European Journal of Endocrinology (2010) 162 169–175 ISSN 0804-4643

CLINICAL STUDY Severe XIST hypomethylation clearly distinguishes (SRYC) 46,XX-maleness from Andreas Poplinski, Peter Wieacker1, Sabine Kliesch and Jo¨rg Gromoll Centre of Reproductive Medicine and Andrology, University Clinics Mu¨nster, Domagkstraße 11, 48149 Mu¨nster, Germany and 1Institute of Human Genetics, Vesaliusweg 12-14, 48149 Mu¨nster, Germany (Correspondence should be addressed to J Gromoll; Email: [email protected])

Abstract Objective: 46,XX-maleness affects 1 in 20 000 live male newborns resulting in and hypergonadotrophic hypogonadism. Although the phenotypes of XX-males have been well described, the molecular nature of the X remains elusive. We assessed the X inactivation status by DNA methylation analysis of four informative loci and compared those to Klinefelter syndrome (KS) and . Design and methods: Patient cohort consisted of ten sex-determining region of the Y (SRYC) XX-males, two (SRYK) XX-males, ten 47,XXY Klinefelter men, six 45,X Turner females and ten male and female control individuals each. Methylation analysis was carried out by bisulphite sequencing of DNA from peripheral blood lymphocytes analysing X-inactive-specific transcript (XIST), phosphogly- cerate kinase 1 (PGK1), ferritin, heavy peptide-like 17 (FTHL17) and short stature homeobox (SHOX). Results: XIST methylation was 18% in (SRYC) XX-males, and thus they were severely hypomethylated compared to (SRYK) XX-males (48%; P!0.01), Klinefelter men (44%; P!0.01) and female controls (47%; P!0.01). Turner females and male controls displayed a high degree of XIST methylation of 98 and 94% respectively. Methylation of PGK1, undergoing X inactivation, was not significantly reduced in (SRYC) XX-males compared to female controls in spite of severe XIST hypomethylation (51 vs 69%; PO0.05). FTHL17, escaping X inactivation, but undergoing cell-type-specific inactivation was similarly methylated in XX-males (89%), KS patients (87%) and female controls (90%). SHOX,anX inactivation escapee located in the , displays similarly low degrees of methylation for XX-males (7%), KS patients (7%) and female controls (9%). Conclusions: XIST hypomethylation clearly distinguishes (SRYC) XX-males from Klinefelter men. It does not, however, impair appropriate epigenetic regulation of representative X-linked loci.

European Journal of Endocrinology 162 169–175

Introduction bitesticular volume to one-tenth that of normal males reflects exclusive , and as residual sperma- Sex chromosomal aberrations are a frequent cause togenesis in the testis has not yet been reported, assisted of male and female infertility. 46,XX-maleness is a rare reproduction techniques (ARTs) are no option for sex chromosomal anomaly that affects 1 in 20 000 reproduction in these patients. Detailed molecular newborn males. Ninety percent of XX-males are studies of the supernumerary X chromosomes in these designated sex-determining region of the Y (SRYC) patients are rare. It is known, however, that X 46,XX-males as translocation of Y chromosomal inactivation in these patients follows a material including SRY triggers testis formation in random pattern with a significant proportion of them these men (1). However, 10% of XX-males lack SRY and exhibiting extreme skewing (3). are thus designated (SRYK) XX-males. In these men, The most frequent sex chromosomal in masculinisation is induced by upregulation of the men is 47,XXY Klinefelter syndrome (KS) with a transcription factor SOX9 that triggers testis formation prevalence of 0.1–0.2% in newborn males and 11% independently from SRY (2). XX-males are charac- among azoospermic infertile men (4, 5). KS patients terised by hypergonadotrophic hypogonadism and display hypergonadotrophic hypogonadism, gynaeco- reduced body height, increased incidence of gyneco- mastia and reduced pubic hair; the testes are small and mastia and maldescended testes compared to 46,XY firm due to spermatogenic impairment (6). Oligozoos- males. In comparison to 46,XY healthy males, these permic ejaculates and residual active foci of spermatogen- men display significantly reduced levels, esis in testicular biopsies have been observed (7, 8), and and severely elevated levels of FSH and LH. Reduction of the use of ART by testicular sperm extraction has been

q 2010 European Society of Endocrinology DOI: 10.1530/EJE-09-0768 Online version via www.eje-online.org

