Hum. Reprod. Advance Access originally published online on October 20, 2005
Human Reproduction 2006 21(2):529-535; doi:10.1093/humrep/dei356
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Heterozygosity mapping by quantitative fluorescent PCR reveals an interstitial deletion in Xq26.2–q28 associated with ovarian dysfunction
Institute of Genetics and Biophysics ‘Adriano Buzzati Traverso’, CNR 80131, Napoli, Italy
1 To whom correspondence should be addressed at: IGB-ABT CNR, Via Pietro Castellino 111, 80131 Napoli, Italy. E-mail: miano{at}igb.cnr.it
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Abstract |
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BACKGROUND: Deletions of Xq chromosome are reported for a number of familial conditions exhibiting premature ovarian failure (POF) and early menopause (EM). METHODS AND RESULTS: We describe the inheritance of an interstitial deletion of the long arm of the X chromosome associated with either POF or EM in the same family. Cytogenetic studies and heterozygosity mapping by quantitative fluorescent PCR revealed a 46,X,del(X)(q26.2–q28) karyotype in a POF female, in her EM mother, and also in her aborted fetus with severe cardiopathy. Applying a microsatellite approach, we have narrowed the extension of an identical interstitial deletion located between DXS1187 and DXS1073. These data, in line with other mapped deletions, single out the proximal Xq28 as the region most frequently involved in ovarian failure. We also propose that other factors may influence the phenotypic effect of this alteration. Indeed, skewed X inactivation has been ascertained in EM and POF to be associated with different X haplotypes. CONCLUSION: Our analysis indicates that Xq26.2–q28 deletion is responsible for gonad dysgenesis in a family with EM/POF. The dissimilar deletion penetrance may be due to epigenetic modifications of other X genes that can contribute to human reproduction, highlighting that ovarian failure should be considered as a multifactorial disease.
Key words: heterozygosity mapping/premature ovarian failure syndrome/quantitative fluorescent PCR/XCI/Xq26.2–Xq28
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Introduction |
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Premature ovarian failure (POF) and early menopause (EM) conditions are present in a broad spectrum of gonad dysgenesis, from a complete cessation of ovarian function to an intermittent follicle maturation failure. They are often present in members of the same family in association with alterations of the Xq chromosome (Vegetti et al., 2000
; Zinn, 2001). This intra-familial variability may represent a different degree of expression of the same genetic disease explained by genetic heterogeneity and/or by different environmental factors. The complex spectrum of these conditions is also enhanced by the huge repertoire of POF with loss of different genome portions that may cause defects in oogenesis or impairment of follicle maturation (Schlessinger et al., 2002). Moreover, POF patients are a fascinating disease group because those carrying a structural abnormality of the X chromosome exhibit extremely skewed XCI (X chromosome inactivation) due to cell selection (Sato et al., 2004). During the past few years, accurate mapping of X chromosome anomalies has been successfully achieved by quantitative fluorescent PCR (QF-PCR) amplification of chromosome-specific short tandem repeats (STR) (Donaghue et al., 2003). More precisely defined region(s) on Xq have been mapped in various POF diseases (Maraschio et al., 1996; Marozzi et al., 2000) although neither a clear relationship between the position of breaks within this region nor the severity of POF has been established. Findings derived from investigation of X/autosome balanced translocations and Xq terminal deletions have allowed identification of two independent loci within the Xq arm that seem to be involved in ovarian function (Marozzi et al., 2000). These loci are located at Xq26–q28 (POF1) and at Xq13.3–q22 (POF2) respectively. Which genes cause POF conditions is not completely understood. In the POF2 locus, translocation mapping studies have revealed three different genes interrupted in families with POF, DIAPH2 (Diaphanous Drosophilae homologue 2; Sala et al., 1997), DACH2 (Drosophilae dachshund homologue 2; Prueitt et al., 2002; Bione et al., 2004) and POF1B (Bione et al., 2004). In the POF1 locus no disease gene has been ascertained, although the association between FMR1 premutations and POF disease has suggested that this gene acts as a POF risk factor with an estimated relative risk of 
20% (Murray et al., 2000; Sullivan et al., 2005). Because these disorders are highly frequent in the female population (1%, Sato et al., 2004), finding POF genes may improve our knowledge of reproductive capacity, allowing the prediction of impending menopause and the implementation of strategies to advance conception. In the present study, we report on a female with POF and her mother with EM with the genotype 46,X,del(Xq26.2–q28). We have constructed a detailed map of a cytogenetically visible deletion of Xq present in both females, using QF-PCR and microsatellite analysis, to assess the X chromosome DNA dosage and to determine accurately the extension of the monosomy. Differences in the X chromosome inactivation patterns have been evaluated in association with EM/POF conditions.
