Background: The role of extremely skewed X-chromosome inactivation (XCI) has been questioned in the pathogenesis of recurrent spontaneous abortion (RSA) but the results obtained were conflicting. Aims: We therefore investigated the XCI patterns in peripheral blood DNA obtained from 80 patients who had RSA and 160 age-matched controls. Methods: Pregnancy history, age, karyotype, and disease information was collected from all subjects. The methylation status of a highly polymorphic cytosine-adenine-guanine repeat in the androgen-receptor (AR) gene was determined by use of methylation-sensitive restriction enzyme HpaII and polymerase chain reaction. Results: Skewed XCI (&gt; 8 5% skewing) was observed in 13 of the 62 patients informative for the AR polymorphism (20.9%), and eight of the 124 informative controls (6.4%) (P = 0.0069; χ 2 test). More importantly, extremely skewed XCI, defined as &gt; 90% inactivation of one allele, was present in 11 (17.7%) patients, and in only two controls (P = 0.0002; χ 2 test). Conclusions: These results support the interpretation that disturbances in XCI mosaicism may be involved in the pathogenesis of RSA. © 2006 The Authors Journal compilation © 2006 The Royal Australian and New Zealand College of Obstetricians and Gynaecologists

Acar, A.

Akyerli, C.B.

Bagislar, S.

Cengiz, B.

Ceylaner G.

Ceylaner, S.

Ozcelik, T.

Soylemez F.

Ustuner I.

