Mother and Daughter With 45,X/46,X,r(X)(p22.3q28)and Mental Retardation: Analysis of theX-Inactivation Patterns

Mari Matsuo,1 Koji Muroya,2 Kenji Nanao,3 Yukihiro Hasegawa,3 Hiroshi Terasaki,4Kenjiro Kosaki,2 and Tsutomu Ogata2*1Department of Pediatrics, Tokyo Women’s Medical University, Tokyo, Japan2Department of Pediatrics, Keio University School of Medicine, Tokyo, Japan3Endocrinology, Metabolism, and Genetics Unit, Tokyo Metropolitan Kiyose Children’s Hospital, Kiyose, Japan4Mitsubishi Kagaku Bio-clinical Laboratories, Inc., Tokyo, Japan

We report on a mother and daughter bothwith a 45,X/46,X,r(X)(p22.3q28) karyotypeand mental retardation. Fluorescence insitu hybridization (FISH) and microsatelliteanalyses for 14 loci/region at Xp22.3 andseven loci/region at Xq28 indicated that thering X chromosome was missing a roughly12-Mb region from Xp22.3 with the break-point between DXS85 and DXS9972, and an-other region of less than 100 kb from Xq28with the breakpoint distal to the region de-fined by the FISH probe c8.2/1. X-inactiva-tion analysis, using the methylation statusof the AR gene (exon 1) as an indicator,showed that the normal and ring X chromo-somes in the X,r(X)(p22.3q28) cell lineagewere randomly inactivated. The Xp22.3 de-leted region partially overlaps with the re-gional intervals of MRX19, MRX21, MRX24,MRX37, MRX43, and MRX49 associated withheterozygote manifestation. Therefore, it islikely that one or more of these MRX genes,subject to X-inactivation, are lost from thering X chromosome, and that reduced ex-pression of the MRX gene(s) caused by ran-dom X-inactivation has resulted in mentalretardation in the mother and daughter.Am. J. Med. Genet. 91:267–272, 2000.© 2000 Wiley-Liss, Inc.

KEY WORDS: ring X chromosome; familialcases; MRX gene; random X-in-activation; reduced expression

INTRODUCTION

A ring X chromosome is often associated with mentalretardation (MR) [Fryns et al., 1990]. In particular, MRis prevalent in patients with small or tiny ring X chro-mosomes [Van Dyke et al., 1991]. Molecular studies insuch patients have indicated that MR is primarilycaused by functional X disomy, resulting from deletionor impaired expression of XIST [Jani et al., 1995]. MRis also manifested by patients with preferentially inac-tivated ring X chromosomes [Collins et al., 1994]. Inthese patients, a gene for X-linked nonspecific MR(MRX) may be mutated on the structurally normal Xchromosome, or there may be a coincidental abnormal-ity.

In this article, we report on a mother and daughterwith a large ring X chromosome and MR, and discussthe mechanism leading to MR in terms of random X-inactivation.

CLINICAL REPORT

This family has been reported previously [Uehara etal., 1997]. In brief, the mother became pregnant at age32 years, and the daughter was delivered at 38 weeksof gestation by cesarean section due to fetal distress. Atbirth, her length was 41 cm (–4.2 SD), weight 1.8 kg(–3.4 SD), and OFC 28.5 cm (–3.5 SD). The 36-year-oldfather was clinically normal, with a height of 162 cm(–1.5 SD).

At age 1 month the daughter was referred to KiyoseChildren’s Hospital because of a cardiac murmur.Physical examination showed a feeble infant withdown-slanting palpebral fissures. Ullrich-Turner signswere absent except for mild webbed neck. Cardiac cath-eterization and aortography demonstrated coarctationof the aorta, and aortic angioplasty was performed withleft subclavian flap at age 3 months. Subsequently, shehad growth failure and developmental retardation. Atage 31⁄2 years her height was 85.2 cm (–2.8 SD), weight10.5 kg (–2.2 SD), and OFC 43.7 cm (–3.0 SD). She washyperactive and expressionless. She spoke severalsingle words but was unable to make two-word sen-

Grant sponsors: Pediatric Research from the Ministry of Healthand Welfare of Japan, Pharmacia and Upjohn Fund for Growthand Development Research.