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Materials and methods |
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Subjects
According to common protocol, POF and EM are defined as hypergonadotrophic amenorrhoea with FSH measurements
20 IU, LH >15 IU and cessation of menses for a duration of 6 months, at age 
40 years (POF), or between the ages of 41 and 44 years (EM) (Anasti, 1998; Vegetti et al., 2000). A 26 year old Italian woman, II:2, was referred because of periods of amenorrhoea alternating with regular menstruation. She had menarche at 10 years, was of normal height (160 cm) and had none of the Turner syndrome traits. She was pregnant with a malformed female fetus, and therapeutic abortion was decided at 14 weeks. Tissue samples of the abortion were unavailable for histological analysis and we cannot report the phenotype accurately. The mother of II:2, I:1, had menarche at 12 years and had another daughter (II:1) with regular menstruation. At 42 years of age, I:1 entered menopause. Both female subjects, I:1 with EM and II:2 with POF, underwent complete clinical assessment, which included medical analysis, and gynaecological and obstetric history considering the outcome of pregnancies.
Chromosomal analysis
Peripheral blood samples were obtained from the two probands of the family DG (I:1 and II:2). A cell culture of the fetus biopsy was also obtained (III:1). Tissue culturing was carried out using the standard protocol. Metaphase chromosome spreads were prepared from phytohaemagglutinin-stimulated lymphocytes, and the chromosomes were GTG-banded by standard methods (Lemieux et al., 1990). Routine resolution banding techniques were also performed on metaphase chromosomes. More than 30 cells were analysed for each karyotype.
QF-PCR and genotyping
DNA was extracted from peripheral blood lymphocytes by high salt extraction (Miller et al., 1988). The DNA concentration was measured with a spectrophotometer, and samples were diluted with distilled water to a concentration of 20 ng/µl. DNA samples were analysed in QF-PCR protocol (Donaghue et al., 2003). This method is based on a PCR amplification that incorporates a fluorochrome into the samples, which are then visualized and quantified using an automated DNA sequencer. The marker set was based on Marshfield Screening Set 11 and consisted of 15 polymorphic microsatellites (STR) with an average spacing of 1 cM and an average heterozygosity of 0.75. Four additional markers, B_53a12/4, B_53a12/5, B_53a12/15 and B_XY_22, were developed ad hoc and used to increase the information content for the distal Xq28–XqPar region. The genetic frequency of the new STR markers was provided from male equimolar DNA pools of normal large multiethnic CEPH (Foundation Jean Dausset-Centre d’Etude du Polymorphisme Humain) families (data unpublished). A heterozygosity index was established and marker positions were verified using human public databases (University of California, Santa Cruz (UCSC) http://genome.ucsc.edu/, GDB http://www.gdb.org/, Table I). Either the forward or the reverse oligonucleotide primer was labelled with 6-FAM, HEX or NED fluorescent dyes (Amersham Biosciences, Buckinghamshire, UK). PCR were performed in a 10 µl volume containing 20 ng of template DNA, 0.8 µmol of each oligonucleotide primer, 200 mmol of each dNTP, and 0.4 IU of AmpliTaq Gold (Amersham Biosciences, Buckinghamshire, UK), in 1xPCR buffer with 1–2 mmol MgCl2 (Applied Biosystems, Foster City CA, USA). DNA was initially denatured at 94°C for 7 min and was then subjected to 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s, followed by a final extension step of 30 min at 72°C. Twenty-four to 26 PCR cycles were carried out as standard practice for QF-PCR analysis. The internal size standard TAMRA 350 was added to 2 µl of PCR products and 24 µl of formamide and then was submitted to capillary electrophoresis in an automatic sequencer (MEGABace1000 Amersham Biosciences, Buckinghamshire, UK). Alleles were assigned with Genetic Profiler 2.0 software (Amersham Biosciences, Buckinghamshire, UK).
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X inactivation
The primers we used flank a sequence containing a highly polymorphic CAG repeat and two HpaII sites in the AR gene (Fusco et al., 2004). When the undigested control DNA is used as the PCR template, both alleles become amplified, whereas using the HpaII-digested DNA as template, only the inactive allele, which is methylated and therefore resistant to restriction digestion with HpaII, will be amplified. When DNA obtained from the control male is digested with HpaII, no PCR product is obtained. In a sample obtained from the female control, if the X chromosome is subject to random inactivation, both alleles should be present, whereas in the case of skewed inactivation, only the allele that is preferentially inactivated will be amplified. XCI degree threshold patterns are classified as random (XCI 70%), non-random (70% XCI 80%) or skewed (XCI 90%).