Downloaded from Bioscientifica.com at 10/01/2021 09:01:58PM via free access 170 A Poplinski and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2010) 162 successful in several reported cases (9–11). Although transcriptional regulation by DNA methylation. More- endocrinological features of these men including reduced over, it aims to gain new useful information about the testosterone and LH levels have been described (3), the epigenetic nature of the X chromosomes in XX-maleness. precise molecular role of the supernumerary X chromo- some has not been elucidated in KS yet. A sex chromosomal anomaly affecting women is the Subjects, materials and methods 45,X Turner syndrome. It has an incidence of 1:2000, and is caused by the complete or partial absence of the Patients second gonosome (12). Decreased body height, ovarian dysgenesis, accelerated oocyte apoptosis and oestrogen Ten hitherto untreated (SRYC) 46,XX-male patients deficiency leading to severely impaired fertility are and ten 47,XXY Klinefelter patients were chosen for classical symptoms in these women (13). epigenetic characterisation from the cohort that was Of particular importance for X chromosomal gene genetically, endocrinologically and clinically described dosage compensation between the sexes is the X in detail by Vorona et al. (2007). Patients’ ages ranged chromosome inactivation. It is initiated embryonically from 14 to 46 for XX-males and from 20 to 42 for by the expression of the X-encoded X-inactive-specific Klinefelter patients. transcript (XIST) that is exclusively transcribed from the The male control group consisted of ten men who inactive (14, 15). Expression of XIST served as semen donors in the Centre of Reproductive triggers silencing of the X chromosome in cis and leads Medicine and Andrology. They had no chromosomal to DNA methylation of promoter regions of several aberrations, and semen analysis proved these men to be inactivated X-linked loci (16, 17). This silencing process normozoospermic. is incomplete on the inactive X chromosome as about The female control group consisted of ten women 15% of the genes on the inactive X chromosome are with normal ovarian function as determined by expressed and designated ‘escapees’ (18, 19). XIST repeated hormone assessments and ultrasonographic expression is subjected to a strict transcriptional control demonstration of regular ovulation. They represented through DNA methylation. Hypomethylation of XIST the proven healthy female partners in couples seeking leads to the overexpression of XIST and inappropriate advice for infertility, with husbands presenting with inactivation of X-linked genes (20). decreased sperm parameters. X chromosomal genes can be classified into different All patients and volunteers provided written informed categories depending on the mechanism of transcrip- consent, and agreed to the assessment of genetic tional regulation. These are represented by the material as approved by the Ethics Committee of the following genes in this study. University Clinics Mu¨nster and the State Medical Board. Phosphoglycerate kinase 1 (PGK1) belongs to the genes subjected to X inactivation, and its status is thus Genetic and cytogenetic characterisation directly affected by XIST expression. It is silenced by DNA methylation on the inactivated X chromosome and is Cytogenetic analysis was performed on lymphocytes as expressed from the active X chromosome, and can serve described by Vermeulen et al. (1999) (26). For (SRYC)- as an indicator of X chromosome inactivation (21, 22). and (SRYK)-46,XX males and 47,XXY Klinefelter Short stature homeobox (SHOX) is regulated in a patients, 30 metaphase cells were assessed by fluor- developmental manner and is of particular importance escence in situ hybridisation (FISH) analysis using a SRY for the phenotypic appearance of women with 45,X probe (Vysis, Hoofddorp, The Netherlands). Turner syndrome; its expression is essential in growth plates and is in tight correlation with body height (23). DNA isolation It is located in the pseudoautosomal region of the gonosomes and escapes X inactivation on the inactive X Genomic DNA was extracted from peripheral lympho- chromosome. cytes from 1 ml EDTA blood using the FlexiGene Kit A third gene on the X chromosome, ferritin, heavy (Qiagen) according to the manufacturer’s instructions. peptide-like 17 (FTHL17), has been shown to be expressed solely in spermatogonia and silenced in Bisulphite conversion somatic cells (24, 25). Hence, it represents those X-linked genes that display cell-type-specific expression. Two micrograms of genomic DNA in a volume of 50 ml The aim of this study was to delineate the epigenetic were incubated with 5.5 ml 3 M NaOH for 15 min at status of the X chromosome in XX-males in comparison 37 8C. After denaturation at 95 8C for 2 min, DNA was with Klinefelter patients and women with Turner placed on ice. Five hundred microlitres of bisulphite syndrome to analyse the impact of the sex chromosomal solution (5 M sodium bisulphite and 100 mM hydro- constitution on X inactivation. It utilises represen- quinone (Sigma–Aldrich)) were added, and the reaction tative X chromosomal loci to depict the impact of mixture was incubated at 55 8C for 16 h. Converted different sex chromosomal constitutions on differential DNA was desalted using the DNA cleanup Wizard