Bilkent University Institutional Repository

Australian and New Zealand Journal of Obstetrics and Gynaecology 2006; 46: 384–387384 © 2006 The AuthorsJournal compilation © 2006 The Royal Australian and New Zealand College of Obstetricians and GynaecologistsBlackwell Publishing Asia Original ArticleX-inactivation and recurrent spontaneous abortionExtremely skewed X-chromosome inactivation patterns in women with recurrent spontaneous abortionSevgi BAGISLAR,1* Isik USTUNER,2* Bora CENGIZ,2 Feride SOYLEMEZ,2 Cemaliye Boylu AKYERLI,1 Serdar CEYLANER,3 Gulay CEYLANER,3 Aynur ACAR4 and Tayfun OZCELIK11Department of Molecular Biology and Genetics, Faculty of Science, Bilkent University, Ankara, Turkey, 2Department of Obstetrics and Gynaecology, Ankara University School of Medicine, Ankara, Turkey, 3Genetics Section, Zekai Tahir Burak Women’s Hospital, Ankara, Turkey, 4Department of Medical Biology, Selcuk University Meram Medical Faculty, Konya, Turkey AbstractBackground: The role of extremely skewed X-chromosome inactivation (XCI) has been questioned in the pathogenesisof recurrent spontaneous abortion (RSA) but the results obtained were conflicting.Aims: We therefore investigated the XCI patterns in peripheral blood DNA obtained from 80 patients who had RSAand 160 age-matched controls.Methods: Pregnancy history, age, karyotype, and disease information was collected from all subjects. The methylationstatus of a highly polymorphic cytosine-adenine-guanine repeat in the androgen-receptor (AR) gene was determinedby use of methylation-sensitive restriction enzyme HpaII and polymerase chain reaction.Results: Skewed XCI (> 85% skewing) was observed in 13 of the 62 patients informative for the AR polymorphism(20.9%), and eight of the 124 informative controls (6.4%) (P = 0.0069; χ2 test). More importantly, extremely skewedXCI, defined as > 90% inactivation of one allele, was present in 11 (17.7%) patients, and in only two controls(P = 0.0002; χ2 test).Conclusions: These results support the interpretation that disturbances in XCI mosaicism may be involved in thepathogenesis of RSA.Key words: androgen receptor, mosaicism, mutation, recurrent abortion; X-chromosome inactivation.IntroductionRecurrent spontaneous abortion (RSA) is an importantmedical problem that affects 1–2% of couples wishing to havechildren. It is defined as loss of three or more consecutivepregnancies prior to 20 week of gestation.1 The aetiologicfactors that have been implicated in RSA include anatomicalabnormalities (15%), infectious agents (1–2%), hormonalimbalances (20%), immunologic factors (20%) and geneticdisorders (2–5%).2,3 However, a large proportion of couples(37–79%) receive no explanation for their pregnancy losses.Based on the observation that an Xq28 deletion is associatedwith a high spontaneous abortion rate and familial skewedX-chromosome inactivation (XCI) in a large family,4 malelethal X-linked mutations that cause skewed XCI in femalecarriers was proposed as an aetiologic factor in RSA.X-chromosome inactivation is a physiologic event that takesplace during early embryonic development and results in thetranscriptional silencing of one of the two X-chromosomesin females.5 The inactive X can be either paternally ormaternally derived in different cells of the same individual,and once the decision as to which X to inactivate is made in aparticular cell, all the clonal descendants of that cell abide bythe decision. Therefore, females who are heterozygous forX-linked genes are mosaics of two cell types, with either thepaternal or the maternal alleles active. The choice of whichX to inactivate is generally a random event.6 However, underextraordinary circumstances, exclusive or almost exclusiveinactivation of one X-chromosome which leads to skewedXCI may be observed.7,8 According to the ‘male lethalX-linked mutations as a cause of recurrent abortions’ model,4This manuscript was presented in European Human Genetics Conference, 12–15 June 2004, Munich, Germany as a poster presentation.