J7Med Genet 1998;35:539-544

Uniparental and functional X disomy in Turnersyndrome patients with unexplained mentalretardation and X derived marker chromosomes

Tohru Yorifuji, Junko Muroi, Masahiko Kawai, Ayumi Uematsu, Hiroshi Sasaki,Toru Momoi, Masayuki Kaji, Chutaro Yamanaka, Kenshi Furusho

AbstractWe analysed parental origin and X inacti-vation status of X derived marker(mar(X)) or ring X (r(X)) chromosomesin six Turner syndrome patients. Two ofthese patients had mental retardation ofunknown cause in addition to the usualTurner syndrome phenotype. By FISHanalysis, the mar(X)/r(X) chromosomesof all patients retained the X centromereand the XIST locus at Xql3.2. By poly-morphic marker analysis, both patientswith mental retardation were shown tohave uniparental X disomy while theothers had both a maternal and paternalcontribution of X chromosomes. By RT-PCR analysis and the androgen receptorassay, it was shown that in one of thesementally retarded patients, the XIST onthe mar(X) was not transcribed andconsequently the mar(X) was not inacti-vated, leading to functional disomy X. Inthe other patient, the XIST was tran-scribed but the r(X) appeared to be activeby the androgen receptor assay. Ourresults suggest that uniparental disomy Xmay not be uncommon in mentally re-tarded patients with Turner syndrome.Functional disomy X seems to be thecause of mental retardation in thesepatients, although the underlying molecu-lar basis could be diverse. In addition,even without unusual dysmorphic fea-tures, Turner syndrome patients withunexplained mental retardation need tobe investigated for possible mosaicismincluding these mar(X)/r(X) chromo-somes.(JMed Genet 1998;35:539-544)

Keywords: Turner syndrome; uniparental X disomy; Xinactivation

Turner syndrome is one of the most commonchromosomal anomaly syndromes character-ised by a variety of symptoms including shortstature, gonadal dysfunction, cardiac anoma-lies, renal anomalies, and dysmorphic featuressuch as neck webbing, cubitus valgus, higharched palate, and widely spaced nipples. Itaffects 1 in 2500-10 000 live born females' andnearly half of the patients have 45,X karyo-types, while the others have structurally abnor-mal X chromosomes or have mosaickaryotypes.' Except for short stature, which isan almost constant feature, the clinical presen-

tation of Turner syndrome is highly variable.However, mental retardation is usually notincluded as a feature of this syndrome.

It has been reported that Turner syndromepatients with 45,X/46,X,r(X) karyotypes mayhave unusually severe phenotypes, which in-clude mental retardation, soft tissue syndactyly,or abnormal facies.25 In many of these patients,it has been shown that the ringX chromosomeslack theX inactivation centre (XIC) at Xql 3.2.6This leads to functional disomy of the proximalpart of the X chromosome, which may havecontributed to the severe phenotypes observedin these patients. This "loss of XIC functionaldisomy theory" was challenged by the report ofMigeon et al,7 which showed that some of ther(X) chromosomes of patients with a severephenotype retained the XIST locus, which is astrong candidate for XIC itself.8'0 However,even in patients whose r(X) retained XIST, noor very weak expression of these XIST geneswere observed, thus the "functional disomytheory" still seems valid. Migeon et all' alsoreported a mentally retarded, dysmorphicpatient with a 45,X/46,X,del(X)(q21.3-qter)/46,X,r(X) karyotype whose del(X) retained theXIST locus but did not express XIST, whichfurther supported the "functional disomytheory". In this patient it was shown that thenormal X, del(X), and r(X) were all derivedfrom the maternal X chromosome. Althoughthe causal relationship between uniparental dis-omy X and functional disomy X remainsspeculative, the proposed mechanism is that thedel(X) and r(X) originated after the X countingprocess at the morula stage, thereby escapingthe X inactivation process despite the presenceof two X chromosomes in a cell.There is, however, some evidence against the

"functional disomy theory". For example,Dennis et al'2 reported three patients withsevere phenotypes and in two of them the r(X)chromosomes were late replicating while in oneof them, the XIST gene was transcribed. Theyalso pointed out that patients with 45,X/46,X,r(Y) karyotypes had similarly severe phe-notypes and concluded that the mechanismleading to a severe phenotype could be a non-specific effect of chromosomal mosaicismrather than functional disomy. In addition, inthe original report of Kushnick et al,2 only onethird of the r(X) chromosomes were early rep-licating while the others were late replicating.Thus the mechanism leading to mental retar-dation and multiple anomalies has still notbeen completely uncovered.