*Correspondence to: Dr. Tsutomu Ogata, Department of Pedi-atrics, Keio University School of Medicine, 35 Shinanomachi,Shinjuku-ku, Tokyo 160-8582, Japan. E-mail: t-ogata@po.iijnet.or.jp

Received 20 May 1999; Accepted 4 January 2000

American Journal of Medical Genetics 91:267–272 (2000)

© 2000 Wiley-Liss, Inc.

tences or to follow oral instructions. She respondedwell to small sounds coming from behind her. Sphinc-ter control was not acquired. Motor development ap-peared relatively well preserved. At age 4 years, herdevelopmental quotient was estimated as 70, with ver-bal development being more severely affected than mo-tor development.

The mother was 35 years old on the last examina-tion. She measured 135 cm (–4.6 SD) and had regularmenses. Ullrich-Turner somatic signs were apparentlyabsent. She had some difficulty in daily conversation,although she performed housework and reared thedaughter. She had a permissive personality and wasnot hyperactive. More detailed examinations includingintelligence quotient was refused by her husband.

METHODSConventional and Molecular

Cytogenetic Studies

Chromosome preparations were obtained from cul-tured peripheral blood lymphocytes of the daughterand her parents. G-banding analysis was performed on100 metaphases each from the daughter and themother and on 20 metaphases of the father. High-resolution G-banding was also carried out on the twofemales. Fluorescence in situ hybridization (FISH)analysis was performed for 50 lymphocyte metaphasespreads of the daughter, using probes for the Xp/Yptelomeric region (cY29) [Ning et al., 1996], KAL (Oncor,Gaithersburg, MD), DXS85 (782) [Hofker et al., 1985],DXZ1 (Oncor), XIST (Oncor), DXS52 (St14-1) [Alcalayand Toniolo, 1988], DXS253E (P3) [Oberle et al., 1985],and the Xq/Yq telomeric region (c8.2/1) [Ning et al.,1996]. FISH analysis was also carried out for 100 in-terphase nuclei of buccal mucosa cells in the daughter,using the probes for the Xp/Yp telomeric region andDXZ1. Chromosomal location of the examined loci/regions is shown in Table I. The cY29 probe was labeledwith biotin and was detected by avidin conjugated tofluorescein isothiocyanate, and the remaining probeswere labeled with digoxigenin and were detected byrhodamine antidigoxigenin.

Microsatellite Analysis

Genomic DNA was extracted from peripheral bloodleukocytes of the daughter and the parents, and wasamplified by polymerase chain reaction (PCR) for 11loci on Xp22.3 and four loci on Xq28 (Table I). Ampli-fication was performed in a reaction volume of 50 mlcontaining 0.3 mg genomic DNA, 20 pmol fluorescentlylabeled forward primer, 20 pmol unlabeled reverseprimer, 0.2 mM dNTPs, and 2 U Taq polymerase. Theprimer sequences and the PCR conditions have beenreported in Cox et al. [1998] and in Genome Database.The size of the PCR products was determined on anautosequencer (ABI PRISM™ 310) using GeneScan™.

X-Inactivation Analysis

Methylation status of the AR gene (exon 1) was ana-lyzed, according to the method of Allen et al. [1992]. In

brief, 0.3 mg of leukocyte genomic DNA was amplifiedby PCR with a fluorescently labeled forward primerand an unlabeled reverse primer flanking the polymor-phic CAG repeat and two methylation sensitive HpaIIsites before and after HpaII digestion, and the PCRproducts were examined for marker size and area un-der curve on the autosequencer. The primer sequencesand PCR conditions were as previously described[Allen et al., 1992].

RESULTS

The lymphocyte karyotype was 45,X[73]/46,X,r(X)(p22.3q28)[27] for the daughter (Fig. 1), 45,X[97]/46,X,r(X)(p22.3q28)[3] for the mother, and 46,XY forthe father. FISH analysis for lymphocyte metaphasespreads showed that the ring X chromosome was posi-tive for DXZ1, XIST, DXS52, DXS253E, and the Xq/Yqtelomeric region, and negative for the Xp/Yp telomericregion, KAL, and DXS85 (Fig. 2, Table I). Furthermore,FISH analysis for buccal mucosa cells showed thatDXZ1 was present in a single copy in 11 interphasenuclei and in two copies in 89 interphase nuclei, andthat the Xp/Yp telomeric region was present in a singlecopy in all 100 nuclei analyzed. Thus, the karyotype ofbuccal smear cells was interpreted as 45,X[11]/46,X,r(X)(p22.3q28)[89].