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(Promega) following the standard instructions. To the Methylation analysis by bisulphite desalted DNA, 5.5 ml of 3 M NaOH were added, and it sequencing was incubated for 15 min followed by ethanol precipi- tation; bisulphite-treated DNA was resuspended in 20 ml PCR products were cloned into pGEM T vector aqua dest. (Promega) and competent Escherichia coli were heat- shock transformed with the resulting plasmids. The plasmids of at least ten clones were isolated and Amplification of differentially methylated sequenced for methylation analysis. regions Regions of interest were amplified from 4 ml bisulphite Methylation analysis by dye-terminator DNA. PCR mixture (25 ml) contained 1! reaction sequencing buffer (Qiagen), 1.5 mM MgCl , 100 mM of each dNTP 2 The extent of methylation was determined by direct (Promega), primers in the concentration given below sequencing of PCR fragments by Qiagen GmbH. All (Eurofins MWG, Ebersberg, Germany) and 1.5 U Taq sequencing reaction mixes were based on the BigDye 3.1 Polymerase (Qiagen, 5 U/ml). PCR conditions were Terminator chemistry (Applied Biosystems, Carlsbad, 3 min at 95 8C followed by 40 cycles of 94 8C; 30 s, CA, USA). Template amounts of 5 ng per 100 bases of primer annealing (see below for temperature; 30 s), fragment length and 10 pmol primer per reaction were 72 C, 30 s and a final extension at 72 C for 10 min. 8 8 used. Reaction mixtures were cycled in a GeneAmp PCR PCR products were purified using the High Pure PCR System 9700 (Applied Biosystems) and purified using Product Purification Kit (Roche) according to the DyeEx (Qiagen). Data collection was carried out on a manufacturer’s manual. 3730!l DNA Analyser (Applied Biosystems) equipped with 50 cm capillary arrays and POP-7 polymer. Identification of potentially methylated After data collection, the raw data channels from the regions generated result files (.ab1) were processed using custom-built software developed at Qiagen. Data proces- The sequence of the first exons and the region 1000 bp sing involved the compensation of the different migration upstream of the desired genes were retrieved from Entrez properties of the four dyes, baseline correction, peak Gene (http://www.ncbi.nlm.nih.gov/sites/entrez) and detection and base calling. For each base, the area was for the identification of CpG islands, the online program calculated from the corresponding peak to measure the MethPrimer (Li & Dahiya 2002 (27); http://www. base’s quantity. Bases that correspond to variable urogene.org/methprimer/) was used. This tool yields positions of CpG sites were identified by comparison of the location of possible CpG islands. Minimum require- the obtained sequence with a reference sequence. The ments for a CpG island were a ratio of observed/expected ratios of methylated versus unmethylated species (C/T in CpGs O0.6 and a percentage of CG O50%. Primers forward reads and G/A in reverse reads) were reported. were picked around CpG-rich areas. Primer sequences for XIST were adapted from Song et al. (2007) (28). Data analysis and plotting Primer sequences (50–30) and PCR conditions For bisulphite sequencing data, the ratio of methylated sites to unmethylated sites in all sequenced clones XIST_fwd: GTAGAGAATGATTTTGTAGTTAAGT- yielded the mean degree of methylation of a given TAAGG region. For dye-terminator sequencing, the mean of all XIST_rev: ATCTTACCTCCCTAATTTAACTTAACAC. analysed sites yielded the mean degree of methylation. Location: Xq13.2 (M97168) For visual presentation of the data and for statistical Product size: 133 bp; annealing temperature: 60 8C analysis, GraphPad Prism (GraphPad Software, La Jolla, (K0.1 8C/cycle) CA, USA) was used. FTHL17_fwd: TTTTTGTTATTTTAGTTGTTGTTTT FTHL17_rev: ACTTCTAAACATACTCCATTTTATC. Location: Xp21.2 (AF285592) Product size: 285 bp; annealing temperature: 59 8C Results SHOX_fwd: GGGATTTTTTGTAGTTTTAGTTTTGT Severe hypomethylation of XIST in SRY(C) SHOX_rev: ACCCTAATTACATTAACCCCAATAC. XX-males Location: Xp22.33 and Yp11.32 (U82668) Product size: 321 bp; annealing temperature: 58 8C We determined the DNA methylation status of XIST in PGK1_fwd: GTTTTTTTTAAGGTAGTTTTGGGTTTG (SRYC) 46,XX-males to assess the impact of their sex PGK1_rev: CRCTATTAACCACAACCCATC. chromosomal constitution on the epigenetic state of the Location: Xq13.3 (L00159) X chromosome. We therefore analysed a 133 bp Product size: 318 bp; annealing temperature: 60 8C. fragment (from C871 to C1003; according to Song

