JMed Genet 1996;33:767-771

A duplication of distal Xp associated withhypogonadotrophic hypogonadism, hypoplasticexternal genitalia, mental retardation, andmultiple congenital abnormalities

Louise Telvi, Alexandra Ion, Jean-Claude Carel, Isabelle Desguerre, Monique Piraud,Anne Marie Boutin, Josue Feingold, Gerard Ponsot, Marc Fellous, Ken McElreavey

Laboratoire deCytogenetique, HopitalSaint Vincent de Paul,82 avenue DenfertRochereau, 75674 ParisCedex 14, FranceL TelviA Ion

Serviced'Endocrinologie,HBpital Saint Vincentde Paul, Paris, FranceJ-C Carel

Service deNeuropediatrie,HBpital Saint Vincentde Paul, Paris, FranceI DesguerreG Ponsot

Service de Biochimie,HBpital Brousse, Lyon,FranceM Piraud

Service deNeuropediatrie,Hopital La RocheGuyon, La RocheGuyon, FranceA M Boutin

INSERM U155, United'EpidemiologieGenetique, Paris,FranceJ Feingold

ImmunogenetiqueHumaine, InstitutPasteur, Paris, FranceM FellousK McElreavey

Correspondence to:Dr Telvi.

Received 7 February 1996Revised version accepted forpublication 23 April 1996

AbstractAn unusual familial case ofthree sibs witha partial duplication of distal Xp se-quences is described. The proband, an 18year old boy, showed mental retardation,severe dysmorphic features, hypogonado-trophic hypogonadism (HHG), and hypo-plastic external genitalia. His karyotypewas 46,Y,inv dup(X)(p22.1-->p22.32). Theproband has two sisters each with thesame inv dup(Xp) chromosome. Both sis-ters presented with short stature but wereotherwise phenotypically normal. The ab-normal X chromosome was inactive in themajority of cells examined. Southern blotdosage analysis indicated a duplication ofdistal Xp sequences. The proximal break-point is located between DXS28 andDXS41, and is therefore at least 2 Mb dis-tal to the DSS locus. The relationshipbetween the phenotype and the Xp dupli-cation is discussed.(7 Med Genet 1996;33:767-771)

Key words: hypogonadotrophic hypogonadism; partialXp disomy; mental retardation.

Duplication of distal Xp sequences is a rarechromosomal rearrangement. In XY subjects,Xp duplications are usually associated withmental retardation, multiple severe congenitalabnormalities, and in some cases 46,XYgonadal dysgenesis, which can lead to femalegender assignment.1-910 This suggests that twoactive copies of an Xp gene in some way inhibitnormal testicular determination. An analysisby Bardoni et al" of 46,XY phenotypic femalescarrying microscopic and submicroscopic Xpduplications defined a region of 160 kb atXp21.3 that is associated with male to femalesex reversal. This locus, termed DSS (dosagesensitive sex reversal), maps to the sameposition at Xp21.3 as the disorder of adrenalgland development, adrenal hypoplasia con-genita (AHC). The gene responsible for Xlinked AHC and HHG has been recently iden-tified as DAX-1 (DSS-AHC critical region onthe X chromosome, gene 1).12 DAX-1 mapswithin the DSS interval and encodes a memberof the nuclear receptor superfamily. Loss offunction mutations ofDAX-1 cause both AHCand HHG. " Although no gene has been shownto be responsible for male to female sex

reversal, DAX-1 is a good candidate genebased on its position and pattern of expression.We report an unusual familial case of three

sibs, one boy and two girls, each harbouring adistal Xp duplication. The karyotype of the boywas 46,Y,inv dup(X)(p22.11->p22.32). Hepresented with multiple congenital abnormali-ties, mental retardation, hypoplastic externalgenitalia, and HHG. His sisters were pheno-typically normal females except that eachpresented with short stature. Southern blottingdosage analysis indicated that the boy carried asingle copy of the DSS region but wasduplicated for Xp sequences located at least 2Mb distal to this position. The DAX-1 openreading frame was sequenced and found to beidentical to that of a normal male. Multiplecongenital abnormalities and mental retarda-tion may be caused by the presence of twoactive copies ofXp genes or alternatively by thedisruption of a gene(s) at the duplicationbreakpoint. Several hypotheses are presentedto explain the occurrence of HHG andhypoplastic external genitalia in the proband:(1) the presence of two active copies of a genelocated distal to the DSS region, (2) thedisruption of a gene located at the duplicationbreakpoint, or (3) a possible long rangeposition effect disrupting DSS expression.