Representative results of the microsatellite analysisare shown in Figure 3, and the data are summarized inTable I. In the daughter, two peaks with a differentmagnitude were detected for each of DXS9972,DXS9979, DXS9985, DXS1043, DXS15, and DXYS154and single peaks only were detected for the remainingnine loci. A single peak was detected for all the locistudied in the parents. For the six loci present in twopeaks in the daughter, the large peaks were identical tothe corresponding paternal peaks, and those forDXS9985, DXS15, and DXYS154 were derived from

Fig. 1. The normal X chromosome (left) and the ring X chromosome(right) of the daughter. Breakage and reunion have occurred at Xp22.3 andXq28 (arrow).

268 Matsuo et al.

the father; by contrast, the small peaks for DXS9972,DXS9979, DXS9985, DXS1043, and DXS15 were dif-ferent in the product size from the corresponding ma-ternal peaks, although the small peak for DXYS154was equal in the product size to the maternal peak forthat locus. For the remaining nine loci present in asingle peak in the daughter, all the peaks were identi-

cal in the product size to the corresponding paternalpeaks, and there was no peak common to the daughterand the mother for DXS10008, DXS10000, DXS10001,DXS9977, DXS9988, and DXS9992.

The results of the X-inactivation analysis are shownin Figure 4. Before HpaII digestion, PCR amplificationyielded 279 bp and 285 bp peaks in the daughter, a 285

Fig. 2. FISH analysis of thedaughter. A: Metaphase spreads hy-bridized with the probe 782 (DXS85).DXS85 is present on the normal Xchromosome (arrow) and absent fromthe ring X chromosome (arrowhead).B: Metaphase spreads hybridizedwith the probe c8.2/1 defining a re-gion ∼100 kb from the Xq telomere.The region is preserved on both thenormal X and the ring X chromo-somes (arrows).

TABLE I. Presence or Absence of Examined Loci/Region on theRing X Chromosome†

Locus MethodChromosomal

location

Results

ResultsFather Daughter Mother

cY29 FISH Xp22.3 (PAR1) NE Negative NE AbsentKAL FISH Xp22.3 NE Negative NE AbsentDXS85 FISH Xp22.3 NE Negative NE AbsentDXS10008 MS Xp22.3 167 167 171 UncertainDXS10000 MS Xp22.3 151 151 149 UncertainDXS10001 MS Xp22.3 163 163 159 UncertainDXS9977 MS Xp22.3 189 189 191 UncertainDXS9988 MS Xp22.3 207 207 205 UncertainDXS9992 MS Xp22.3 116 116 118 UncertainDXS9975 MS Xp22.3 134 134 134 UncertainDXS9972 MS Xp22.3 234 234, 242* 234 PresentDXS9979 MS Xp22.3 316 316, 318* 316 PresentDXS9985 MS Xp22.3 162 162, 168* 170 PresentDXS1043 MS Xp22.3 149 149, 151* 149 Present

DXZ1 FISH Centromere NE Positive NE PresentXIST FISH Xq13 NE Positive NE Present

DXS52 FISH Xq28 NE Positive NE PresentDXS15 MS Xq28 156 136*, 156 154 PresentDXS253E FISH Xq28 NE Positive NE PresentDXYS154 MS Xq28 (PAR2) 242 234*, 242 234 Presentc8.2/1 FISH Xq28 (PAR2) NE Positive NE PresentDXYS225 MS Xq28 (PAR2) 213 213 213 UncertainDXYS227 MS Xq28 (PAR2) 127 127 127 Uncertain

†FISH, fluorescence in situ hybridization; MS, microsatellite; NE, not examined; PAR1, theshort arm pseudoautosomal region and PAR2: the long arm pseudoautosomal region. TheArabic numbers represent the sizes of the PCR products in bp; the asterisks (*) indicate smallpeaks. The locus order is based on the reports of Nelson et al. [1995], D’Esposito et al. [1997],and Cox et al. [1998].

45,X/46,X,r(X) and MR 269

bp peak in the father, and a 288 bp peak in the mother.After HpaII digestion, PCR amplification still producedtwo peaks in the daughter with a ratio of area undercurve being 45%:55%, and gave no discernible peak inthe parents.