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Downloaded from Bioscientifica.com at 10/01/2021 09:01:58PM via free access 172 A Poplinski and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2010) 162 et al. 2007 (28)) containing five potential methylation sites (Fig. 1a) in ten SRY(C) XX-males compared to 47,XXY KS patients and ten female controls. A single CpG site methylation analysis revealed that KS patients and female controls displayed highly similar degrees of XIST methylation (Fig. 2a). The mean methylation per CpG site in KS patients was between 42 and 47%, which is comparable to female controls who displayed a XIST methylation of 46–48%; these values correlate with the presence of two X chromo- somes. The degree of methylation was highly consistent over the five analysed CpG sites of XIST. The distribution of the overall mean XIST methylation per individual showed the same heterogeneous pattern in KS men and female controls, ranging from 20 to 70% in both groups (Fig. 2c). In contrast, a strong deviation from the expected XIST methylation was observed in (SRYC) XX-males. They displayed XIST methylation from 16.5 to 19.3% and are thus severely hypomethylated compared to KS patients and female controls (P!0.001). On an individual basis they showed considerable variation of XIST methylation ranging from a complete unmethylation in one patient to 45% methylation in another, similar to 46,XX female controls and 47,XXY KS men (Fig. 2c). We aimed to assess the influence of the Y chromo- somalmaterialthatSRY(C) XX-males carry as translocation to one of their X chromosomes, and to further characterise the impact of different sex chromo- somal constitutions on XIST methylation. We therefore compared XIST methylation of SRY(C) XX-males to that of SRY(K) XX-males, 46,XY male controls and 45,X Turner patients. (SRYK) XX males display XIST methylation of 45–50%, depending on the CpG site; this represents a significantly elevated degree of XIST C ! methylation compared to SRY( ) XX-males (P 0.001) Figure 2 Mean methylation of a 133 bp fragment in the 50 region and is rather comparable to female controls. of XIST as determined by bisulphite sequencing of DNA from Normal males have, on average, a high degree of XIST lymphocytes. Error bars represent S.E.M. Significance was methylation ranging from 88 to 96% depending on the calculated by one-way ANOVA followed by a Bonferroni’s post-hoc ! CpG site (Fig. 2a) and they showed a very homogeneous test. A statistical significance of P 0.001 is indicated by ‘*’. (a) XIST methylation of (SRYC) 46,XX-males compared to that of 47,XXY Klinefelter males and female controls. Bars give mean XIST methylation of patients with different at individual CpG sites; (b) XIST methylation of (SRYC) 46,XX-males compared to that of (SRYK) 46,XX-males, 46,XY male controls and 45,X Turner females; (c) individual XIST methylation of patients with different karyotypes. Each dot represents the mean XIST methyl- ation over the analysed five CpG sites of one individual patient. Lines indicate mean and S.E.M.

high degree of XIST methylation (Fig. 2b), similar to 45,X women with highly methylated XIST levels of 97–99% (Fig. 2b).