Department ofPaediatrics, KyotoUniversity Hospital, 54Shogoin Sakyo, Kyoto606, JapanT YorifujiJ MuroiM KawaiA UematsuK Furusho

Kurashild CentralHospital, JapanH Sasaki

Japanese Red CrossSociety WakayamaMedical Centre, JapanT Momoi

Shizuoka Children'sHospital, JapanM Kaji

Tenri Hospital, JapanC Yamanaka

Correspondence to:Dr Yorifuji.

Received 29 August 1997Revised version accepted forpublication31 December 1997

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Table 1 Summary of the results of karyotyping, FISH analysis for the XIST locus, RT-PCR analysis for XIST transcription, and polymorphic markeranalysis of the patients. Alleles are arbitrarily named according to their sizes, and mar(X) /r(X) alleles are shown in bold. Since the telomeric ends of themar(X)/r(X) chromosomes were not always shown by the Giemsa banding, the second allele of the chromosome is shown in parentheses when thepolymorphic marker could be absentfrom the mar(X)/r(X) of the patients

Polymorphic marker

Name DXS1003 ALAS2 ARMental XIST XIST Type (DI) (DI) (TRI)

No retardation Karyotype (mosaic ratio) (FISH) (RT-PCR) Location (XpJ1.3) (Xp11.21) (Xql2)

1 Yes 45,X/46,X,+mar(X) (?pll?ql3.2) (89:11) Pos Neg Proband 4,(4) 3,(3) 1,1Mother 3,4 3,4 1,2Father 4 4 3

2 Yes 45,XJ46,X,r(X) (?p22. 1?q23.3) (88:12) Pos Pos Proband 2,2 1,1 1,1Mother 3,3 2,2 1,1Father 2 1 1

3 No 45,X/46,X,r(X) (p22.1q25) (89:9) Pos Pos Proband 2,2 4,9 2,2Mother 2,2 4,4 1,2

4 No 46,X,+mar(X) (?pll?ql3) Pos Pos Proband 4 5 1,1Mother 3,4 4,5 1,1Father 1 2 1

5 No 45,X/46,X,+mar(X) (?p21?q23) (56:54) Pos Pos Proband 1,3 2,2 1,1Mother 1,2 1,2 1,1

6 No 45,X/46,X,r(X)/46,X,dic r(X)/47,X,r(X),dic r(X) (46:51:2:1) Pos Pos Proband 3,4 1,1 1,2Father 3 1 1

DI, dinucleotide repeat; TRI, trinucleotide repeat; TETRA, tetranucleotide repeat; RFLP, restriction fragment length polymorphism.

In this study, we performed molecular andcytogenetic studies on six Turner syndromepatients with X derived marker or r(X)chromosomes. Two of the patients had unex-plained mental retardation and both haduniparental isodisomy of the X chromosome.The X inactivation status of these patients wasalso analysed.

Materials and methodsSUBJECTSThe study subjects consisted of six JapaneseTurner syndrome patients with at least one cellline with an X derived marker (mar(X)) or ringX chromosome (r(X)). Two of these patients(patients 1 and 2) had unexplained mentalretardation. Another four patients (patients3-6) were Turner syndrome patients withoutmental retardation. The patients regularlyvisited the growth clinic of one of our hospitals.All the patients were recruited after givinginformed consent. The study protocol wasapproved by the ethical review board of theDepartment of Paediatrics, Kyoto UniversityHospital. The chromosomal origins of thesesmall chromosomes were previously identifiedby fluorescent in situ hybridisation (FISH)analysis using an X centromere specific probe(DXZ-1) (data not shown).

