4J Med Genet 1992; 29: 400-406

A complex rearrangement associated with sex

reversal and the Wolf-Hirschhorn syndrome: a

cytogenetic and molecular study

Katrina Coles, Moira Mackenzie, John Crolla, John Harvey, Julia Starr, FrancesHoward, Patricia Jacobs

AbstractWe report a male infant referred withmultiple congenital abnormalities con-

sistent with the Wolf-Hirschhorn syn-

drome. Cytogenetic analysis showed a

chromosome complement of 46,XX witha deletion of 4pl5.2-÷4pter and its re-

placement by material of unknownorigin. The patient was positive for a

number of Yp probes including SRY, thetestis determining factor, and in situhybridisation localised the Yp materialto the tip of the short arm of one Xchromosome. Using pDP230, a probe forthe pseudoautosomal region, and M27p,which recognises a locus in proximal Xp,the material translocated on to 4p was

identified as originating from the shortarm of the paternal X chromosome.The most reasonable explanation for

this complex rearrangement is two sep-

arate exchange events involving bothchromatids of Xp during paternal meio-sis. An aberrant X-Y interchange gave

rise to the sex reversal and an X;4 trans-location resulted in additional, appar-

ently active Xp material and a deletion of4p which produced the Wolf-Hirschhornphenotype.

Wessex RegionalGenetics Laboratory,Salisbury DistrictHospital, Odstock,Salisbury SP2 8BJ.K ColesM MackenzieJ CrollaJ HarveyJ StarrP Jacobs

Frimley ParkHospital, PortsmouthRoad, Frimley,Camberley, Surrey.F Howard

Correspondence toDr Jacobs.Received 4 October 1991.Revised version accepted8 November 1991.

The Wolf-Hirschhorn syndrome is a rare

chromosome disorder associated with a partialddletion of the short arm of chromosome 4.It is now a well defined clinical entity withcharacteristic facial and other anomaliesaccompanied by profound growth and de-velopmental delay. In spite of severe malfor-mations patients may survive well beyondinfancy.'

Lurie et al2 suggested that about 13% ofcases of the Wolf-Hirschhorn syndrome resultfrom translocations, with inheritance in famil-ial cases equally likely to be from the mother or

father. Recent reports suggest that in cases

resulting from de novo deletions or rearrange-ments of 4p the abnormality usually involvesthe paternal chromosome.3The incidence of phenotypic males with a

46,XX karyotype is approximately 1/20 000males.4 Most cases are the result of an aberrantX-Y interchange during paternal meiosis,resulting in the transfer ofYp material, includ-ing the testis determining gene SRY, to thedistal short arm of the X chromosome, as

shown by Southern blot analysis followed byin situ hybridisation."'

Mattei et al8 estimated the incidence ofX;autosome translocations as 1 to 3 per 10 000live births with approximately one-third ofcases being unbalanced. Over half the pub-lished reports of unbalanced X;autosometranslocations have a normal X chromosomereplaced by the derived X. Of the remainder,most are the result of adjacent II or 3:1segregation, in approximately equal numbers.It is unusual to find the autosomal derivativewith two normal sex chromosomes, resultingfrom adjacent I segregation. Mattei et a18 citedthree cases out of a total of 44 unbalancedX;autosome translocations. In each case themother carried the balanced translocation,which allowed recognition of the derived chro-mosome. However, it is not uncommon to findpatients carrying a rearranged autosome wherethe origin of the translocated material cannotbe identified. Such abnormal chromosomesmay contain X chromosome material makingthe recognition of de novo unbalanced X;auto-some translocations, carrying the autosomalderivative in place of a normal autosome, diffi-cult. Thus, such patients may be more com-mon in the population than suggested by thedata of Mattei et al.8This report describes such a patient, with an

unbalanced X;4 translocation giving rise to theWolf-Hirschhorn syndrome, and, in addition,sex reversal resulting from an aberrant X-Yinterchange.