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Results |
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Abnormal X determining
A 26 year old woman (D.G. II:2) with POF was referred to our laboratory for genetic counselling at 14 weeks of gestation. Abnormal ultrasound findings revealed a fetus (III:1) with cystic hygromas and congenital complex cardiopathy with one ventriculus (Figure 1). After a therapeutic abortion, conventional cytogenetic procedures were performed on the fetus sample (III:1), the POF proband (II:2) and her mother, reported as EM female (I:1). Chromosome metaphases were obtained from peripheral blood lymphocyte cultures of both females and the fetus biopsy. GTG staining showed the presence of a microscopically visible deletion of the long arm of the X chromosome (Xq) in the three samples in which the karyotype designation was 46X, del(X)(q26–qter) (Figure 2). The finding of the abnormal X chromosome in the two females with ovarian dysfunction is completely in line with the literature data that Xq deletions are responsible for the POF phenotypes (Marozzi et al., 2000
). We did not have the clinical and molecular evidence to ascribe the fetus malformation to the Xq26–q28 deletion. Significantly, we cannot exclude the possibility that the severe symptoms are associated with an X inactivation mechanism.
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QF-PCR and STR mapping
Detailed deletion mapping was performed by QF-PCR, a technique used in the accurate diagnosis of the sex chromosome status and its imbalances (Donaghue et al., 2003). The aim of this analysis was to assess the fine mapping of the Xq deletion choosing polymorphic STR within the genetic region involved in the visible cytogenetic alteration, in order to detect the amount of fluorescent activity produced by the X chromosome markers (Table I). In a public database, we chose 15 informative STR with a high heterozygosity index among the tri/tetra/penta/hexanucleotide repeat markers already characterized in the Xq26–Xqter region. We enriched the density of polymorphic tags in distal Xq28 with the integration of four new polymorphic STR developed in our laboratory (B_53a12/4, B_53a12/5, B_53a12/15, B_XY_22; Table I). Using the selected markers, we genotyped in QF-PCR reactions the DNA of female 46,X,del(Xq26.2–qter) (II:2 Figure 3A) and of two control samples, CEPH 1347M02F (XX) and 1347C16M (XY). In this case, the peaks of fluorescent activity of an X chromosomal marker outside the deleted region of the POF female were identical to the activity of the same marker in a normal female (ratio 1:1). Moreover, the peaks of fluorescent activity of an X chromosomal marker inside the deletion corresponded to one allele and were identical to the activity of the same marker in a normal hemizygous male (ratio 1:1). According to the hemizygosity of each STR in the CEPH control male (1347C16M) with known genotypes, the 1:1 ratio was detected for the contiguous DNA polymorphisms DXS1187–DXS1254–DXS8094–DXS1192–DXS1205–DXS8106–DXS8043–DXS8091–DXS8069–DXS1684–DXS8061–DXS15–DXS1073 (Figure 3B), documenting that the tested female with the deletion was monoallelic for those markers. The heterozygosity correlation between the POF female and the CEPH female control (1347M02F) established that the POF locus was bi-allelic for the markers DXS1047 and DXS1108, flanking the mono-allelic DXS1187 and DXS1073 that are the proximal and distal STR to the deletion respectively (Figure 3B). The deletion is therefore interstitial, located in a region of 22 Mb from Xq26.2 to Xq28. Based on the estimated physical distances reported in the X-chromosome consensus map (UCSC Release May 2004 at http://genome.ucsc.edu/; Figure 4) the proximal and distal breakpoints are, respectively, 130 600 and 153 200 kb from ptel.
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Evaluation of the POF1 deletions
By comparing other POF deletions in the region with our findings, we have reviewed the DNA loss located outside the POF2 locus (Xq13.3–q22) (Table II). Xq interstitial and terminal deletions including POF1 (Xq26–q28) were found from q23 to q28 (Marozzi et al., 2000); from q25 to qter (Davison et al., 1998); from q22.3 to q27 (Vegetti et al., 2000); from q27 to q28 (Rossetti et al., 2004); and from q27.3 to q28 (Eggermann et al., 2005). A comparison of these findings suggests that the minimal POF1 region could be located between DXS1200 (Eggermann et al., 2005) and DXS15 (Rossetti et al., 2004) reducing the critical region roughly to 5 Mb (UCSC Release May 2004; Figure 4). The absence of any correlation between the deletion size and the type of ovarian failure is in agreement with the hypothesis that a restricted Xq region is responsible for the POF/EM phenotype.