KARYOTYPE DETERMINATIONS AND FISH ANALYSIS

Karyotypes on peripheral blood leucocyteswere determined by conventional or high reso-lution trypsin-Giemsa staining. The FISHanalysis using X centromere (DXZ- 1) andXIST specific probes was performed as recom-mended by the supplier of these probes (OncorInc, MD). Part of the karyotyping and theFISH analyses were performed at the SRLLaboratories Inc (Japan).

DNA ISOLATION

Genomic DNA was isolated essentially asdescribed by Miller et al."3

POLYMORPHIC MARKER ANALYSISThe types of polymorphic markers and theirchromosomal locations are listed in table 1.The sequences of the primers for DXS227,

DXS441, DXS453, DXS559, DXS1003,DXS1 162, and HPRT1 were obtained throughthe GDB Genome Data Base at http://gdbwww.gdb.org. The sequences of the prim-ers for ALAS2, AR, and PGK are the same asthose described by Cox et al,'4 Allen et al,'9 andGilliland et al, 16 respectively. One of eachprimer pair was end labelled with 32p, then PCRwas performed in 25 pl reaction mixtures con-taining 50 ng of genomic DNA, 10 mmol/lTris-HCl (pH 8.3), 50 mmol/l KCI, 25 mmol/lMgCl2, 0.1% Triton X- 100, 200 [tmol/l of eachdNTP, 0.5 units of Taq DNA polymerase (Toy-obo, Japan), and 25 pmol of each primer. Thecycling parameters for each locus were thesame as those listed in the above references.For the analysis of di-, tri- and tetranucle-

otide repeat polymorphisms, 3 p1 of the PCRproducts were electrophoresed through 6%denaturing polyacrylamide gels. Then the gelswere exposed to Fuji x ray films (NewRX). Forthe PCR-RFLP analysis of the PGK gene,PCR was performed without 32p primerlabelling. Three microlitres of the PCR prod-ucts were digested with BstXI (New EnglandBiolab, MA), then electrophoresed through a10% polyacrylamide gel. The gel was stainedwith ethidium bromide and observed under aUV light.

RT-PCRMononuclear cells were isolated fromheparinised peripheral blood by Ficoll/Paquegradient centrifugation, then total RNA wasisolated by the method described byChomczynski et al.'7 The total RNA (2 pg) wasreverse transcribed in 50 tl reactions contain-ing 50 mmol/l Tris-HCl (pH 8.3), 75 mmol/lKCl, 3 mmol/l MgCl2, 10 mmol/l DTT, 500[tmol/l each dNTPs, 50 [tg/ml oligo(dT)92,8,and 50 units M-MLV reverse transcriptase(Gibco BRL, MD) at 37°C for one hour. Fourmicrolitre aliquots of the reverse transcriptionmixtures were used to amplify segments of thetranscripts from the XIST gene and thehypoxanthine-guanine phosphoribosyltrans-ferase (HPRT) gene. The reaction buffers werethe same as those for the polymorphic marker

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while the amplifications were in the logarithmicphase. 8

Table 1 continued

DXS453 DXS559 DXS227 DXS1162 DXS441 PGK HPRT1(DI) (DI) (DI) (DI) (DI) (RFLP) (TETRA)(Xql2) (Xql 2) (Xq 13. 1) (Xql 3. 1) (Xql3.2) (Xq 13.3) (Xq26. 1)

1,1 1,1 4,4 3,3 3,3 2,(2) 11,1 1,3 4,5 3,3 3,3 1,2 1,22 2 5 2 3 2 42,2 2,2 5,5 3,3 1,1 1,1 11,2 4,4 4,5 2,3 3,3 1,1 2,22 2 5 3 1 1 11,3 1,1 5,5 1,1 6,6 11,1 1,1 5,5 1,1 6,6 1,11,2 2,2 5,5 2,3 2,3 11,3 2,3 4,5 1,3 3,3 1,22 2 5 2 2 31,1 1,1 1,5 2,5 1,1 21,1 1,1 5,5 5,6 1,2 1,11,1 1,3 1,6 21 1 1 2

