Low-level X chromosome mosaicism in women with sporadic premature ovarian failure

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Reproductive BioMedicine Online (2011) 22, 399 403 www.sciencedirect.com www.rbmonline.

Medical Centre, Ljubljana, Slovenia * Corresponding author. E-mail address: ksenija.gersak@mf.uni-lj.si (K Gersak).

She is a specialist in obstetrics and gynaecology, a chief of Chair of Obstetrics and Gynaecology and lecturer on the postgraduate education programme for biomedicine, University of Ljubljana, and

1 Reproductive BioMedicine Online (2011) 22, ARTICLE Low-level X chromosome mosaicism in women with sporadic premature ovarian failure K Gersak *, A Veble Department of Obstetrics and Gynecology, Institute of Medical Genetics, University Medical Centre, Ljubljana, Slovenia * Corresponding author. address: ksenija.gersak@mf.uni-lj.si (K Gersak). Prof Ksenija Gersak, MD, PhD graduated from the Faculty of Medicine, University of Ljubljana. She is a specialist in obstetrics and gynaecology, a chief of Chair of Obstetrics and Gynaecology and lecturer on the postgraduate education programme for biomedicine, University of Ljubljana, and assistant medical director for research at the University Medical Centre, Ljubljana. Her main research interests are reproductive physiology and reproductive genetics. Abstract Low-level X chromosome mosaicism and its clinical relevance are still under debate. It could be interpreted as a technical artefact, genuine mosaicism or as being age-related. This study evaluated the contribution of X chromosome mosaicism in phenotypically normal women with sporadic premature ovarian failure (POF). During , 114 patients with POF and 64 age matched controls were karyotyped. Thirteen patients (11.4%) had true X chromosome mosaicism (>10% of aneuploid cells) and 12 had (10.5%) low-level X-mosaicism (between 6 10% of aneuploid cells). The mean age of women with true and low-level mosaicism was 26.0 ± 5.65 years and ± 3.87 years, respectively (P < 0.001). In the control group the incidence of cells with an abnormal number of X chromosomes was 1 3%. The results have practical implications for genetic counselling and fertility treatment. To search and confirm the low-level mosaicism, a higher number of metaphases should be analysed or additional fluorescence in-situ hybridization analysis must be performed. Although peripheral blood does not reflect the situation in ovarian tissue well, it is presumed that there are different aetiological causes for true and low-level X chromosome mosaicism. The possible causes and reproductive significance of low-level X chromosome mosaicism are discussed. RBMOnline ª 2011, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: ageing, premature ovarian failure, X chromosome loss, X chromosome mosaicism Introduction X chromosome mosaicism, balanced translocations involving the X chromosome, Xq deletions and other variants of X chromosome abnormalities are usually associated with abnormal sexual development and reproductive performance, such as primary or secondary amenorrhoea, infertility, recurrent abortions and premature ovarian failure (POF). POF is defined as menopause before age 40 and occurs in 1 2% of women. Causes of POF are of genetic, autoimmune, iatrogenic and environmental origin. Genetic causes of POF probably comprise about one-third to one-half of all cases (Bione and Toniolo, 2000; Goswami and Conway, 2005; Santoro, 2001; Simpson, 2008; van Dooren et al., 2009). The frequency of X chromosome mosaicism in women with the sporadic form of POF has been estimated to be between /$ - see front matter ª 2011, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. doi: /j.rbmo