*Sevgi Bagislar and Isik Ustuner are equally contributing first authors.Correspondence: Professor Tayfun Ozcelik, Department of Molecular Biology and Genetics, B-242, Bilkent University, Bilkent, Ankara 06800, Turkey. Email: tozcelik@fen.bilkent.edu.trDOI: 10.1111/j.1479-828X.2006.00622.xReceived 09 January 2006; accepted 19 April 2006.X-inactivation and recurrent spontaneous abortion© 2006 The Authors 385Journal compilation © 2006 The Royal Australian and New Zealand College of Obstetricians and Gynaecologists; 46: 384–387X-chromosomal mutations that are cell lethal or associatedwith cell-growth disadvantage could be tolerated in femalesas a result of the XCI process. This means that in those cellsin which the mutant X-chromosome is the active X, cell deatheventually occurs during development, and the XCI patternbecomes skewed. However, at the phenotypic level, the carrierfemale lives and exhibits some (if not no) symptoms. However,in males, as there is only one X-chromosome that is alwaysactive, the putative cell lethal mutation becomes a male lethaltrait, and thus contributes to the aetiology of RSA.In the studies conducted to test the hypothesis that skewedXCI may be involved in the pathogenesis of RSA, bothsupporting9–13 and refuting14,15 results have been obtained(Table 1). We therefore investigated the peripheral blood XCIpatterns of controls and females who experience recurrentpregnancy loss to delineate the molecular basis of RSA in theTurkish population.Materials and methodsPatientsEighty Caucasian women diagnosed with RSA, and 160apparently healthy female controls, were genotyped to deter-mine whether women with a history of RSA have a greaterfrequency of extremely skewed XCI than control subjects.The ethics review board of the participating institutionsapproved the study protocol. Pregnancy history, age, anddisease information, accompanied by informed consent, wasobtained from all subjects. We included those cases withthree or more clinically recognised spontaneous abortions ofunknown cause. A positive urine pregnancy test or serum βhCGand evidence of the loss of conception products accompaniedby vaginal bleeding, or ultrasonographic findings of fetaldemise with subsequent reduction in serum βhCG, were usedto confirm spontaneous abortion. While ectopic or molarpregnancies were excluded, only those abortions that haveoccurred before week 20 of gestation were included in the study.Clinical evaluation of the cases excluded anatomic, cytogenetic,infectious, immunologic, hormonal, and social/exposureas causes of spontaneous abortion.2,3 Also abnormal hystero-salpingogram, antiphospholipid antibodies, or connective tissuedisorder were included in the exclusion criteria. While all ofthe RSA cases were karyotyped using peripheral bloodsamples, this was not done in the control group because of thelack of an indication for cytogenetic studies. Gravida 2, para2 women with no history of pregnancy loss comprised thecontrol group. The mean age of the cases and controls were30.4 ± 5 (mean ± SD; range = 22–42 years), and 32.4 ± 4.9years (range = 21–47 years), respectively.X-chromosome inactivation studyDNA was extracted from 10 mL venous blood samples ofpatients and controls using Nucleospin DNA isolation kit(Macherey-Nagel, Dueren, Germany). Genotyping of apolymorphic site in the androgen receptor gene (AR) wasperformed and quantified to assess the XCI patterns asdescribed.16 The degree of skewing was estimated by an assaybased on a methylation-sensitive HpaII restriction site locatednear the AR gene. This site is methylated on the inactive X,and unmethylated on the active X-chromosomes. When thegenomic DNA is cleaved with HpaII prior to polymerase chainreaction (PCR), only the methylated AR allele, which representsthe inactive X-chromosome, is amplified. A trinucleotidecytosine-adenine-guanine (CAG) repeat polymorphism locatedwithin the amplified region is used to distinguish between thetwo alleles. For each patient and control two separate PCRs,with or without HpaII treatment, were performed using thesame set of primers. Male DNA with cytogenetically verified46XY karyotype was used as control for complete digestion.All of the PCR products, before and after digestion, wereseparated by employing two independent detection methods.One is electrophoretic separation of the alleles in 4% MetaPhoragarose (FMC Bioproducts, Rockland, ME, USA) andethidium bromide staining. The other is separation of radio-active PCR products (α-[33P]-dCTP) (PerkinElmer, Wellesley,MA, USA) on 8% sequencing gels. Densitometric analysis ofthe alleles was performed using the Multi-Analyst softwareversion 1.1 (Bio-Rad Laboratories Inc., Hercules, CA, USA).The results of the densitometric analyses included normali-sation of the ratios based on the non-digested samples bydividing the allele ratio of the digested sample by the ratio ofthe non-digested sample from the same specimen. The useof this ratio corrects for preferential amplification of oneallele, which often occurs for the shorter microsatellite allele.The results from control and test groups were compared bythe χ2 test with Yates’ correction.ResultsX-chromosome inactivation status was found to be informativein 77.5% of both the cases (62/80) and the controls (124/160).Only those individuals whose alleles resolve