analysis. For the amplification of the XISTtranscripts, a pair of primers, one in exon 1 (5'-TGCCCTACTAGCTCCTCGGA-3'), theother in exon 2 (5'-ACACATGCAGCGTGGTATCT-3'), were used to amplify a 88 bpsegment. Cycling parameters were: denatura-tion at 94°C for one minute, annealing, andextension at 60°C for one minute. Similarly, a296 bp segment of the HPRT transcript was

amplified using a pair of primers, one in exon3 (5'-TGAACGTCTTGCTCGAGATG-3'),the other in exon 6 (5'-TCTGCATTGT-T-lTGCCAGTG-3'). Cycling parameters were:

denaturation at 94°C for one minute, anneal-ing, and extension at 58°C for 20 seconds. ThePCR products were electrophoresed through a10% polyacrylamide gel, stained with ethidiumbromide, and observed under UV light. Theamount of transcript was semiquantitated bydensitometric analysis of the RT-PCR products

Figure 1 RT-PCR analysis of the expression of the XISTgene. Results for normalfemale control (F), normal malecontrol (M), and patients 1-4 are shown (molecular weightmarker ¢)(x) 174 DNA digested with HaeIII). For eachlane, RT-PCR products of the XIST gene (88 bp, lowerband) were electrophoresed together with the products of thehypoxanthine guanine phosphoribosyltransferase (HPRT)gene (296 bp, upper band). Numbers beneath each laneshow the relative intensity of the XIST bands as measuredby the densitometric analysis. Numbers in parentheses showthe expected percentage of the XIST genes on mar(X)/r(X)chromosomes. The pictures are overexposed for reproductionpurposes and the semiquantitation was performed at ashorter exposure time.

ANDROGEN RECEPTOR ASSAY

Human androgen receptor assay wasperformed basically as described previously.'8Briefly, genomic DNA was digested either withRsaI alone or with RsaI plus HpaII.Then, 100 ng of the digested DNA was used asthe template for amplification. The sequencesof the primers were 5'-TCCAGAATCTGTTCCAGAGCGTGC-3' (HUMARA- 1)and 5'-GCTGTGAAGGTTGCTGTTCCTCAT-3' (HUMARA-2). One ofthe primer pair(HUMARA-1) was labelled with 32p, then PCRwas performed in the same buffer as forpolymorphic marker analysis. The cycling pa-rameters were as follows: initial denaturation at94°C for five minutes then 30 cycles ofdenaturation at 94°C for one minute, annealing,and extension at 60°C for 1 minute. Threemicrolitre aliquots of the PCR products wereelectrophoresed on a 6% denaturing polyacryla-mide gel and autoradiographed on Fuji x rayfilms. In order to show the presence ofDNA inthe HpaII/RsaI digests, a 204 bp genomicsegment (exon 5 of the TBG gene) at Xq2 1-q22was amplified using a pair of primers, 5'-GCTGCCCATAAGGCT-3' and 5'-CTACGCTTCCGTTGGGTTC-3'. The cycling pa-rameters were the same as those for theandrogen receptor assay. The PCR productswere electrophoresed through a 10% polyacryla-mide gel, stained with ethidium bromide, andobserved under a UW light.

ResultsPATIENT PROFILES

Patient 1 was born after an uneventfulpregnancy and delivery with a birth weight of3210 g. Shortly after birth, muscular hypotoniaand feeding difficulty were noted. Musclebiopsy on day 10 showed normal histology. Herhypotonia improved gradually although herdevelopmental milestones were delayed withhead control at 4 months, sitting at 10 months,and walking alone at 1 year 10 months. Herdevelopmental delay persisted with DQsaround 60-65. At the age of 3, she came to ourclinic because of short stature. At this time,chromosome analysis of peripheral bloodleucocytes showed a 45,X/46,X,+mar (50:50)karyotype. Growth hormone treatment wasstarted at the age of 6. Currently she is 16 yearsof age and still on growth hormone therapy.Her height is 145 cm (mean -2.45 SD) and sheshows no signs of puberty. She has Turner syn-drome characteristics such as increasednumber of naevi, widely spaced nipples, andchronic otitis media. Except for mental retar-dation, she does not have any other featuresunusual for Turner syndrome. Karyotypeanalysis was repeated at the age of 16 andfound to be 45,X/46,X,+mar(X) (89:11).