Figure 1 Localisation and number of CpG sites of the analysed X-linked genes. CpG-rich regions (CpG islands) were identified by DNA methylation state of representative X Methprimer, and PCR primers were designed to amplify the chromosomal genes indicated regions. Given coordinates indicate the position of the analysed fragment in relation to the transcription start site. Lollipops We next addressed the question of whether sex represent CpG methylation site within the fragments. (a) XIST, C 133 bp, five CpG sites; (b) PGK1, 318 bp, 38 CpG sites; chromosomal anomalies, in particular (SRY )-XX- (c) FTHL17, 285 bp, 24 CpG sites; (d) SHOX, 321 bp, 28 CpG sites. maleness associated with XIST hypomethylation, result

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Downloaded from Bioscientifica.com at 10/01/2021 09:01:58PM via free access EUROPEAN JOURNAL OF ENDOCRINOLOGY (2010) 162 Epigenetics of the X in XX-maleness 173 in an abnormal epigenetic state of X chromosomal loci. degree of SHOX methylation as male controls and KS Hence, we analysed three representative X chromo- men (7%), while in 45, X women a comparably low somal genes, PGK1, FTHL17 and SHOX,forthe degree of methylation was detected (4.8%). presence of CpG islands using MethPrimer (27) and designed suitable PCR primers. PGK1 was chosen as an indicator of X chromosome Discussion inactivation harbouring a CpG island in its promoter. We analysed a 318 bp fragment from K338 to K21 X chromosome inactivation represents an essential relative to the transcription start site (Fig. 1b) compris- mechanism to balance the X chromosomal gene dosage ing 38 CpG methylation sites in (SRYC) XX-males, KS between the sexes. Sex chromosomal aberrations, males and 45, X women controlled by healthy men and however, constitute a major thread to this finely tuned women. (SRYC) XX males and KS men displayed a non- process. We present the first descriptive and comparative significant (PO0.05) reduction of PGK1 methylation to study of the DNA methylation status of the X chromo- 51 and 56% respectively (Fig. 3a) compared to female some in XX-maleness, KS and Turner syndrome. The study of general molecular features of controls who had 69% PGK1 methylation. PGK1 XX-maleness is always limited by the naturally small methylation was 3.4% in male controls; this value study cohort due to the extreme rarity of this syndrome. was significantly lower compared to that in female It is especially difficult to get hold of samples from controls (P!0.001), but comparable to PGK1 methyl- (SRYK) XX-males as only about 10% of 46,XX-males ation in 45,X Turner women (0.4%). lack a translocation of Y chromosomal material to one FTHL17 represents X-linked genes that do not of their X chromosomes (2, 29, 30). However, our undergo X inactivation, but are regulated in a cell- cohort of ten (SRYC) XX-males represents one of the type-specific manner. We hypothesised that FTHL17 largest cohorts ever reported (31). We could clearly might be a suitable candidate to indicate whether this demonstrate that hypomethylation of XIST is a common differential regulation is impaired by the abnormal sex feature of these men with a significant reduction of XIST C chromosomal constitution in SRY( ) XX-males and methylation from about 50 to 20% on average when other sex chromosomal aberration. compared to female controls and KS men. Analysis of the coding sequence of FTHL17 revealed Despite this epigenetic aberration, three relevant a predicted CpG – island in the first exon (Fig. 1c) and a X-linked genes representing X inactivation (PGK1), 285 bp fragment from C24 to C308 relative to the escape from X inactivation (SHOX) and cell-type-specific transcription start site comprising 24 CpG methylation inactivation (FTHL17) were normally methylated at sites was chosen for subsequent analysis. Male and their CpG islands when compared to those in female female controls showed a high degree of FTHL17 controls and Klinefelter patients, indicating comparably methylation of 91 and 90% respectively, indicative of a normal X inactivation. high inactivation status (Fig. 3b). No difference in As XIST RNA expression is coupled to its DNA FTHL17 methylation was found in (SRYC) XX-males methylation status (20), it would obviously be a vital (89%), KS men (87%) or 45, X women (93%). investigation to compare actual XIST expression in SHOX is located in the pseudoautosomal region of the XX-males to that in KS men and female controls. This X and escapes X inactivation; it therefore represents the would yield the information whether XIST methylation so-called escapees. We analysed a 321 bp fragment from still dictates XIST expression in (SRYC) XX-males. K375 to K55 relative to the transcription start site However, this study has been performed retrospectively of SHOX comprising 28 CpG sites (Fig. 1d). Female and owing to the poor availability of a sufficient number and male controls showed a low degree of SHOX of fresh blood samples from XX-males it is not feasible methylation with 9 and 7% respectively (Fig. 3c). at the moment to back-up our epigenetic findings with (SRYC) XX-males displayed, on average, a similar expression data.

