Fluorescent in-situ hybridization and sequence-tagged sites for ...

Molecular Human Reproduction vol.3 no.5 pp. 439–443, 1997

Fluorescent in-situ hybridization and sequence-tagged sites for delineation of an X:Y translocation in a patient with secondary amenorrhoea B.Delon1, H.Lallaoui2, C.Abel-Lablanche2, A.Geneix3, V.Bellec1 and M.Benkhalifa1,4 1Cytogenetique

et FIV, LMM. Me´rieux Foundation, 94, rue Chevreul, 69007 Lyon, 2Cytoge´ne´tique, LABS, route de Canta ´ ´ Gallet, Nice, and 3Laboratoire de Cytoge´ne´tique Me´dicale, Faculte´ de Me´decine, Clermont- Ferrand, France

4To

whom correspondence should be addressed

We describe a phenotypically normal female with secondary amenorrhoea due to a translocation of genetic material involving the long arm of chromosome X (Xq28) and the long arm of chromosome Y (Yq11). We used fluorescent in-situ hybridization to localize the breakpoint on the Xq. The Y chromosome breakpoint was identified using polymerase chain reaction (PCR) detection of sequence-tagged sites (STS) specific for interval 5 at Yq11.21. The relationship between this X:Y translocation and premature ovarian failure is discussed. Key words: secondary amenorrhoea/STS/X chromosome /X:Y translocation/Y chromosome

Introduction Rapid technical developments in classical and molecular cytogenetics within the last 20 years have improved the ability of phenotype/karyotype correlation studies to detect structural abnormalities of the X chromosome and facilitated gene mapping. Sarto et al. (1973) compared karyotypes and phenotypes in women with X:autosome translocations and delimited a ‘critical region’ on Xq (Xq13-Xq26) which must be intact to allow for normal ovarian development. Wyss et al. (1982) reviewed 149 cases of X structural abnormalities and proposed that genes involved in ovarian development were located on the proximal part of Xp and on the distal part of Xq. Goldman et al. (1982) correlated phenotypes with karyotypes in six patients with ovarian dysgenesis and X chromosome deletions. They concluded that Xq deletions were in agreement with a relatively normal phenotype except for primary and secondary amenorrhoea. However, breakpoints within band Xq22 did not interfere with normal gonadal function (Madan et al., 1981; Therman et al., 1990) and the ‘critical region’ seemed to be further divided into two segments, Xq13-q22 and Xq22-q26. Tharapel et al. (1993) reported a woman with a Xq26-qter deletion and secondary amenorrhoea and suggested that a gene involved in premature ovarian failure (POF1) was located at Xq26-q27. Powell et al. (1994) reported a patient with secondary amenorrhoea and a balanced X:autosome translocation: 46,X, t(X;6) (q13.3 or q21;p12). They postulated that a second gene (POF2) responsible for ovarian function was located at Xq13.3-q21.1. The majority of X:Y translocations involve the transposition of Yq material on Xp with breakpoints at Xp22 and Yq11 (Gabriel-Robez et al., 1990). Molecular analysis of different DNA probes located at Xp22 and Yq11 revealed that these two regions share sequence homology and suggested that homologous recombination between these sequences may be involved in the aetiology of these X:Y translocation events © European Society for Human Reproduction and Embryology

(Ballabio et al., 1989; Yen et al., 1991). Xq and Yq interchanges are less frequent. To our knowledge only three females with Xq:Yq translocations have been reported (Koo et al., 1977, Cameron et al., 1984, Kelly et al., 1984). Here, we describe a woman with secondary amenorrhoea due to a translocation between the long arm of chromosome X (Xq27–28) and the long arm of chromosome Y (Yq11.21). The breakpoint on the X chromosome was localized using G banding and fluorescent in-situ hybridization (FISH) with a sub-chromosome painting probe which hybridized to the Xq24-qter region. On the Y chromosome, the breakpoint was localized using polymerase chain reaction (PCR) detection of Y-specific sequence-tagged sites (STS) in interval 5 at Yq11.21.