2 400 K Gersak, A Veble 3% and 11% (Devi and Benn, 1999; Lakhal et al., 2010; Wong and Lam, 2005; Wu et al., 1993). When an abnormal number of sex chromosomes is seen in a low percentage of cells, the result could be interpreted as a technical artefact, genuine mosaicism or being age-related (Russell et al., 2007; Wise et al., 2009). Low-level X chromosome mosaicism and its clinical relevance are still under debate. The aim of the present study was to: (i) evaluate the contribution of X chromosome mosaicism in phenotypically normal women with sporadic idiopathic POF; and (ii) discuss the reproductive significance of low-level X chromosome mosaicism. Materials and methods The study included 114 women with sporadic idiopathic POF out of 197 women with amenorrhoea referred to the Department of Obstetrics and Gynaecology, Division of Medical Genetics, in the period between 1999 and The diagnosis of POF was based on the following criteria: (i) at least 6 months of amenorrhoea; (ii) age at menopause of <40 years; and (iii) two consecutive determinations of serum FSH higher than 40 IU/l. Karyotyping was performed within a 12-month period after the last menses. Women with phenotypic features suggestive of Turner syndrome, primary amenorrhoea or gonadal dysgenesis (65 women) were excluded. Also 18 women with structural abnormalities in one or more chromosomes (balanced translocation with X chromosome involved, X chromosome abnormalities), blood lymphocyte microchimerism, Fragile X premutation, mutations in the FOXL2 or inhibin INH genes were excluded (Gersak et al., 2004, 2010a,b; Harris et al., 2002; Shelling et al., 2000). Sixty-four women with a regular menstrual cycle (28 32 days) with no history of uterine malformations or chronic vascular, renal and autoimmune diseases were included in the control group. Thyroid evaluation (thyroid-stimulating hormone, T3, T4 and/or antithyroid antibodies), adrenal hormones and a comprehensive biochemistry panel (including calcium, phosphorus, electrolytes, cholesterol and fasting glucose) results were normal. All women gave their informed consent. Cytogenetic studies were carried out on peripheral blood samples, cultured for approximately 72 h. For each chromosome analysis, 50 GTG-banded cells were analysed, three of which were karyotyped (Hook, 1977). If the initial cytogenetic analysis revealed any cells with sex chromosome hypo- or hyperploidy, at least 100 cells were counted and analysed. True mosaicism was considered as presence of more than 10% of aneuploid cells whereas low-level mosaicism was defined as 6 10% of aneuploid cells. Confirmation of the mosaicism by fluorescent in-situ hybridization (FISH) on peripheral blood lymphocytes and buccal mucosal cells was not performed in all patients. Results The mean age of the women did not differ between the POF and the control groups (30.20 ± 5.39 years, ± 5.38 years, respectively). X chromosome mosaicism was found in 25 (21.9%) out of 114 women with POF. Thirteen patients (13/114, 11.4%) had true sex chromosome mosaicism (Figure 1). Among them, in four patients an abnormal number of sex chromosomes was seen in more than 40% of analysed cells. Twelve patients (12/114, 10.5%) had low-level X chromosome mosaicism (Figure 1). The mean age of women with true and low-level X chromosome mosaicism was 26.0 ± 5.65 years and ± 3.87 years, respectively (P < 0.001). Four women with low-level mosaicism had already delivered five offspring (four girls and one boy) and one woman with true X chromosome mosaicism had delivered one healthy boy. One live-born girl with true sex chromosome mosaicism was born to the mother with low-level X chromosome mosaicism. In one patient with true X chromosome mosaicism, pregnancy was initiated by an assisted reproduction technique (stimulation of ovulation). This study found cells with an abnormal number of X chromosomes in 25 out of 64 healthy women with regular menstrual cycles. The incidence of X chromosome loss was 1% to 3% (Figure 2). In two women, 1 2% of 47,XXX cells were found. No structural chromosome abnormalities were identified in the control group. Discussion This study evaluated the contribution of true and low-level X chromosome mosaicism to POF in phenotypically normal women with sporadic POF by routine G-banding chromosome analysis of at least 50 metaphases. Obviously FISH may be the most appropriate method of confirming suspected numerical mosaicism (Shaffer and Tommerup, 2005), particularly for detecting low-level X chromosome mosaicism (Lakhal et al., 2010; Devi and Benn, 1999). But according to the International System for Cytogenetic Nomenclature, numerical and structural abnormalities still have to be excluded at a banding level appropriate to the referral s guidelines (Gardner and Sutherland, 2003; Guttenbach et al., 1995; Shaffer and Tommerup, 2005). At least 50 cells have to be analysed to exclude the presence of 6% mosaicism with a 0.95 level of confidence (Hook, 1977). With an evaluation of 20 metaphases, only a mosaicism greater than 14% can be found with the same confidence. This study found mosaicism in 21.9% of patients by G-banding chromosome analysis, if the true and low-level mosaicism are regarded as identical abnormal results. Wu et al. (1993) reported five out of 61 (8.2%) POF cases with X chromosome mosaicism. In a Hong Kong group of 312 women with secondary amenorrhoea, 11 cases with karyotype 45,X/46,XX and three with mosaic triple/poly X (Wong and Lam, 2005) were found. Lakhal et al. (2010) detected 34 (5.9%) patients with homogeneous or mosaic X-chromosome aneuploidy out of 568 with secondary amenorrhoea. But in all patients, karyotype analysis using R-banding was performed on only 20 metaphases. By contrast, Portnoi et al. (2006) identified no 45, X/46,XX or 46,XX/47,XXX chromosome mosaicisms in any of their POF patients or controls. Compared with POF studies, more data is available on X chromosome mosaicism in women with primary amenorrhoea or Turner syndrome (Birkebaek et al., 2002; Cortés-Gutiérrez et al., 2007;