Case reportsThe proband was born on 2.2.77 to a 22 yearold gravida 2 and her 23 year old husband. Thepregnancy was uneventful. At delivery at 41weeks' gestation, weight, length, and headcircumference were 2410 g, 45 cm, and 33 cm,respectively.At 17 years the boy could walk but not

speak. He showed profound mental retardationwithout motor deficit (fig 1). Physical examin-ation showed severe dysmorphic features in-cluding microcephaly (-3 SD), long face, blueeyes with mottled irides, downward slantingpalpebral fissures, hypertelorism, epicanthus,bilateral ptosis, short eyelashes, bulbous nose,short philtrum, high arched palate, retrog-nathia, low set ears, low hairline, large andshort neck, bulging thorax, low set, widelyspaced, retracted nipples, lumbar lordosis, dor-sal kyphoscoliosis, and valgus knees. He hadspindle shaped fingers with clinodactyly of thefifth fingers and his feet were flat with bilateralvalgal and talocalcaneal fusion. The skin was

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dry and keratotic with congenital cutis laxa andmajor ligamentous laxity. He bit his fingers.Axial and limb hypotonia and short stature (-8SD) were noted (fig 2).

Neurological examination showed a pyrami-dal syndrome. A CT scan and MRI analysisshowed cerebellar cortico-subcortical atrophy.X ray examination showed asymmetry of thepelvis, subluxed femoral heads, and collapse ofthe tibial condyles. However, there were nosigns of chondrodysplasia punctata. Theproband had mixed deafness caused by perfo-

Figure1 The face of the patient at 17years of age.(Photographs reproduced with consent.)

...

Figure 2 The patient at 17years of age.

Table 1 The levels of steroid sulphatase activity observedin proband (II. 2), his two sisters (II. 3, II. 4), and hisbrother (II. 7)

STS leucocytes STSfibroblasts

Proband II.2 82.3 831Sister IL.3 39.4Sister II.4 26.5Brother I1.7 32Normal levels in males 13.9-32.1 175-637Normal levels in females 19.3-40.4

The results are expressed in nkat.kg-1.

rated tympanic membranes and lesions of thecochlea. Optical examination showed epi-blepharon, cicatricial blepharoconjunctivitis,and pillary coloboma. Cardiac ultrasoundshowed ballooning of the mitral valve, probablya consequence of the connective tissue hyper-extensibility. Electroencephalogram and elec-tromyography were normal.At 17 years the external genitalia were hypo-

plastic with a micropenis (25 mm), leftcryptorchidism with inguinal testis, and foldingof the scrotal skin. The testes were small (10 x7 mm). A pelvic ultrasound showed twokidneys. There was no facial, pubic, or axillaryhair.The two sisters were born in 1978 and 1979,

respectively (fig 3). They showed short stature(-3 SD) as the only phenotypic abnormality.One of the sisters showed clinical and biologi-cal puberty at 13.5 years.

CYTOGENETIC ANALYSISBanding patterns were analysed using theRHG and RTBG techniques on chromosomesfrom lymphocyte cultures.'4 Chromosomeanalysis of the proband showed a 46,Y,invdup(X)(p22.11-p22.32) chromosome com-plement (fig 4). Both sisters 11.3 and II.4 hadthe same duplicated Xp chromosome. Theirfather (1.2) and their phenotypically normalbrother had 46,XY karyotypes. X chromosomereplication studies were performed on lym-phocytes from 11.3 and II.4. The inv dup(Xp)chromosome was found to be late replicating in51 out of 52 cells (98%) and in 53 out of 55cells (96%) in the two sisters, respectively. Themother was not available for analysis as she haddied.A cell line from the male patient is banked in

our department.

ENDOCRINOLOGYAt 17 years the proband had a serum FSH levelof < 0.5 IU/l (normal range 0.6-4.8 IU/l)which rose after administration of LHRH to1.5 IU/l (normal 1.2-6.1). Basal LH levels were< 0.5 IU/l (normal 0.7-2.2 IU/l) and re-mained undetectable following LHRH stimu-lation. Testosterone levels rose from 0.3 ng/ml(normal 1.2-7.0 ng/ml) to 2.6 ng/ml afteradministration of hCG. Antimullerian hor-mone level was low for the age of the patient:1.7 ng/ml (normal 25 ± 15 ng/ml) (Dr NJosso, Paris). Levels of dehydroepiandroster-one sulphate (SDHA) were found to benormal. Growth hormone (GH) levels werefound to be normal before and after ornithinestimulation.

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A family with inv dup(Xp)

111 2 3 4 5 6 7

*1 (i) Dup (Xp) N Normal karyotype

Abortion Dead

/f Proband

Figure 3 The pedigree of the family. Affected subjects are indicated by solid symbols.

STEROID SULPHATASE (STS) ACTIVITYThe activity of STS was measured by a radio-chemical method using dehydroepiandroster-one sulphate as substrate, according to thetechnique of Piraud et al."5 STS activity, codedby the STS gene located at Xp22.32, wasfound to be increased in fibroblasts andlymphocytes from the proband. The level ofSTS in lymphocytes from the two sisters wasfound to be within the normal range (table 1).

