cell line and a 46,X,i(Y)(p10). The AZF STS analysis on their buccal cells showed that a part of the long arms of the Y chromosome was actually present on the ‘isochromosome’ of both patients, since all AZFa markers were positive. However the AZFb + c markers were absent. The AZFa FISH analysis confirmed this observation since it was positive on the Y re-arranged chromosomes in almost all the 46,XY metaphases analysed (55/56 in patient A and 27/27 in patient B). Somatic karyotypes were therefore redefined as follow: 45,X[14]/46,X,idic(Y)(pter- > q11.23::q11.23- > pter)[86].ishYp11.3(SRYx2) for patient A, and 45,X[83]/46,X,idic(Y)(pter- > q11.23::q11.23- > pter) [31].ish Yp11.3 (SRYx2) for patient B.
In patient C, the initial somatic karyotype showed that, in 10% of the mitosis, the Y chromosome was lost, whereas in 90% of the mitosis, one or two copies of a Y ring chromosome were detected. An initial FISH analysis with alpha-satellites probes of the centromere and the Yp11.3 (SRY) region showed that the ring Y chromosome breakpoints were located in p11.3 on the short arm, and q11 on the long arm. The STS analysis showed the presence of the AZFa region and a deletion including the distal part of AZFb and the whole AZFc region (Table 1). The Yq11 breakpoint could therefore be located in the distal part of the AZFb region. The AZF FISH analysis not only confirmed this result but also suggested a sequential increase of the Y deletion (Table 1). Indeed, in the AZFb FISH experiment, among the 46,XY metaphases positive for the centromeric probe (n = 47), 41 (87%) were positive for AZFb and 6 (13%) were negative.
Hence, combining the molecular and FISH analysis of the AZF region, the initial karyotype of patient C was refined as 45,X[10]/46,X,r(Y)(p11.3q11.23)[89]/47,X,r(Y), + r(Y) (p11.3q11.23)[1].ishr(Y)(p11.3q11.23) (DYZ3+, SRY+) and a mosaicism for the size of the AZF deletion within the 46,X,r(Y) population, was evidenced.
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Discussion |
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In the literature, most cases of Y rearrangements, including idic(Y) and r(Y), are reported, as here, in a mosaic form, usually in association with a 45,X cell line. Other cases of patients with re-arranged and AZF-deleted Y chromosome associated with a somatic mosaicism of the sex chromosomes are summarized in Table 2. The associated sex chromosomes mosaicism is likely due to the instability of the re-arranged Y, which can be lost through cell divisions (Alvarez-Nava and Puerta, 2006
; Patsalis et al., 2005; Siffroi et al., 2000). The proportion of 45,X cells is highly variable between individuals, ranging from 1% to 90%, and can also differ between the different cell lineages within the same individual. These mosaic karyotypes are associated with a wide spectrum of clinical phenotypes, ranging from male infertility with normal sexual characteristics, ambiguous external genitalia, or female with typical or atypical Turner syndrome (Alvarez-Nava et al., 2003; DesGroseilliers et al., 2006; Le Bourhis et al., 2000). A simple relationship between the percentage of 45,X cells among blood lymphocytes and the patient's phenotype has not been found. In the present study, the three patients all shared a normal male phenotype, despite the high level of 45,X cell lines observed in patient B.
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An important issue regarding infertile male carriers of a re-arranged Y is the relationship between the abnormal Y and the infertility phenotype. Here, the presence or absence of one or more of the three AZF regions is of crucial importance.
This study provides the first FISH analysis of the AZF regions in patients with complex sex chromosome mosaicism. In patient C, a mosaicism for the AZFb deletion was found in six metaphases (13%) among the 47 analysed. This result is of great interest, as it suggests that the instability of the Y deleted chromosome, could also be involved in an extension of the deletion during cell divisions, and possibly in the transmission of a larger deletion to the next generation. This variation in the size of AZF deletion could be the result of the particular behavior of the ring chromosomes during mitosis. Indeed, the occurrence of sister chromatid exchanges could result in the formation of dicentric ring chromosomes, which would then undergo unequal partition during successive mitotic divisions inducing the formation of rings of different sizes (Miller and Therman, 2001).
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Acknowledgements |
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We would like to gratefully acknowledge Roberte Pelletier and Christine De Robertis for their technical expertise. A.K.F. was recipient of a grant of ‘Poste d'accueil INSERM’ and SR of a ‘contrat d'interface’ INSERM. We wish to thank the Chromosome Y Mapping Core group of the Sanger Institute (Cambridge, UK) (http://www.sanger.ac.uk/) for providing the BAC clones used in this study, and Drs Stora de Novion and J. Lespinasse for their contribution to the biological data of the patients.
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Submitted on January 9, 2007; resubmitted on March 11, 2007; accepted on April 11, 2007.
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