3 Low-level X chromosome mosaicism and POF 401 Figure 1 The incidence of X aneuploid cells in women with sporadic idiopathic premature ovarian failure (POF). Only patients with more than 5% of X aneuploid cells are presented. Figure 2 The incidence of cells with X aneuploidy in the control group. If the initial cytogenetic analysis revealed any cells with sex chromosome hypo- or hyperploidy, at least 100 cells were counted and analysed. In 25 out of 64 healthy women the incidence of X chromosome loss was 1 3%. Sybert and McCauley, 2004; Wong and Lam, 2005). Karyotyping and some mutation analyses of samples taken from patients with phenotypic features suggestive of Turner syndrome, primary amenorrhoea or gonadal dysgenesis are part of the centre s ongoing study. In the present study, true X chromosome mosaicism was found in 11.4% of women with sporadic idiopathic POF and low-level mosaicism in 10.5%. There is no consensus on the definition of low-level mosaicism, which has been considered as the presence of <10% of abnormal cells, <6% of abnormal cells or even as a concept which should not be reported at all (Morel et al., 2002; Shaffer and Tommerup, 2005). According to the present results, it is presumed that there are at least two different subgroups of patients with X chromosome mosaicism. The mean age of women with true and low-level X chromosome mosaicism was significantly different (26.0 ± 5.65 years and ± 3.87 years, respectively). Although peripheral blood does not reflect the situation in other tissues well, i.e. in ovarian tissue, the onset of POF occurred earlier in women with more than 10% of aneuploid cells. In all patients, karyotyping was performed within a 12-month period after the last menses. The major difficulty that affects studies aimed at revealing the effects of mosaicism is the definition of a non-pathogenic level of aneuploidy in a specific tissue. Therefore control studies of unaffected individuals are required. However, in the low-level subgroup of women with sporadic POF, this study detected a significantly higher level of X chromosome loss than in the age-matched controls (P < 0.01). Healthy women aged years show monosomy X at a rate from 2.5% to 3.1% (Guttenbach et al., 1995; Russell et al., 2007). The present analysis reached a similar result. It is presumed that there are different aetiological causes for true and low-level X chromosome mosaicism. True X blood mosaicism probably reflects the cytogenetic characteristics of ovarian tissue. It could be the result of mitotic errors that appear during the cleavage stage of an early embryo, probably during blastogenesis and can involve all three germ layers. The nature of the mosaicism is not known. Some indirect observations suggest that it may originate from a process of trisomy rescue and that it increases with maternal age (Kuliev and Verlinsky, 2004). In a mosaic ovary, aberrant X chromosome pairing and impaired genetic control of chromosomal nondisjunction may cause premature germ cell death and thus decrease the number of germ cells and accelerate oocyte atresia as well as cause post-natal destruction of germ cells (Gardner and Sutherland, 2003; Kuo and Guo, 2004). One obvious explanation could also lie in the haploinsufficiency of loci on the X chromosome (Simpson, 2008).