Materials and methods Clinical report The patient was a 28 year old infertile woman who was otherwise phenotypically normal. Menses began at age 12 but were described as light. She had used oral contraceptives from age 17–25 years, and then became amenorrhoeic for 5 months followed by light periods which lasted for 15 days. Cycles remained irregular (between 15 days and 3 months). An exploratory hysteroscopy was performed at age 25 years and showed an endometrial polyp. At laparoscopy the right ovary appeared normal with a Graafian follicle. At age 27 she became amenorrhoeic with an elevated gonadotrophin concentration and a negative progestogen test. Since phenotypically normal females who carry Y chromosomal material are at an increased risk of developing tumours, a gonadectomy was recommended. Cytogenetic analysis Metaphase spreads were obtained from phytohaemagglutin-stimulated peripheral blood lymphocytes. Chromosome analysis by R and G banding was performed using classical protocols. The karyotype was found to be 46, X,der(X) (Figure 1a). The DNA synthesis patterns of the normal and translocated X in peripheral blood were analysed using bromodeoxyuridine (Dutrillaux et al., 1976).

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Figure 1. In-situ hybridization of the patient’s lymphocyte metaphase spread. (a) Chromosome analysis by G banding. (b) Subchromosomal fluorescent in-situ hybridization (FISH) of Xq24-q28 with X-300 probe. The arrow points to the supplementary material at the end of Xq. (c) FISH with a centromeric probe of X chromosome (DXZ1; Oncor Inc) revealed with tetramethylrhodamine B isothiocynate (red) and Y chromosome painting (Cambio probe) revealed with fluorescein isothiocynate (green).

Fluorescent in-situ hybridization The preparation of Xq24-qter and total X painting probes, as well as FISH procedures, were performed according to Antonacci et al. (1995). Briefly, DNA from a somatic hybrid cell line which retained the terminal portion of human Xq was amplified using an Alu-primer according to the Inter–Alu–PCR protocol of Liu et al., 1993. PCR products were labelled with biotin-16-dUTP or digoxigenin-11-dUTP by random priming. FISH procedures were performed in hybridization solution and washing was with 60% formamide. For each slide, 100 ng of probe was prehybridized for 2 h at 37°C with 10 mg of Cot-1 DNA. Slides were then hybridized overnight at 37°C and the probe detected with three layers of antibodies. Slides were counterstained with 496’ diamino-2 phenylindole (DAPI) or with propidium iodide (PI). The translocation was identified by FISH with two Inter–Alu– PCR probes: whole X and Xq24-qter chromosome painting probes respectively named HY136C (Mariano Rocchi, Bari, Italy) and X-

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300. The DXZ1 probe (centromeric; Oncor Inc, Gaithersburg, MA, USA), the total Y painting (Cambio probe, Cambridge, UK) and DYZ3 probes (centromeric; Oncor Inc) were used according to the manufacturer’s procedures (Figure 1).

DNA analysis DNA was isolated from a peripheral blood sample and PCR was performed using five primer pairs localized within intervals 5 and 6 on the long arm of chromosome Y and one on Yp. The primer pair which we used allowed amplification of sequence-tagged sites (STSs). We had chosen to amplify the STS’s:sY81 which allowed the detection of DYS271 locus (5A), sY117 (DYS209;5F), sY131 (DYS222;6A), sY149 (DYS1;6D), sY156 (DYS239;6F) (Vollrath et al., 1992) and SRY on Yp (Ao et al., 1994) (Figure 2). Approximately 550 ng of DNA were amplified by simple PCR or multiplex PCR. Initial denaturation of mix PCR was achieved without Taq polymerase at 95°C for 5 min in a water bath. A total of 35 cycles of PCR followed

X:Y translocation in a patient with secondary amenorrhoea

Figure 2. Genetic deletion interval map of the human Y chromosome (Vergnaud et al., 1986). Locus designation sequence-tagged sites used for polymerase chain reaction (PCR) amplification of patient genomic DNA, and size of PCR products. the addition of Taq polymerase; each cycle consisted of 1 min of denaturation at 95°C, 1 min of hybridization at 60°C and 1 min of extention at 72°C. Cycles were followed by 10 min at 72°C. The amplification of SRY and DYS1 loci was made by multiplex PCR in order to reduce the number of manipulations. The other loci were amplified separately. The PCR products were separated by electrophoresis in a 2% agarose gel containing ethidium bromide (0.5 mg/ml) and the bands visualized under UV illumination (Figure 3).