Patient 2 was born at 38 weeks' gestationwith a birth weight of 2420 g. The pregnancyand delivery were uneventful. At 1 year 3months, she had white spots over her left thighand left abdomen which disappeared graduallyover several years. Her mental development

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was moderately delayed. At the age of 15 years11 months, she came to our clinic because ofshort stature (141.9 cm, mean -3.64 SD),obesity, and mental retardation. Apart fromshort stature, she had cubitus valgus, a low hairline, chronic otitis media, and impaired glucosetolerance. She had breast development at Tan-ner stage 3 and she did not have other dysmor-phic features unusual for Turner syndrome.Chromosome analysis on peripheral blood leu-cocytes showed a 45,X/46,X,r(X) (88:12)karyotype. Although she did not have formalIQ testing, her mental delay was in the educa-ble range.

Patients 3 to 6 were control patients withoutmental retardation. Karyotyping was per-formed at ages 18 (patient 3), 4 (patient 4), 1 1(patient 5), and 14 (patient 6). These patientshad features of Turner syndrome, although,generally, the degree of dysmorphism wasmilder than typical Turner syndrome patients.

Figure 2 Androgenreceptor assay of the Xinactivation status. Resultson patients 1 (A), 2 (B), 3(C), and 4 (D) are shown.M and P are maternal andpaternal DNA, respectively,which are amplified withoutHpaII digestion. Eachpatient's DNA was

amplified with (+) or

without (-) previousHpaII digestion. WithHpaII digestion, an

inactivated allele is

preferentially amplified,while in the absence ofdigestion, both alleles are

equally amplified.

KARYOTYPE AND FISH ANALYSIS

Peripheral blood karyotypes of the patients andthe results of FISH analysis using an XISTspecific probe are summarised in table 1.Where available, estimated breakpoints of themar(X)/r(X) chromosomes are also shown.The mar(X)/r(X) chromosomes of all patientsretained the XIST locus on FISH analysis(data not shown).

PARENTAL ORIGIN OF MARKER CHROMOSOMES

Since marker chromosomes of all patientsretained the X centromere and the XIST locusat Xql3.2, polymorphic markers within thisinterval were used to determine the parentalorigins of the mar(X)/r(X) chromosomes. Inaddition, several other polymorphic markersoutside the region were also examined. In orderto examine if the assays could detect 1 to 10ratio mosaicism, mixtures of two different maleDNAs at 1 to 20 ratios were used as sensitivitycontrols.As shown in table 1, mar(X)/r(X) chromo-

somes were maternal in patients 1, 5, and 6 andpaternal in patients 2, 3, and 4. Interestingly,patient 1 had only a maternal contribution ofXchromosomes, while patient 2 had only a

paternal contribution, showing that patient 1had maternal isodisomy X and patient 2 hadpaternal isodisomy X. Paternity of patient 1was confirmed by using three other highlypolymorphic markers on autosomes (D7S440,D15S11, and APOB, data not shown). Allother patients had both a maternal andpaternal contribution ofX chromosomes. Sen-sitivity test PCR showed that the allele of a

male DNA could be detected in the presence ofa 20-fold larger amount of a second male DNAexcept when the minor allele was two base pairslonger in dinucleotide repeat polymorphisms,in which case the second fainter band was oftenobscured in the ghost band of the major band(data not shown).