4 402 K Gersak, A Veble The impact of low-level X chromosome mosaicism on ovarian or menstrual function is a poorly described phenomenon and the pathogenesis is not clear. Current data suggest that the process of aneuploidization has the potential to produce tissue- and organ-specific chromosomal mosaicism during both embryonic differentiation and post-natal development (Iourov et al., 2008). Chromosomal mosaicism has the potential to mediate intercellular diversity and is suggested to be a key process that accelerates ageing. It was observed in ovarian germline tissues and in the brain. Low-level X mosaicism in the ovaries and in peripheral blood might also be caused by this process. The diseases associated with chromosomal mosaicism also include complex neuropsychiatric and immune diseases (Iourov et al., 2008; Persani et al., 2009). X monosomy is more frequent in T and B lymphocytes than in other blood cell populations in women with systemic sclerosis and autoimmune thyroid disease (Invernizzi et al., 2005). Recently published data focused on different human pathogenic conditions (Wise et al., 2009) and suggest future biomedical research. On the other hand, accelerated ageing can be linked with the limited oocyte pool and/or their suboptimal state of development due to mutations in genes that primarily affect follicle function. It is well known that both the short and long arms of the X chromosome contain genes important for ovarian function (Duzcan et al., 2003; Portnoi et al., 2006). Oocyte quality can also be influenced by deficiency or overexpression of specific gene products on the X chromosome (Rizzolio et al., 2009). Genes that are primarily expressed in the ovaries are BMP15, GDF9 and GPR3. Bone morphogenetic protein 15 and growth/differentiation factor 9 are members of the transforming growth factor b superfamily. They are involved in oocyte maturation and follicular development (Di Pasquale et al., 2004; Dube et al., 1999; Hreinsson et al., 2002; Kovanci et al., 2007; McGrath et al., 1995). G-protein coupled receptor 3 is a transmembrane receptor that is involved in signal transduction. It maintains meiotic arrest in mammalian oocytes and is thought to be a communication link between oocytes and the surrounding somatic tissue (Mehlmann et al., 2004; Song et al., 1996). In conclusion, routine cytogenetic analyses should be performed for women with unexplained sporadic POF even when there are no other clinical features suggestive of chromosome abnormality. This standpoint has practical implications for genetic counselling and fertility treatment. To search and confirm the low-level mosaicism a higher number of metaphases should be analysed or additional FISH analysis must be performed. Although peripheral blood does not reflect the situation in ovarian tissue well, it is presumed that there are different aetiological causes for true and low-level X chromosome mosaicism. According to this assumption, further clinical studies are required. Acknowledgements The authors would like to thank the laboratory and clinical staff of the Institute of Medical Genetics for their excellent technical assistance, K. Writzl, MD, PhD for advices on final revision and Mr B.M. Gersak for revision of the English text. References Bione, S., Toniolo, D., X chromosome genes and premature ovarian failure. Semin. Reprod. Med. 18, Birkebaek, N.H., Crüger, D., Hansen, J., Nielsen, J., Bruun-Petersen, G., Fertility and pregnancy outcome in Danish women with Turner syndrome. Clin. 