Figure 3 Methylation analysis of X-linked PGK1, FTHL17 and SHOX. Columns represent the mean methylation of all analysed CpG sites of all individuals in a given group. Error bars indicate S.E.M., and significance was calculated by one-way ANOVA followed by a Bonferroni post-hoc test. (a) PGK1; (b) FTHL17; (c) SHOX.

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It has been described earlier that XIST expression explains why the latter are exclusively infertile due to becomes dispensable for X inactivation once it is the absence of male fertility-related regions on the Y established (32). Silencing of the inactive X chromo- chromosome, including AZF (39), whereas the former some is then overtaken by histone modifications, such sometimes display residual active foci of spermatogen- as extensive H3K27 methylation that is conferred by the esis (7, 8). The severe XIST hypomethylation of (SRYC) Polycomb repressive complex 2 (33). However, if this XX-males indicates that there might be more hitherto would confer relaxed stringency to the DNA methyl- unidentified epigenetic differences. ation status of XIST, female controls and KS men could In summary, we provide strong evidence that XIST as well display severely changed XIST methylation hypomethylation in (SRYC) XX-males is a substantial levels. As this is not the case, other hitherto unknown epigenetic hallmark of this chromosomal anomaly. factors must account for the massive hypomethylation It associates the anthropomorphic and biochemical of XIST that remain elusive. One important finding in distinction of (SRYC) XX-maleness from KS with a our study is that the two (SRYK) XX-males displayed characteristic epigenetic pattern. Both Klinefelter female-like 50% XIST methylation. This finding patients and (SRYC) XX-males share the characteristic assigns a crucial role to the Y–X translocation in pattern of hypergonadotrophism indicating a severely (SRYC) XX-males in the aetiology of severe XIST impaired endocrine feedback regulation and an in hypomethylation. general normal inactivation of X-linked genes. The As proposed and indicated earlier (34), KS men in this fact that presumably only XIST methylation dis- study undergo appropriate X chromosome inactivation. tinguishes both entities points to a hitherto unknown The striking epigenetic similarities of the X chromo- impact of the on X inactivation. Thus, somes of KS patients and women in terms of future studies will have to elucidate the impact of the methylation status and variation suggest that insuffi- X–Y-chromosomal crosstalk on the endocrine status ciency and apoptosis of Leydig cell in KS men (7) could and phenotypic characteristics in correlation to the be due to the ‘female’ expression pattern in these cells sex chromosomal constitution. that could compromise their function. Although no functional proof of escapee transcripts can be given Declaration of interest here, the epigenetic analysis suggests that KS men, like women, express genes from the second X that escapes X The authors certify that they have no affiliation with or financial involvement in any organisation or entity with a direct financial inactivation. We thus strengthen the hypothesis that interest in the subject matter or materials discussed in the manuscript. these ‘escapees’, which are present in women and KS Any research support is identified. men, but not in normal men, are major contributors to the phenotypes in KS (35). Funding Our data support the concept of inter-individual variation of X inactivation. Earlier studies demonstrated This study was supported by the Deutsche Forschungsgemeinschaft that X-inactivated genes show substantial variation of DFG GR 1547/8-1. expression between different women accompanied by variable retention of epigenetic silencing mechanisms Acknowledgements (36). Earlier findings that females show considerable We thank Nicole Terwort for excellent technical assistance. Michael heterogeneity in levels of X-linked expression (19) have Zitzmann, Elena Vorona and Andreas Schu¨ring are thanked for recently been backed up by the report that both clinical characterisation of patients. X-inactivated genes and escapees show significant variation of expression (37). The wide variation of XIST methylation in all XX-carrying patients and References controls in this study reflects the general heterogeneity of X-linked expression in individuals with two 1 Huang WJ & Yen PH. Genetics of spermatogenic failure. Sexual X chromosomes. Development: Genetics, Molecular Biology, Evolution, Endocrinology, A number of characteristics that distinguish (SRYC) Embryology, and Pathology of Sex Determination and Differentiation 2008 2 251–259. XX-males from KS men have been described before. 2 Kojima Y, Hayashi Y, Mizuno K, Sasaki S, Fukui Y, Koopman P, These include anthropomorphic features such as Morohashi K & Kohri K. Up-regulation of SOX9 in human reduced body height and an increased incidence of sex-determining region on the Y chromosome (SRY)-negative XX maldescended testis, elevated LH levels, significantly males. Clinical Endocrinology 2008 68 791–799. 3 Vorona E, Zitzmann M, Gromoll J, Schuring AN & Nieschlag E. reduced creatinine levels and extremely skewed inacti- Clinical, endocrinological, and epigenetic features of the 46,XX vation of the androgen receptor located on the male syndrome, compared with 47,XXY Klinefelter patients. X chromosome (3). Karyotypically, the difference Journal of Clinical Endocrinology and Metabolism 2007 92 between these syndromes is that KS men possess a 3458–3465. C 4 Van Assche E, Bonduelle M, Tournaye H, Joris H, Verheyen G, complete Y chromosome, whereas (SRY ) XX-males Devroey P, Van Steirteghem A & Liebaers I. only carry an SRY-containing fragment translocated of infertile men. Human Reproduction 1996 11 1–24 (discussion to one of their X chromosomes (38). This partially 25-6).