Case reportThe patient was born at 41 weeks to a 21 yearold mother who already had two normal sons;the father was aged 24. Birth weight was2400 g, head circumference 30 2 cm, andlength 44 cm. The placenta weighed 550 g.At birth he was growth retarded, had micro-

cephaly with frontal bossing, haemangioma ofthe forehead, and apparent left proptosis as theright eye was micropthalmic with an iris and achoroidal coloboma; the left eye had a verysmall choroidal coloboma. He also had abroad, beaked nose, blocked nasolacrimalducts, a very short philtrum, everted upper lip,downturned corners of the mouth, a smallmandible, and a cleft palate. The left ear wasfloppy and misshapen and the right ear poster-iorly rotated, the long narrow chest had nip-ples at different heights, and he had finger-likethumbs. Genitalia were obviously male, but hehad a short penis with a hypospadias and asmall scrotum, though gonads were palpable.He also had a sacral sinus, umbilical hernia,

400

A complex rearrangement associated with sex reversal and the Wolf-Hirschhorn syndrome

and low anal tone. Kidney and brain ultra-sound were normal as were ECG and theheart. A third fontanelle was palpable at birth.Although the patient was initially referred

with ?CHARGE syndrome, the phenotypewas later agreed to be consistent with a clinicaldiagnosis of Wolf-Hirschhorn (4p -) syn-drome.At 7 weeks the patient smiled, at 5 months

he laughed and turned his head to his mother'svoice, and by 9 months he could sit well andget both hands to the midline but could notreach or hold. At 17 months his weight was6 5 kg, head circumference 42 cm, and length70 cm, all three well below the 3rd centile. Hecould roll from back to side and sat well withsupport. He would play with both handstogether but would not transfer, that is, 5month stage of development (fig 1).By 3 years of age he was in the 1 year skills

and size range: head circumference 44 cm,weight 7 78 kg. He was sitting alone, casting,nodding for 'yes' and shaking his head for 'no',but had no words. He developed bilateral glueears and has occasional fits which are wellcontrolled with carbamazepine.

Materials and methodsCYTOGENETICSChromosome studies were performed on peri-pheral blood lymphocytes. Three day cultureswere released from an excess thymidine blockwith deoxycytidine enriched medium and har-vested by standard methods to produce pro-metaphase chromosomes which were then Gbanded.9 Replication banding was producedby the incorporation of 30 pg/ml bromode-oxyuridine (BudR) for six hours before har-vesting. Metaphases were stained with 0-1%acridine orange in phosphate buffer andviewed under epifluorescence. One hundredcells were scored to establish replication status.

IN SITU HYBRIDISATIONChromosome preparations were made fromperipheral blood lymphocytes, skin fibro-blasts, and lymphoblastoid cell lines, usingstandard cell culture techniques. BudR wasadded before harvesting in order to achievelate replication banding. Metaphase spreadswere hybridised in situ according to a modifi-cation of the method of Buckle and Craig.10Probes were labelled with tritiated dATP anddCTP to counts of 1 2 x 108dpm/4g DNA(probe p75/79) and 3-6 x 107 dpm/4g DNA(probe pDP230). After exposure for two tofour weeks the chromosomes were stained inHoechst 33258, exposed to UV for one hour,and stained with Giemsa to give replication Rbanding. The probes used were p75/79 (locusDXYS25),6 which maps to Yp, proximal toSRY the putative TDF, and pDP230 (locusDXYS20)," a pseudoautosomal probe recog-nising sequences at the tips of the X and Ychromosomes.

Figure 1 The patient aged 2 years and the externalgenitalia.

MOLECULAR STUDIESDNA was extracted from whole blood by a saltprecipitation technique,'2 digested with appro-priate restriction enzymes, separated by agar-ose gel electrophoresis, and transferred to ny-lon membranes. For the hybridisation DNAprobes were labelled with 32P by randomprimer techniques.'3 Details of the probes usedare given in the table.

Several Y probes were used, spanning thecentromere and short arm. Pseudoautosomaland X specific probes were used to determine

401

Coles, Mackenzie, Crolla, Harvey, Starr, Howard, J3acobs

the parental origin of the X chromosomes.M271 is an X probe recognising a sequencethat is non-methylated on inactive and methy-lated on active X chromosomes; after digestionwith MspI and its methylation sensitive iso-schizomer HpaII, the inactivation status of Xchromosomes can be determined. A number ofprobes for the short arm of chromosome 4were used but only two were found to beinformative and are included in the table.

ResultsCYTOGENETICSG banded chromosome analysis showed thepatient to have a complement of 46,XX withan abnormality of the short arm of one chro-mosome 4. The terminal segment was deleted,with a breakpoint at 4pl5.2, and additional,unidentified material translocated on to thedeleted chromosome (fig 2). Replication band-ing showed that this material was early replic-ating, suggesting that it was active. The

Probes used.