Results Cytogenetic analysis revealed the presence of supplementary material at the telomeric end of one of the X chromosomes in our patient (Figure 1a). Both parents karyotypes were normal. Results of the FISH with the Xq24-qter painting probe, suggested the breakpoint to be localized at the tip of Xq. Indeed, the size of the hybridized sequences between the translocated and normal X chromosomes were highly similar in the patient (Figure 1b). This observation led us to assume that the deleted fragment on the translocated X chromosome was very small and we estimated the breakpoint to be Xq2728. X inactivation studies in 30 cells revealed that the late replicating X chromosome was the abnormal X in all cases. DAPI staining revealed a very bright band on the abnormal Xq suggesting that the supplementary material could be composed of heterochromatin from Yq12. A total Y painting probe confirmed that the translocation implicated the long arm of chromosome Y. Heterochromatin and euchromatin portions of the Y chromosome are not evenly hybridized with this probe. The heterochromatic region is weakly painted, whereas euchromatic sequences exhibit strong fluorescence. The presence of a bright fluorescent band on the translocated Y fragment suggested that it was made up in part of euchromatic DNA (Figure 1c). Results of FISH on the probe DYZ3 indicated

that the translocated fragment lacked its centromere. In order to confirm that the X-Y chromosomal interchange involved euchromatic DNA and to further localize the breakpoint on Yq, we analysed genomic DNA from the patient by PCR. Five loci in addition to SRY were selected because they are localized in the euchromatic region of Yq (Figure 2). According to Vergnaud et al. (1986) who have constructed a deletion map of the human Y chromosome composed of seven deletion intervals, the euchromatic part of Yq was assigned to intervals 5 and 6. These were further divided into sub-intervals from 5A to 5Q and from 6A to 6F (Ma et al., 1992). The loci DYS271 and DYS209 correspond to intervals 5A and 5M respectively. The loci DYS222, DYS1 and DYS239 coincide with intervals 6A, 6D and 6F respectively (Figure 2). The SRY locus is proximal to the pseudoautosomal region on Yp (Yp11.3). Since in our patient, the DNA sequence corresponding to the SRY locus was not translocated onto the X chromosome, we used it as an internal control for the reaction of PCR. Results of the PCR products are shown in Figure 3. The data indicate that loci DYS209, DYS222, DYS1 and DYS239 were presented in our patient’s genomic DNA. Loci SRY and DYS271 were not identified (or amplified). According to Vollrath et al. (1992) STSs can be classified as Y specific or as ‘male–female common’. The ‘male–female common’ STSs could be the result of Y region endowed with weaker sequence homology with the X chromosome or with autosomal DNA. These shared STSs would be responsible for the finding of bands in addition to the Yq locus specific one. Likewise, PCR results in female controls indicated the presence of nonspecific DNA amplifications. The male control showed a definate amplification pattern at all six loci (Figure 3). These results indicate that part of interval 5 and all of interval 6 were translocated onto the X chromosome. We postulate that the breakpoint occurred between sub-intervals 5A and 5M, at 441

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Figure 3. Agarose gel electrophoretic separation of polymerase chain reaction (PCR) products from sequence-tagged sites on the Y chromosome. Patient’s DNA analysis showed amplification corresponding to male control loci: DYS1, DYS239, DYS222 and DYS209. Channel 1: amplification of locus SRY (275 bp) and DYS1 (132 bp) shows both bands in male and non-specific multiples bands in female control. Channel 2: amplification of locus DYS239 (950 bp) shows band at 950 bp plus one non-specific band near 140 bp in male, and only the non-specific band at 140 bp in female control. Channel 3: amplification of locus DYS222 (143 bp) results in a unique band in male, and no band in female control. Channel 4: amplification of locus DYS209 (262 bp) results also in a single band in male, and no band in female control. Channel 5: amplification of locus SYS271 (209 bp) shows a definite 209 pb signal in male control alone. W 5 PCR control (without DNA); L 5 ladder.

Yq11.21. In accordance, the following karyotype was assigned: 46,X,der(X)t(X;Y)(q28;q11.21).