adequately fordensitometric analysis were included in the study. Extremelyskewed XCI was present in 11 (17.7%) cases and in two (1.6%)controls (P = 0.0002) (Table 2). Extremely skewed XCI,defined as > 90% inactivation of one allele, is observed in ∼1–7%of females aged 25 or younger, and in ∼2–16% of women aged60 years or older.17,18 When XCI values between 85 and 89%Table 1 Studies of skewed X-chromosome inactivation andrecurrent spontaneous abortion (RSA)StudyNo. (%) observed with > 90% skewingRSA ControlLanasa et al. 199910 7/48 (14.6) 1/67 (1.5)Sangha et al. 199911 14/76 (18) 6/111 (5)Uehara et al. 200112 7/42 (16.7) 2/36 (5.6)Beever et al. 200313 25/207 (12) 7/102 (7)Sullivan et al. 200314 7/106 (6.6) 4/102 (3.9)Kim et al. 200415 1/45 (2.2) 5/54 (9.3)S. Bagislar et al.386 © 2006 The AuthorsJournal compilation © 2006 The Royal Australian and New Zealand College of Obstetricians and Gynaecologists; 46: 384–387were also considered, skewed XCI was observed in 13 of the 62(20.9%) informative cases, and eight of the 124 (6.4%) controls(P = 0.0069). These results indicate that extremely skewedXCI is more frequent in RSA cases than in controls, and raisesthe important question as to what causes this observation.DiscussionSkewed XCI could originate from primary (bias in the initialchoice) or secondary (selection following random XCI)causes.8 Two different hypotheses have been proposed toexplain the aetiology of skewed XCI in women who experienceRSA: (i) male lethal X-linked mutations4,19 and (ii) areduction of the size of the follicular pool13 associated withskewed XCI and trisomic pregnancies. A gender bias towardsfemale offspring among liveborn children and maternalinheritance of skewed X-inactivation was documented inconjunction with the first hypothesis. However, a significantexcess of boys among live births was observed in females withskewed XCI who were ascertained on the basis of trisomicpregnancies. Among our 11 patients with extreme skewing(> 90%), only three patients had live births and the observedfemale : male ratio is 3 : 1 (Table 3). Deviation from theexpected female : male ratio of 1 : 1 raises the possibility thatmale lethal X-linked mutations could be an aetiologic factorin our RSA cases, at least in the group of patients with extremeskewing. In addition to deleterious X-linked mutations,X-autosome translocations, normal ageing, twining, confinedplacental mosaicism, reduction of the size of the follicularpool, monoclonal expansion of peripheral blood cells, andenvironmental insults, such as chemotherapy, are examplesof secondary causes. Some of these factors are unlikely to beinvolved in our patients: for example only those patients witha normal karyotype were included in the study, excludingthe potential contribution of X-autosome translocations.Twining, because of negative history, and ageing, because ofthe mean age of diagnosis at 30.4 ± 5 years, are also highlyunlikely. A history or present diagnosis of haematologicmalignancies, which may be associated with monoclonalexpansion of peripheral blood cells, or exposure to environ-mental insults, such as chemotherapy, was not documentedin any study subject. With respect to the primary causes ofskewed XCI, we do not think dysfunction of gene(s) thatregulates XCI20 could be implicated in the aetiology of RSAas dosage compensation for X-linked genes is not physiolog-ically required in XY cells and therefore is not expected tobe lethal in male fetuses. Our findings also leave us with thequestion of why some studies did not show a statisticallysignificant association between RSAs and skewed XCIpatterns.14,15 It is important to note that a heterogeneous groupof aetiologic factors is known to contribute to the developmentof RSAs,2,3 and that small sample size15 may influence theoutcome of statistical analyses.We plan to direct our future efforts towards the ascertain-ment of extended pedigrees to assess the parental origin of theinactive X-chromosome. In addition, detailed high-resolutiongenomic analysis by newer molecular techniques, such ascomparative genomic hybridisation microarray analysis,21could prove to be very valuable at least in a subgroup of patientswho may harbour X-chromosomal submicroscopic deletions.AcknowledgementsWe thank Iclal Buyukdevrim-Ozcelik for critical reading ofthe manuscript. Supported by grants from the Scientificand Technical Research Foundation of Turkey TÜBITAK–SBAG 3334, International Centre for Genetic Engineeringand Biotechnology – ICGEB-CRP/TUR04-01, and BilkentUniversity Research Fund (to Dr Ozcelik).References1 Wilcox AJ, Weinberg CR, O’Connor JF et al. Incidence ofearly loss of pregnancy. N Engl J