XIST EXPRESSION

The expression of the XIST gene was analysedby RT-PCR analysis of peripheral blood leuco-cytes. RT-PCR for the HPRT transcripts was

used as an internal control. The results aresummarised in table 1 and fig 1 shows therepresentative results. Although HPRT expres-sion was similar in all subjects, XIST expres-sion was not observed in patient 1 indicatingthat the mar(X) of patient 1 is not expressingXIST. As expected, XIST expression was notobserved in a male control. For other patients,when the RT-PCR products for XIST weresemiquantitated by the densitometric analysis,the intensity of the bands correlated well withthe expected percentage of XIST genes onmar(X)/r(X) chromosomes. Interestingly, theintensity of bands from patients 2 and 3 wassimilar. Since both of these patients had a simi-lar ratio of mosaicism (table 1), these resultssuggest that the expression of the XIST geneon the r(X) of patient 2 is comparable to that ofpatient 3.

X INACTIVATION STATUS OF THE MARKER/RINGCHROMOSOMESWe then analysed the X inactivation status ofthe mar(X)/r(X) chromosomes. Since replica-tion is sometimes hard to interpret for a verysmall marker chromosome, we used the andro-gen receptor assay originally described by Allenet al.'5 This assay is based on the fact that themethylation status around the polymorphicCAG repeat in the androgen receptor gene(Xq 12) correlates well with the inactivationstatus of the chromosome. When the Xchromosome is inactive, the HpaII sites adja-cent to the CAG repeat are methylated. There-fore, when the genomic DNA is digested withmethylation sensitive HpaII before amplifica-tion of a DNA segment spanning the HpaIIsites and the CAG repeat, only the allele fromthe inactive X chromosomes is amplified. Bythis assay, when undigested male DNA wasmixed with a 40-fold excess of HpaII digestedmale DNA, the allele of the undigested DNAcould be easily amplified, showing that themethod could detect very low level mosaicism(data not shown).As shown in fig 2A, the mar(X) of patient 1

was not inactivated as expected from the resultsof RT-PCR analysis. Interestingly, when theDNA of patient 2 was digested with HpaII,amplification was very poor (fig 2B). This sug-gests that the r(X) in patient 2 was almostentirely active despite the fact that the XISTgene on r(X) appeared to be expressed by theRT-PCR analysis. For both of these patients, anunrelated genomic segment (exon 5 of theTBG gene) could be easily amplified fromHpaII/RsaI digested DNA showing the pres-ence ofDNA in the digested samples (data notshown). All other patients with normal intelli-gence showed obvious bands derived frominactive mar(X)/r(X) chromosomes (fig 2C,D).

DiscussionIn our series of six patients with mar(X)/r(X)chromosomes, two had unexplained mentalretardation and both of these had uniparentalisodisomy X, while none of the patientswithout mental retardation had uniparentaldisomy X. Although the number of patients

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was small, these results suggest that uniparen-tal origin of these small X chromosomes is nota rare phenomenon. Our cases are similar tothe case described by Migeon et allwith a 45,X/46,X,del(X)(q21.3-qter)/46,X,r(X)karyotype in whom del(X) and r(X) werederived from maternal uniparental disomy.However, unlike their case, our patientspresented only with moderate mental retarda-tion in addition to the usual Turner syndromephenotype and did not have unusual dysmor-phic features. The degree of mental retardationalso appeared less severe in our patients. Thereason for the difference in clinical presenta-tion is unclear although the difference in thelength of X chromosomes which are function-ally disomic or the difference in the ratio ofmosaicism in various organs could be possibleexplanations. Although it is not clear at presentwhether uniparental disomy X is specific formentally retarded patients, our results showedthat this type of chromosomal abnormalityshould be taken into account even in Turnersyndrome patients with only mild mentalretardation. In this regard, it is interesting thatin patient 1 aged 3, the mar(X) constituted50% in the peripheral blood leucocytes,whereas at the age of 16 the percentage fell to11%. This suggests that at an older age thechromosomal mosaicism could be undetect-able by the usual karyotyping of peripheralblood. Turner syndrome patients with somedegree of mental retardation probably requirekaryotyping of a large number of peripheralblood leucocytes to detect mar(X)/r(X) chro-mosomes and, when detectable, parental originand X inactivation status of these chromo-somes need to be analysed.As a mechanism leading to a severe pheno-