Discussion We report here a combination of both classical and molecular cytogenetic techniques to investigate a phenotypically normal female individual with secondary amenorrhoea and a translocation between chromosomes X and Y. FISH, using total chromosome painting, centromeric, as well as specific locus probes, is a powerful tool for prenatal and postnatal cytogenetic studies. In contrast, sub-chromosomal painting probes have not been fully exploited thus far. Using a Xq24-qter subchromosomal painting probe, we estimated the breakpoint to be at Xq27–28 (Figure 1b). PCR studies indicated that on the Y chromosome breakpoint was within Yq11.21 (Figure 3). Yen et al. (1991) studied an X:Y translocation using molecular probes at Xp22.3 that detect homologous sequences on Yq11. In one of these structural abnormalities, they 442

sequenced DNA at the breakpoint and showed the exchange to result from homologous recombination between X and Y sequences. The pseudoautosomal regions located at the terminal end of the short arm of both X and Y chromosomes are homologous. These sequences allow X and Y chromosome pairing at meiosis. Occasional errors in the process of crossingover could favour translocation between Xp and Yp, resulting in XX males harbouring the SRY gene (Mc Elreavey et al., 1992; Taiar et al., 1995). Affara et al. (1996), summarized the homologous regions between the two human sex chromosomes. In their data sequences, Xq27-28 share homology with sequences at Yq11.22, but not Yq11.21. Although the mechanisms promoting translocation are relatively unknown, it would be interesting to study whether this Xq2728;Yq11.21 exchange occurs at sites of sequence similarity. Vergnaud et al. (1986) divided the Y chromosome into seven deletion intervals with intervals 5 and 6 along the euchromatic region of the Yq. Breakpoints analysis in four Yq deleted patients presenting with azoospermia with or without Turner stigmata, suggested that interval 5 is involved in skeletal development and growth (Barbaux et al., 1995). Interval 6 carries genes which are involved in male fertility (Sinclair et al., 1990) and could be totally or partially deleted in men with oligo- or azoospermia (Kotecki et al., 1991; Ma et al., 1992). To our knowledge, there are few reported translocations between the long arm of the X and the long arm of the chromosomeY (Koo et al., 1977; Cameron et al., 1984; Kelly et al., 1984; Lahn et al., 1994). Cameron et al. (1984) reported a young woman with secondary amenorrhoea, an otherwise unremarkable phenotype and a der(X)t(X;Y)(q22;q12). The authors suggested that the ovarian failure may have been due to the monosomy of Xq22-qter, albeit the fact that the abnormal X was found to be inactivated. In individuals with Xq deletions or unbalanced translocations involving the X chromosome, the deleted or derivative X are always normally inactivated. However, many genes escape X inactivation and their presence in a single copy in a female with an X chromosome abnormality is clearly harmful. This possibility is in agreement with the view that two distinct genes clusters on Xq are involved in ovarian activity; one at Xq13-q21 and the other at Xq26-q28 (Tharapel et al., 1993; Powell et al., 1994). Powell et al. (1994), suggested that when translocated or deleted, these two regions give rise to different ovarian failure phenotypes. Patients with breakpoints within Xq13-q25 more often suffer from primary amenorrhoea (48%). Patients with breakpoints within Xq26-q28 are prone to develop secondary amenorrhoea (25%) otherwise they retain a normal gonadal function (Therman et al., 1990). An additional explanation for X:Y translocation comes from an abnormal meiotic pairing, leading to meiotic arrest or Y euchromatin specific problems. However, when the derivative X is inactivated the genetic material in Yq is expected to be inactivated at the same time. Several theories have been put forward to explain how breakpoints at different positions on one of the X chromosomes translate into gonadal failure (Madan, 1983; Therman et al., 1990). One of them is that single or multiple gene deletions will result in reduced dosage. Intact genes are required especi-

X:Y translocation in a patient with secondary amenorrhoea

ally during oogenesis, before X inactivation and during reactivation of the inactivated X chromosome (Therman et al., 1980; Zinn et al., 1992). However, in cases of unbalanced X-autosomal translocations, the autosomal genes cannot affect the phenotype since the abnormal X is inactivated (Therman et al., 1980). Our data provide additional support for the theory of a critical region on Xq involved in ovarian development and function. Although the abnormal X was inactivated some genes that had escaped X inactivation may have been involved.

Acknowledgements We wish to thank Dr M.B.Qumsiyeh, Duke University, USA and Dr S.Mellanc¸on, Montreal University, Canada for critical reading of the manuscript. This work was supported by the bioMe´rieux company, Lyon, France.

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