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5 Low-level X chromosome mosaicism and POF 403 Kuliev, A., Verlinsky, Y., Meiotic and mitotic nondisjunction: lessons from preimplantation genetic diagnosis. Hum. Reprod. Update 10, Kuo, P.L., Guo, H.R., Mechanism of recurrent spontaneous abortions in women with mosaicism of X-chromosome aneuploidies. Fertil. Steril. 82, Lakhal, B., Braham, R., Berguigua, R., et al., Cytogenetic analyses of premature ovarian failure using karyotyping and interphase fluorescence in situ hybridization (FISH) in a group of 1000 patients. Clin. Genet. 78, McGrath, S.A., Esquela, A.F., Lee, S.J., Oocyte-specific expression of growth/differentiation factor-9. Mol. Endocrinol. 9, Mehlmann, L.M., Saeki, Y., Tanaka, S., et al., The Gs-linked receptor GPR3 maintains meiotic arrest in mammalian oocytes. Science 306 (5703), Morel, F., Gallon, F., Amice, V., et al., Sex chromosome mosaicism in couples undergoing intracytoplasmic sperm injection. Hum. Reprod. 17, Persani, L., Rossetti, R., Cacciatore, C., Bonomi, M., Primary ovarian insufficiency: X chromosome defects and autoimmunity. J. Autoimmun. 33, Portnoi, M.F., Aboura, A., Tachdjian, G., et al., Molecular cytogenetic studies of Xq critical regions in premature ovarian failure patients. Hum. Reprod. 21, Rizzolio, F., Pramparo, T., Sala, C., et al., Epigenetic analysis of the critical region I for premature ovarian failure: demonstration of a highly heterochromatic domain on the long arm of the mammalian X chromosome. J. Med. Genet. 46, Russell, L.M., Strike, P., Browne, C.E., Jacobs, P.A., X chromosome loss and ageing. Cytogenet. Genome. Res. 116, Santoro, A., Research on the mechanisms of premature ovarian failure. J. Soc. Gynecol. Investig. 8 (Suppl.), S10 S22. Shaffer, L.G., Tommerup, N., An International System for Human Cytogenetic Nomenclature (Cytogenetic and Genome Research). Karger, Basel. Shelling, A.N., Burton, K.A., Chand, A.L., et al., Inhibin: a candidate gene for premature ovarian failure. Hum. Reprod. 15, Simpson, J.L., Genetic and phenotypic heterogeneity in ovarian failure: overview of selected candidate genes Ann. N. Y. Acad. Sci. 11, Song, Z.H., Modi, W., Bonner, T.I., Molecular cloning and chromosomal localization of human genes encoding three closely related G protein-coupled receptors. Genomics 28, Sybert, V., McCauley, E., Turner s syndrome. N. Eng. J. Med. 351, Wise, J.L., Crout, R.J., McNeil, D.W., Weyant, R.J., Marazita, M.L., Wenger, S.L., Cryptic subtelomeric rearrangements and X chromosome mosaicism: a study of 565 apparently normal individuals with fluorescent in situ hybridization. PLoS. One 10, e5855. Wong, M.S., Lam, S.T., Cytogenetic analysis of patients with primary and secondary amenorrhoea in Hong Kong: retrospective study. Hong Kong Med. J. 11, Wu, R.C., Kuo, P.L., Lin, S.J., Liu, C.H., Tzeng, C.C., X chromosome mosaicism in patients with recurrent abortion or premature ovarian failure. J. Formos. Med. Assoc. 92, Declaration: This work was supported by grant from the Ministry of Higher Education, Science and Technology of the Republic of Slovenia (Grant No. P3 0124). Received 29 April 2010; refereed 3 January 2011; accepted 4 January 2011.

Short Report. B Lakhal a, R Braham b, R Berguigua a, N Bouali a, M Zaouali c, M Chaieb b, RA Veitia d,e,f, A Saad a,g and H Elghezal a,g

Short Report. B Lakhal a, R Braham b, R Berguigua a, N Bouali a, M Zaouali c, M Chaieb b, RA Veitia d,e,f, A Saad a,g and H Elghezal a,g Clin Genet 2010: 78: 181 185 Printed in Singapore. All rights reserved Short Report 2010 John Wiley & Sons A/S CLINICAL GENETICS doi: 10.1111/j.1399-0004.2009.01359.x Cytogenetic analyses of premature