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5 Minks J, Robinson WP & Brown CJ. A skewed view of X 24 Loriot A, Boon T & De Smet C. Five new human cancer-germline chromosome inactivation. Journal of Clinical Investigation 2008 genes identified among 12 genes expressed in spermatogonia. 118 20–23. International Journal of Cancer. Journal International du Cancer 2003 6 Lanfranco F, Kamischke A, Zitzmann M & Nieschlag E. Klinefelter’s 105 371–376. syndrome. Lancet 2004 364 273–283. 25 Wang PJ, McCarrey JR, Yang F & Page DC. An abundance of 7 Aksglaede L, Wikstrom AM, Rajpert-De Meyts E, Dunkel L, X-linked genes expressed in spermatogonia. Nature Genetics 2001 Skakkebaek NE & Juul A. Natural history of seminiferous tubule 27 422–426. degeneration in Klinefelter syndrome. Human Reproduction Update 26 Vermeulen A, Verdonck L & Kaufman JM. A critical evaluation 2006 12 39–48. of simple methods for the estimation of free testosterone in 8 Foresta C, Galeazzi C, Bettella A, Marin P, Rossato M, Garolla A & serum. Journal of Clinical Endocrinology and Metabolism 1999 84 Ferlin A. Analysis of in intratesticular germ cells from 3666–3672. subjects affected by classic Klinefelter’s syndrome. Journal of Clinical 27 Li LC & Dahiya R. MethPrimer: designing primers for methylation Endocrinology and Metabolism 1999 84 3807–3810. PCRs. Bioinformatics 2002 18 1427–1431. 9 Schiff JD, Palermo GD, Veeck LL, Goldstein M, Rosenwaks Z & 28 Song MA, Park JH, Jeong KS, Park DS, Kang MS & Lee S. Schlegel PN. Success of testicular sperm extraction (corrected) and Quantification of CpG methylation at the 50-region of XIST by intracytoplasmic sperm injection in men with Klinefelter syn- pyrosequencing from human serum. Electrophoresis 2007 28 drome. Journal of Clinical Endocrinology and Metabolism 2005 90 2379–2384. 6263–6267. 29 Rajender S, Rajani V, Gupta NJ, Chakravarty B, Singh L & 10 Chantot-Bastaraud S, Ravel C & Siffroi JP. Underlying Thangaraj K. SRY-negative 46,XX male with normal genitals, abnormalities in IVF/ICSI patients. Reproductive Biomedicine Online complete masculinization and infertility. Molecular Human 2008 16 514–522. Reproduction 2006 12 341–346. 11 Greco E, Iacobelli M, Rienzi L, Fabris GF, Tesorio N & Tesarik J. 30 Valetto A, Bertini V, Rapalini E & Simi P. A 46,XX SRY-negative Birth of a healthy boy after fertilization of cryopreserved oocytes man with complete virilization and infertility as the main with cryopreserved testicular spermatozoa from a man with nonmosaic Klinefelter syndrome. Fertility and Sterility 2008 89 anomaly. Fertility and Sterility 2005 83 216–219. 991.e5–991.e7. 31 Aksglaede L, Jorgensen N, Skakkebaek NE & Juul A. Low semen 12 Gravholt CH. Epidemiological, endocrine and metabolic features volume in 47 adolescents and adults with 47,XXY Klinefelter or in Turner syndrome. European Journal of Endocrinology 2004 151 46,XX male syndrome. International Journal of Andrology 2009 32 657–687. 376–384. 