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X inactivation status
XCI analysis was performed in the probands, I:1 with EM and II:2 with POF, and in the CEPH DNA controls, 1347M02F (female) and 1347C16M (male). Using the androgen receptor (AR) fluorescent assay that analyses the methylation status of CpG islands of the AR gene, we found that I:1 and II:2 females were extremely skewed (XCI degree threshold 90%), with different X haplotypes (Figure 1). In particular, female I:1 showed the degree threshold X1 98%; X2 2%, while her daughter II:2 showed X1 1%; X2 99%. Considering the evidence that POF Xq deletions have different lengths, these results are compatible with the hypothesis that haplo-insufficiency is the molecular mechanism for the POF phenotype. The association with different X haplotypes could instead explain the variable penetrance of the Xq26.2–Xq28 deletion. If so, both XCI and POF result from an underlying mutation that is selected against random XCI of ovarian development genes.
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Discussion |
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Pedigrees with EM and POF often segregate with Xq monosomy. They are classified as the result of a variable expression of the same genetic disease causing oocyte maturation failure (Vegetti et al., 2000
). In an Italian family showing both EM and POF conditions, we established the inheritance of an identical Xq26.2–q28 deletion in association with different XCI haplotypes. By comparing classical cytogenetic studies with the QF-PCR heterozygosity mapping method, we ascertained a hemizygous fragment with a single peak for the contiguous markers DXS1187–DXS1073, and a double peak for the flanking STR DXS1047 and DXS1108. The QF-PCR approach was successfully applied to narrow down the karyotyped Xq deletion using new polymorphic markers developed ad hoc, which could also facilitate the identification of thePOF1 gene, as well as the fine mapping of other Xq28 anomalies. In this way, we found that the two ovarian dysfunctions, EM and POF, are associated with an identical interstitial deletion of 22 Mb of DNA located in the POF1 locus, corroborating the genetic role that this portion of the long arm of the X chromosome could play in human reproduction (Figure 4; Marozzi et al., 2000). The EM/POF deletion is rather large and can be tentatively restricted to 5 Mb considering the previously published findings of Eggermann et al. (2005) that described a POF deletion located in the DXS1200–DXS1684 interval which is the minimal critical region so far described. Although this locus contains several putative candidate genes, the genetic mechanism that produces ovarian failure is still controversial. One of the mechanisms might involve a gene dosage effect, such as the lack of expression of genes normally escaping X inactivation (haploinsufficiency). Alternatively, we hypothesize that an X chromosome carrying deletions determines mispairing at meiosis (Schlessinger et al., 2002). In this respect, the fine mapping of the inherited Xq deletion in POF families might be informative in the definition of the minimal region required for such a proposed mispairing mechanism.
Furthermore, it is noteworthy that the identification of a chromosome abnormality within an individual may segregate in other family members who have the same defect. One possible explanation for the differences in the penetrance of the Xq26.2–Xq28 deletion in EM/POF conditions could result from a different inactivation status of the X chromosome. Skewed XCI is frequently observed in women with POF (Sato et al., 2004) and the abnormal X is frequently inactivated in carriers of X-linked deletions (Leppig and Disteche, 2001). The abnormalities are generally well tolerated because of the preferential inactivation of the abnormal X, which can restore, at least in part, a balanced genetic make-up. However, the description of some abnormal phenotypes can be ascribed to failed or partial X inactivation and/or incomplete selection in favour of cells with a normal balance of gene expression. Our study confirms and extends these considerations. In fact, the observation that the EM mother and the POF daughter carrying the Xq26.2–Xq28 deletion are skewed for different X haplotypes, suggests that a direct role for epigenetic factors in determining dissimilar ovarian dysfunction penetrance exists. Alternatively, we cannot exclude the epigenetic contribution of modifying loci unlinked to the Xq to the female fertility. Thus, our findings highlight the pivotal role that the proximal Xq28 gene content could play in determining human menopausal age, and also underlines that other factors, such as epigenetic modifications, may affect ovulation.
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Acknowledgments |
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We thank the family for its cooperation and the Gene Expression Core Laboratory of BioGeM for the technical support in the microsatellite electrophoresis. This project was supported by a FIRB grant from MIUR-Italy.
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References |
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Submitted on July 28, 2005; accepted on September 26, 2005.
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