type in patients with mar(X)/r(X), "loss ofXICfunctional disomy theory" has been acceptedsince the initial description of the patients.However, our results have indicated that thesituation is far more complicated.The mar(X) in patient 1 retained the XIST

locus, but did not express XIST transcripts asshown by the RT-PCR experiment. It was alsoshown that this patient had maternal uniparen-tal isodisomy X. The mechanism leading toshutdown of the expression of XIST on themar(X) remains unclear. One possible mech-anism was proposed by Migeon et al,' that is,the mar(X) originated after the X chromosomecounting at the morula stage. Anotherpossibility is that XIST expression requiresadditional factors, which could be an unknownsequence in the XIC region or factors suppliedby other chromosomes. Given the differencebetween patients 1 and 2, these additional fac-tors could be paternal in origin. It is wellknown that the paternal X chromosome ispreferentially inactivated in marsupials and inextraembryonic tissues of mice.9 20 Similarphenomena have been reported in humans.2 Inaddition, a recent report by Skuse et af2showed that, in humans, maternal and paternalX chromosomes are imprinted differently.The transcription of XIST is known to be

necessary for the cis X inactivation process tostart normally.8 However, whether the expres-

sion of XIST is sufficient for the initiation ofthe X inactivation is still unknown. In ourpatient 2, the clinical phenotype was similar tothat of patient 1, although the patient hadpaternal isodisomy. Like patient 1, her r(X)retained the XIST locus, but, unlike patient 1,this XIST appeared to be transcribed at a simi-lar level to the normally inactivated X chromo-somes. However, by the androgen receptorassay, the methylation status of the r(X) issimilar to that of the active X chromosome.The reason for this apparent discrepancybetween the expression of the XIST gene andthe X inactivation status of r(X) is currentlyunknown. It could be that the expression ofXIST is necessary for an X chromosome to beinactivated, but not sufficient. There could beother factors which cooperate with XIST to cisinactivate the X chromosome. Anotherpossibility is that there are mixed populationsof r(X) in patient 2 although they all appearedthe same on cytogenetic examination. It is pos-sible that a larger population of r(X) is notexpressing XIST and is not inactivated whileother populations of r(X) are expressing XISTand are inactivated. Detailed examination ofeach of the individual r(X) might answer thisquestion.

Overall, our results support the idea thatfunctional X disomy is a cause of mental retar-dation in Turner syndrome patients. However,the mechanism leading to functional disomy isnot as simple as the loss of the XIST locus. Itappears that there are multiple pathways lead-ing to functional X disomy. Furthermore, howit affects normal mental development remainscompletely unknown. Detailed structural andfunctional examination of mar(X)/r(X) chro-mosomes on a number of patients forkaryotype-phenotype correlation might answersome of these questions.

We thank our patients and their families who kindly participatedin this research.

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3 Grompe M, Rao N, Elder FFB, Caskey CT, Greenberg F.45,X/46,X,r(X) can have a distinct phenotype differentfrom Ullrich-Turner syndrome. Am Jf Med Genet 1992;42:39-43.

4 Van Dyke DL, Wiktor A, Palmer CG, et al. Ullrich-Turnersyndrome with a small ring X chromosome and presence ofmental retardation. Am J Med Genet 1992;43:996-1005.

5 Lindgren V, Chen CP, Bryke CR, Lichter P, Page DC,Yang-Feng TL. Cytogenetic and molecular characteriza-tion of marker chromosomes in patients with mosaic 45,Xkaryotypes. Hum Genet 1992,88:393-8.

6 Wolff D, Brown CJ, Schwartz S, Duncan AMV, Surti U,Willard HF. Small marker X chromosomes lack the X inac-tivation center: implications for karyotype/phenotype cor-relations. Am 7 Hum Genet 1994;55:87-95.

7 Migeon B, Luo S, Stasiowski BA, et al. Deficient transcrip-tion of XIST from tiny ring X chromosomes in femaleswith severe phenotypes. Proc Natl Acad Sci USA 1993;90:12025-9.

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10 Lee J, Jaenisch R. Long-range cis effects of ectopicX-inactivation centres on a mouse autosome. Nature 1997;386:275-9.

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