13 Gravholt CH. Turner syndrome in adulthood. Hormone Research 32 Wutz A & Jaenisch R. A shift from reversible to irreversible X 2005 64 86–93. inactivation is triggered during ES cell differentiation. Molecular 14 Clemson CM, McNeil JA, Willard HF & Lawrence JB. XIST RNA Cell 2000 5 695–705. paints the inactive X chromosome at interphase: evidence for a 33 Shibata S, Yokota T & Wutz A. Synergy of Eed and Tsix in the novel RNA involved in nuclear/chromosome structure. Journal of repression of Xist gene and X-chromosome inactivation. EMBO Cell Biology 1996 132 259–275. Journal 2008 27 1816–1826. 15 Panning B, Dausman J & Jaenisch R. X chromosome inactivation 34 Stabile M, Angelino T, Caiazzo F, Olivieri P, De Marchi N, is mediated by Xist RNA stabilization. Cell 1997 90 907–916. De Petrocellis L & Orlando P. Fertility in a i(Xq) Klinefelter patient: 16 Csankovszki G, Nagy A & Jaenisch R. Synergism of Xist RNA, DNA importance of XIST expression level determined by qRT-PCR in methylation, and histone hypoacetylation in maintaining X ruling out Klinefelter cryptic mosaicism as cause of oligozoos- chromosome inactivation. Journal of Cell Biology 2001 153 permia. Molecular Human Reproduction 2008 14 635–640. 773–784. 35 Ross NL, Wadekar R, Lopes A, Dagnall A, Close J, Delisi LE & 17 Heard E. Delving into the diversity of facultative heterochromatin: Crow TJ. Methylation of two Homo sapiens-specific X–Y the epigenetics of the inactive X chromosome. Current Opinion in homologous genes in Klinefelter’s syndrome (XXY). American Genetics & Development 2005 15 482–489. Journal of . Part B, Neuropsychiatric Genetics: the 18 Brown CJ & Greally JM. A stain upon the silence: genes escaping X Official Publication of the International Society of Psychiatric Genetics inactivation. Trends in Genetics 2003 19 432–438. 2006 141B 544–548. 19 Carrel L & Willard H. X-inactivation profile reveals extensive 36 Anderson CL & Brown CJ. Variability of X chromosome variability in X-linked gene expression in females. Nature 2005 inactivation: effect on levels of TIMP1 RNA and role of DNA 434 400–404. methylation. Human Genetics 2002 110 271–278. 20 Panning B & Jaenisch R. DNA hypomethylation can activate Xist 37 Yin S, Wang P, Deng W, Zheng H, Hu L, Hurst LD & Kong X. expression and silence X-linked genes. Genes and Development 1996 Dosage compensation on the active X chromosome minimizes 10 1991–2002. transcriptional noise of X-linked genes in mammals. Genome 21 Hansen RS & Gartler SM. 5-Azacytidine-induced reactivation of Biology 2009 10 R74. the human X chromosome-linked PGK1 gene is associated with a 0 38 de la Chapelle A. The etiology of maleness in XX men. Human large region of cytosine demethylation in the 5 CpG island. PNAS Genetics 1981 58 105–116. 1990 87 4174–4178. 39 Simoni M, Tuttelmann F, Gromoll J & Nieschlag E. Clinical 22 Matarazzo MR, De Bonis ML, Strazzullo M, Cerase A, Ferraro M, consequences of microdeletions of the Y chromosome: the Vastarelli P,Ballestar E, Esteller M, Kudo S & D’Esposito M. Multiple extended Munster experience. Reproductive Biomedicine Online binding of methyl-CpG and polycomb proteins in long-term 2008 16 289–303. gene silencing events. Journal of Cellular Physiology 2007 210 711–719. 23 Blaschke RJ & Rappold G. The pseudoautosomal regions, SHOX and disease. Current Opinion in Genetics & Development 2006 16 Received 1 September 2009 233–239. Accepted 6 October 2009

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