DISCUSSION

The mother and daughter shared a large ring X chro-mosome. Although microsatellite analysis failed toshow peaks common to the daughter and the mother, itappears that loci on the ring X chromosome were de-tectable as the small peaks in the daughter with thering X chromosome in 27% of lymphocytes, but wereundetectable in the mother with the ring X chromo-some only in 3% of lymphocytes. As for maternal fer-tility, it should be pointed out that the mosaic cell ratioin lymphocytes does not reflect that in oocytes. Sincelymphocytes repeatedly undergo mitosis, 45,X cells arecontinuously produced due to mitotic instability of thering X chromosome and, therefore, increase with age.By contrast, most of her oocytes could have a46,X,r(X)(p22.3q28) rather than 45,X karyotype, sincefemale germ cells cease mitosis and enter meiosis dur-ing fetal life. This would explain why the mother hadnormal menses and was fertile.

The mother and daughter were both mentally re-tarded, with impaired verbal development uncommonto Ullrich-Turner syndrome [Rovet, 1993]. In this con-text, the results of X-inactivation analysis are notewor-thy. The presence of two peaks after HpaII digestion inthe daughter suggests that the normal and ring X chro-mosomes in the X,r(X)(p22.3q28) cell lineage were ran-domly inactivated, since PCR products are not ex-pected from HpaII-digested 45,X cells (the apparentlack of peaks derived from the ring X chromosome inthe mother would be due to the scantiness of cells withthe ring X chromosome). Although variable methyl-ation at the HpaII sites or incomplete HpaII digestion

might be possible, the results of the parents imply thatPCR products are not obtained from cells with a singleX chromosome. Thus, although MR might be a coinci-dental feature in the mother and the daughter, it ap-pears that the ring X chromosome is missing an MRXgene(s) subject to X-inactivation, and that reduced ex-pression of the MRX gene(s) caused by random X-inactivation results in the development of MR. Thisnotion postulates that a sizable portion of brain cellshave the ring X chromosome. This is probable, becausebrain cells almost complete mitotic division during fe-tal life. In support of this, the ring X chromosome of thedaughter was much more frequent in slow-dividingbuccal mucosa cells than in fast-dividing lymphocytes.By contrast, loss of a gene escaping X-inactivationwould not cause MR. If such a gene is deleted, it shouldbe present in a single active copy irrespective of theX-inactivation pattern, as in patients with Ullrich-Turner syndrome.

The Xp22.3 deletion may be relevant to the develop-ment of MR. Since the Xp22.3 breakpoint was deter-mined between DXS85 and DXS9972 (Table I), the ringX chromosome is missing a roughly 12-Mb region fromXp22.3 [Nelson et al., 1995] (although there was nodiscernible peak common to the daughter and themother for the six loci from DXS10008 to DXS9992, theresults would not be informative: peaks derived fromthe ring X chromosome, if they are present, would notbe recognized in the mother, so that it is uncertainwhether the daughter is homozygous or hemizygous forthe six loci). The deleted region partially overlaps withthe regional intervals of six MRX loci defined by link-age analysis (MRX19 [Donnelly et al., 1994], MRX21[Kozak et al., 1993], MRX24 [Martinez et al., 1995],MRX37 [Bar-David et al., 1996], MRX43 [cited in Lubset al., 1999], and MRX49 [Claes et al., 1997]). Mild MRin an obligate carrier female(s) in each family is con-sistent with the MRX gene(s) being subject to X-inactivation. Thus, if the MRX gene(s) is deleted fromthe ring X chromosome, this could cause MR underrandom X-inactivation. In this case, the MRX gene(s) ismapped distal to DXS9972 on the basis of the presentstudy. Furthermore, the MRX gene(s) should residedistal to the gene for microphthalmia with linear skindefects syndrome (MLS) located between DXS9988 andDXS1043, because the daughter was free from MLSfeatures under random X-inactivation [Ogata et al.,1998]. Although another MRX gene has been assignedbetween DXS1060 and DXS1139 in the terminal partof the X-differential region at Xp22.3, the MRX genehas been suggested to escape X-inactivation [Muroya etal., 1996].

The Xq28 deletion might also be involved in the de-velopment of MR. Since the ring X chromosome pre-served the region defined by c8.2/1 (Table I), the ring Xchromosome is missing at most 100 kb DNA sequencesin the second pseudoautosomal region [D’Esposito etal., 1996]. However, two genes, SYBL1 and IL9R, havebeen located in the 100-kb region. Although IL9R re-siding distal to DXYS227 escapes X-inactivation[D’Esposito et al., 1997], SYBL1 containing DXYS225between its exons 5 and 6 has been demonstrated toundergo X-inactivation and implicated in nervous sys-

Fig. 3. Misrosatellite analysis for DXS9972. A large 234 bp and a small242 bp peaks are detected in the daughter, whereas a 234 bp peak only isdetected in the parents. Since the ring X chromosome is present in 27% oflymphocytes in the daughter and only in 3% of lymphocytes in the mother,it is likely that DXS9972 on the ring X chromosome is detected as the small242-bp peak in the daughter but is undetected in the mother.