Med 1988; 319: 189–194.2 Stephenson MD. Frequency of factors associated with habitualabortion in 197 couples. Fertil Steril 1996; 66: 24–29.3 Branch DW. Management of recurrent early pregnancy loss.2001; ACOG Practice Bulletin No. 24. Washington: AmericanCollege of Obstetricians and Gynecologists.4 Pegoraro E, Whitaker J, Mowery-Rushton P, Surti U, LanasaM, Hoffman EP. Familial skewed X inactivation: A moleculartrait associated with high spontaneous-abortion rate maps toXq28. Am J Hum Genet 1997; 61: 160–170.Table 2 Proportion of the cases and controls with skewed X-chromosome inactivationGroupNo. (%) observed with85–89% skewing> 90% skewing TotalRSA patients (n = 62) 2 (3.2) 11 (17.7) 13 (20.9)Control females (n = 124) 6 (4.8) 2 (1.6) 8 (6.4)For comparison by χ2, P = 0.0002. RSA, recurrent spontaneous abortion.Table 3 Characteristics of the cases with extremely skewed X-chromosome inactivationPatient X-inactivationDate of birthPregnancy historySex of children1 (03–358) > 95 1976 G6,P2 F22 (03–384) > 95 1970 G6,P1 F13 (03–697) > 95 1975 G3,P0 –4 (03–759) > 95 1977 G3,P0 –5 (03–811) > 95 1976 G3,P0 –6 (04–009) > 95 1979 G5,P0 –7 (04–010) > 95 1977 G3,P0 –8 (03–405) 90–95 1972 G4,P1 M19 (03–687) 90–95 1978 G3,P0 –10 (03–914) 90–95 1975 G4,P0 –11 (04–011) 90–95 1975 G5,P0 –12 (03–915) 85–89 1968 G5,P1 M113 (03–916) 85–89 1962 G6,P0 –X-inactivation and recurrent spontaneous abortion© 2006 The Authors 387Journal compilation © 2006 The Royal Australian and New Zealand College of Obstetricians and Gynaecologists; 46: 384–3875 Lyon MF. Gene action in the X-chromosome of the mouse(Mus musculus). Nature 1961; 190: 372–373.6 Migeon BR. X chromosome inactivation: theme and variations.Cytogenet Genome Res 2002; 99: 8–16.7 Lyon MF. X-chromosome inactivation and human geneticdisease. Acta Paediatr (Suppl.) 2002; 91: 107–112.8 Brown CJ. Skewed X-chromosome inactivation: Cause orconsequence? J Natl Cancer Inst 1999; 91: 304–305.9 Lanasa MC, Hogge WA, Hoffman EP. The X-chromosomeand recurrent spontaneous abortion: The significance of trans-manifesting carriers. Am J Hum Genet 1999; 64: 934–938.10 Lanasa MC, Hogge WA, Kubick C, Blancato J, Hoffman EP.Highly skewed X-chromosome inactivation is associated withidiopathic recurrent spontaneous abortion. Am J Hum Genet1999; 65: 252–254.11 Shanga KK, Stephenson MD, Brown CJ, Robinson WP.Extremely skewed X-chromosome inactivation is increased inwomen with recurrent spontaneous abortion. Am J Hum Genet1999; 65: 913–917.12 Uehara S, Hashiyada M, Sato K, Sato Y, Fujimori K,Okamura K. Preferential X-chromosome inactivation in womenwith idiopathic recurrent pregnancy loss. Fertil Steril 2001; 76:908–914.13 Beever CL, Stephenson MD, Penaherrera MS et al. SkewedX-chromosome inactivation is associated with trisomy in womenascertained on the basis of recurrent spontaneous abortion orchromosomally abnormal pregnancies. Am J Hum Genet 2003;72: 399–407.14 Sullivan AE, Lewis T, Stephenson M et al. Pregnancy outcomein recurrent miscarriage patients with skewed X chromosomeinactivation. Obstet Gynecol 2003; 101: 1236–1242.15 Kim JW, Park SY, Kim YM, Kim JM, Han JY, Ryu HM.X-chromosome inactivation patterns in Korean women withidiopathic recurrent spontaneous abortion. J Korean Med Sci2004; 19: 258–262.16 Allen RC, Zoghbi HY, Moseley AB, Rosenblatt HM, Belmont JW.Methylation of HpaII and HhaI sites near the polymorphicCAG repeat in the human androgen-receptor gene correlateswith X-chromosome inactivation. Am J Hum Genet 1992; 51:1229–1239.17 Buller RE, Sood AK, Lallas T, Buekers T, Skilling JS.Association between nonrandom X-chromosome inactivationand BRCA1 mutation in germline DNA of patients withovarian cancer. J. Natl Cancer Inst 1999; 91: 339–346.18 Sharp A, Robinson D, Jacobs P. Age- and tissue-specific variationof X-chromosome inactivation ratios in normal women. HumGenet 2000; 107: 343–349.19 Lanasa MC, Hogge WA, Kubik CJ et al. A novel X chromosome-linked genetic cause of recurrent spontaneous abortion. Am JObstet Gynecol 2001; 185: 563–568.20 Plenge RM, Hendrich BD, Schwartz C et al. A promotermutation in the XIST gene in two unrelated families with skewedX-chromosome inactivation. Nat Genet 1997; 17: 353–356.21 Larrabee PB, Johnson KL, Pestova E et al. Microarray analysisof cell-free fetal DNA in amniotic fluid: a prenatal molecularkaryotype. Am J Hum Genet 2004; 75: 485–491.

Extremely skewed X-chromosome inactivation patterns in women with recurrent spontaneous abortion

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Extremely skewed X-chromosome inactivation patterns in women with recurrent spontaneous abortion

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