ChromosomeLocus localisation Probe Enzyme Reference

DYZ4 Yp and Yq pDP105 TaqI 14DYZ3 Ycen pDP97 EcoRI 14DXYSI Y Ypll pDP34 MspI 15ZFY and ZFX Ypll.3 and Xp2l.3 pMFI EcoRI 16SRY Ypll.3 pY53.3 HindIII 17DXS278 and DYS136 Xp22.32 and Yqll CR1-S232 TaqI 18DXS255 Xp 11.22 M27,3 EcoRI 19DXYS20 Ypll.3 and Xp22.32 pDP230 TaqI 11D4S115 4pl6.3 p252.3 PstI 20D4511 4pl6.3 p157.9 PstI 20DXYS25 Ypll p75/79 6

2

deleted segment of the chromosome 4 includesthe 'critical region' associated with the Wolf-Hirschhorn syndrome.2122 Parental chromo-somes were normal.

MOLECULARThe patient had an XX sex chromosome com-plement but a male phenotype, so the presenceor absence of Y material had to be determined.Southern blot analysis using Y probes showedthat the patient was negative for D YZ4,D YZ3, and DXYSI Y, loci spanning Yql 1 toproximal Yp1 1, and positive for ZFY andSRY, the putative testis determining gene(fig 3). These results indicated that distalYp material was present somewhere in thepatient's karyotype. The localisation of the Ypsequences was determined by in situ hybrid-isation using the Yp specific probe p75/79.Forty-three metaphases were scored and thedistribution of 145 grains showed that 28(19-3%) were on an X chromosome, with 16located at the terminal region Xp2.2-+Xpter(fig 4). In all metaphases examined, only one ofthe X chromosomes was labelled. The patienttherefore had Yp material translocated to thetip of one X chromosome owing to an aberrantX-Y interchange at paternal meiosis I. Thus,with respect to his sex reversal the patient wasa classic XX male.As the additional material on 4p was not ofY

origin, its identity was investigated. Southernblot analysis using the pseudoautosomal probepDP230 showed the patient to have inheritedall his father's bands but only some of hismother's (fig 5). This suggested that he hadinherited three copies of the pseudoautosomal

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This was formally proved by in situ hy-bridisation using the pseudoautosomal probepDP230. Sixty-four metaphases were scoredand the distribution of 155 grains showed that13 (8 4%) were on Xpter and 11 (7%) were on4pter. The patient's karyotype is therefore46,XX, -4, + der(4)t(X;4)(p22. l;p 15.2).The Southern blot analysis and in situ hy-

bridisation with the pseudoautosomal probeshowed that the patient had two copies of thepaternal pseudoautosomal region and one copyof the maternal pseudoautosomal region, andfurthermore that one copy was translocated onto 4p. However, while the X-Y interchangeleading to the translocation ofY material on toone X chromosome must have happened dur-ing the paternal first meiotic division, we hadno formal proof of the parental origin of thenormal X and the X;4 translocation chromo-some. We therefore used probe M270, whichrecognises sequences on proximal Xp, andshowed that the patient had inherited an Xchromosome from each parent (fig 7A). Thus,the normal X must have been maternal inorigin. Furthermore, as M270 recognises asequence which is differentially methylated on

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xregion, two from his father and one from hismother. If the copies from his father's Y andone of his mother's Xs are on the patient's twoX chromosomes, the copy from his father's Xmust also be present in the karyotype. Thisresult suggested that the unidentified materialon 4p was likely to be of Xp origin. Prometa-phase chromosomes were re-examined and thebanding pattern of the translocated segmentwas entirely consistent with it being the distalsegment of the short arm of an X chromosome,specifically Xp22.1 -.Xpter (fig 6).

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Figure 7 (A) Southernblot of probe M27fl. Thepatient has inherited oneallele from each parent.(B) Southern blot of M27flafter digestion withMspI(M) and HpaII(H).The HpaII digests show theabsence of a band in thefather and the reducedintensity of the bands in themother and patient. Thepatient's two bands are ofequal intensity, indicatingrandom X inactivation.

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active and inactive X chromosomes, we wereable to show that the X inactivation pattern inthe patient's peripheral blood cells was ran-dom (fig 7B). As the normal X was maternaland the abnormal X paternal in origin the X;4translocation chromosome must also be pater-nal in origin. This was formally demonstratedby testing the patient and his parents with 12probes for loci on 4p16. Only two probes wereinformative and both showed that the patienthad inherited one allele from his mother, butno allele from his father. This established thatthe derived chromosome 4 was indeed paternalin origin (fig 8).