270 Matsuo et al.

tem function [D’Esposito et al., 1996]. Thus, if SYBL1is impaired, this could affect mental development.

To our knowledge, an inherited ring X chromosomehas been reported in three families, including this fam-ily (Table II). The findings are summarized as follows.First, all females are mosaic with a 45,X cell lineage,which is more frequent in rapidly dividing lymphocytesthan in slowly dividing mucocutaneous cells, and morefrequent in mothers than in daughters. This is consis-tent with mitotic instability of a ring X chromosome.Second, MR is present in females with a large ring Xchromosome which has been shown to undergo randomX-inactivation but is absent in females with relativelysmall ring X chromosomes which are confirmed orprobable to undergo nonrandom X-inactivation. This iscompatible with the hypothesis that MR results fromfunctional nullisomy for an MRX gene(s) in 46,X,r(X)cells with an inactive normal X chromosome. Third, thesize of ring X chromosomes appears to be related to thelength of reproductive years. This is consistent withthe notion that the degree of gonadal dysfunction in sex

chromosome aberrations is inexplicable by the genedosage effect but is closely correlated with the extent ofmeiotic pairing failure (the size of unpaired region)[Ogata and Matsuo, 1995].

In summary, we observed a mother and daughterwith 45,X/46,X,r(X)(p22.3q28) and MR. Although thegenetic mechanism for MR remains to be determined,it is likely that MR is ascribed to reduced expression ofan MRX gene(s) caused by random X-inactivation.

ACKNOWLEDGMENTS

We thank Dr. Y. Fukushima for the cY29 and c8.2/1probes, Drs. M. Osawa, N. Mastuo, and M. Anzo forcritical comments, and Mr. Takashi Ishii for technicalassistance.

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Fig. 4. X-inactivation analysis for the methylation status of the AR gene (exon 1). Before HpaII digestion, a large 285-bp and a small 279-bp peak aredetected in the daughter with 45,X[73]/46,X,r(X)(p22.3q28)[27], a 285-bp peak in the father with 46,XY, and a 288-bp peak in the mother with 45,X[97]/46,X,r(X)(p22.3q28)[3]. After HpaII digestion, two small peaks are still detected in the daughter, with the ratio of area under curve being 45%:55%,whereas no peak is recognizable in the parents. The comparison of area under curve between the 285-bp, 282-bp, and 279-bp peaks in the daughterindicates that the 282-bp peak is a by-product caused by slippage for the 285-bp peak, whereas the 279-bp peak is a true product, because the 279-bp peakis similar in magnitude to the 282-bp peak before HpaII digestion and larger than the 282-bp peak after HpaII digestion. The results are consistent withthe 285-bp peak being derived from the normal X chromosome of paternal origin. Although the 279-bp peak is undetected in the mother, this would bedue to the ring X chromosome being present in only 3% of her lymphocytes.

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Dallapicola et al.[1980]

45,X[23]/46,X,r(X)(p13q27)[19] Skin fibroblastsI-Daughter 45,X[306]/46,X,r(X)(p13q27)[160] Lymphocytes Non-random* No . . . Menarche at 12 years

old45,X[34]/46,X,r(X)(p13q27)[28] Skin fibroblasts

II-Mother 45,X/46,X,r(X)(p22q24) Not described Not examined No 24 years Irregular menses at27 years old

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II-Daughter 45,X/46,X,r(X)(p22q24) Not described Not examined No . . . Aged 15 monthsIII-Mother 45,X[97]/46,X,r(X)(p22.3q28)[3] Lymphocytes Unknown# Yes 32 years Regular menses at 35

years oldThis report

III-Daughter 45,X[73]/46,X,r(X)(p22.3q28)[27] Lymphocytes Random# Yes . . . Aged 4 years45,X[11]/46,X,r(X)(p22.3q28)[89] Buccal smear

cells

†MR, mental retardation; NE, not examined.*The ring X chromosome is late-replicating in all the 285 lymphocytes of the mother and 250 lymphocytes of the daughter.#See the text for details.

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