Southern blot analysis using the probeCRI-S232, which recognises a sequence justproximal to the pseudoautosomal boundary,showed the patient to have only two copies ofthe locus, one paternal and one maternal. Thisdefines the X-Y interchange as being proximalto DXS278, and therefore also the STS locuson the X (fig 9).Our explanation for the observations in this

patient is that two interchange events occurredduring paternal meiosis. The first, involvingan X and a Y chromatid must have taken placeat meiosis I and gave rise to the sex reversal.The second, involving a chromosome 4 and theotherX chromatid, occurred at either meiosis Ior prophase of meiosis II and gave the addi-tional, apparently active Xp material and a

deletion of distal 4p which produced the Wolf-Hirschhorn phenotype. Therefore, both chro-matids of one X chromosome had beeninvolved in separate exchange events duringpaternal meiosis (fig 10).

DiscussionPrevious reports of unbalanced X;autosometranslocations suggest that where the autoso-mal derivative occurs with two X chromo-somes the translocated X material is usuallyinactivated (in addition to a normal X chromo-some) if it includes the inactivation centre onXq.2325 A similar case to ours described a

female with a derived chromosome 6 carryingXp material, specifically Xp2l1-pter, whichwas apparently early replicating in all cells.26The patient showed minor abnormalities anddelayed psychomotor development, while herheight, weight, and head circumference werebelow the 3rd centile. The malformations werepresumed to result from the deletion of chro-mosome 6 (q26-*qter).

Nearly all the clinical features shown by our

patient have been previously reported inassociation with the Wolf-Hirschhorn syn-drome.2127 Only his finger-like thumbs and

nipples at different heights have not been spe-cifically associated with the syndrome, butother finger, thumb, and nipple abnormalitieshave been described.28

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Figure 10 A diagrammatic representation of the two interchange events (paternal meiosis I and their outcome. The X;4 exchange could alternativeoccurred during prophase of meiosis II.

The extra, active Xp material m:phenotypic effect on the patient, ormalities it does bestow fall withmodified by, the spectrum of Wohorn associated features.The only published report of a n

duplication of Xp22 describes a 16with normal male external genitaliamental delay, and multiple minorincluding deformed ears.29 Like oiand that of Hagemeijer et a126 descrilhis weight, height, and head circwere below the 3rd centile, a consisivation with these partial Xp duplicEOther reports of Xp duplication,

plicated by the patients being pherfemale, despite carrying an intact 1some.30-32 Scherer et a130 suggest

duplication of the ZFX gene at Xp21.3 causedthe sex inversion, with two active copies ofZFX overcoming the male determining actionof ZFY. However, Sinclair et al'7 have since

Meiosis shown that SRY is probably the testis deter-mining gene, leaving the roles of ZFX andZFY in sex determination uncertain. Further-more, Schneider-Gadicke et a133 indicated thatZFX escapes X inactivation, in which case, ifthe model of Scherer et a130 were true, ourpatient and all cases of Klinefelter's syndrome,in spite of random X inactivation, should bephenotypically female.The concept of an X linked gene which,

when duplicated to give two active copies,despite the presence of a Y chromosome,

Meiosis II results in a female phenotype, is still valid.Reviewing the published reports of genetic

males with Xp duplications, our case witha duplication of Xp22.1-*p22.32 and thoseof Narahara et a129 and Brondum-Nielsonand Langkjaer34 with duplications of Xp22.1 -+p22.3 and Xpl1.2--p21.2, are phenotypicallymale, while those of Bernstein et al,31 Schereret al,30 and Stern et a132 with duplications ofXp2l- pter, Xp2l.2-+p22.3, Xp2l.2-.p22.2,and Xp2l -+p22.3 are phenotypically female.

Sperm This suggests that such a gene may belocalised to Xp2l.3, the only segment presentin the phenotypic females but not the males.Unlike ZFX, it would be inactive on aninactivated X chromosome and only when pre-sent in two active copies would sex reversalresult.The patient described in this report is an

XX male with the Wolf-Hirschhorn syn-drome. His phenotype is the result of twoseparate exchange events involving both chro-matids of Xp at paternal meiosis, an aberrantX-Y interchange and an X;4 translocation.

ion The two rearrangements involve different Xpbreakpoints and may be linked by a commonclastogenic event or be coincidental. Whateverthe cause, they have resulted in a complicatedsituation which was only resolved by the ap-plication of cytogenetic, in situ hybridisation,and molecular biological techniques.

occurring atwly have

We are very grateful to David Robinson andother staff of the Wessex Regional GeneticsLaboratory for their help with this case.

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Coles, Mackenzie, Crolla, Harvey, Starr, Howard, J'acobs

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