DNA ANALYSISThe testis determining gene SRY was screenedfor mutations by both Southern analysis usingthe probe pY53.3 and by denaturing gradientgel electrophoresis (DGGE), as described else-where.'6 17 To determine the extent of the Xpduplication Southern blot dosage analysis wasperformed using the Xp probes, from thetelomere to the centromere: p782 (DXS85),pdic56 (DXS143), p99.6 (DXS41), C7(DXS28), QST59 (DXS319:DSS).'8 TheDAX-1 gene was directly sequenced followingPCR amplification of genomic DNA. Theprimers used to amplify and sequence the genewere 1AF, 1AaF, 1AaR, 2875, and 1AR.7 In

RH-

RHG

At o*

4-1111

addition, primers 2AR (GTACCCTTAC-CCGGG), 2AF (CTCCCTCCAGACGTG),and 2AaR (CAATCAAGTTAATAGTT) wereused to amplify and sequence the 3' region ofthe gene. Primer pairs 1AF and lAaR amplifya 764 bp product, 2875 and 2AR a 1004 bpproduct, and 2AF and 2AaR a 621 bp product.Amplified PCR fragments were gel purifiedand directly sequenced using Sequenase(USB).Southern blot dosage analysis using Xp

probes indicated that the proband carried twocopies of the Xp loci DXS85, DXS143, andDXS41. Southern analysis indicated thatDXS28 and DXS319 were both present as asingle copy (fig 5). The SRY gene was found tobe identical to that of a normal male by bothSouthern blotting (fig 5) and by DGGE analy-sis (data not shown). The DAX-1 gene wasfound to have a sequence identical to that of anormal male.

DiscussionTo our knowledge very few cases with a mater-nal origin of dup(Xp) have been reported todate.'9 We identified a familial case with acytogenetically detectable duplication of theshort arm of the X chromosome. Southern blotdosage analysis indicated that the proximalbreakpoint of the duplication was locatedbetween DXS28 and DXS41. A significantincrease in STS activity in cells from theproband also indicated functional disomy ofdistal Xp. Since the proband had two sisterswith the same cytogenetic abnormality, whohad short stature as the only phenotypicabnormality, it suggests that the phenotype iscaused by the presence of two active copies ofgene(s) located at Xp22-23. This hypothesis issupported by the observation that the dupli-cated X chromosome was the inactive X chro-mosome in the majority of cells examined fromthe two sisters. STS activity in lymphocytesfrom each sister was also within the normalrange. The DSS region at Xp21.3 was presentas a single copy in the proband. Since DXS28is located 2 Mb distal to the DSS criticalregion, the breakpoint must be located at least2 Mb distal to DSS.

* _'t,.

RTBG RHG

4-T

RTBG

Figure 4 The X chromosomes of the patient and his two sisters showing the rearranged X chromosome after RHG(Reverse-Heat-Giemsa banding) and RTBG (Reverse-Thymidine-BrdU-Giemsa banding).

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Approximately 21 cases of cytogeneticallyvisible duplications of distal Xp have beenreported to date.'..9 20 Both sporadic andfamilial cases have been described. The dupli-cated material may be located on the Xchromosome, on an autosome, or on the longarm of the Y chromosome. Multiple congenitalabnormalities and mental retardation havebeen described in XY subjects carrying an Xpduplicated fragment. In 11 patients withXp21.3 duplication including the DSS region,ambiguous or female external genitalia hasbeen observed,l1 7-9 20 and in seven of thesecases gonadal dysgenesis was con-firmed.' 4 8 9 11 When analysed, the SRY genewas found to be normal in these subjects.46,XY subjects who have duplications of Xp

usually present with mental retardation andmultiple congenital abnormalities and if theDSS region is duplicated there may also begonadal dysgenesis. The occurrence of HHGin these cases is rare. There are three possiblehypotheses which may explain the presence ofHHG and associated hypoplastic externalgenitalia in our case. First, HHG and associ-ated hypoplastic external genitalia may becaused by two active copies of a gene located atdistal Xp. Mutations involving the Kallmannsyndrome gene at Xp22 were associated withHHG and other abnormalities in a 46,XY sub-ject. However, duplications of this gene areunlikely to give rise to HHG since the majorityof subjects with duplications of Xp includingthe region of the Kallmann gene have normalFSH and LH levels.A second hypothesis is that the chromo-

somal breakpoint of the duplication may

directly interrupt a gene, thereby causingHHG. Bardoni et at5 described two 46,XY sub-jects who carried duplications of Xp sequencesdistal to the DSS region and whose phenotyperesembled the boy described in this paper. Inone subject (case 4, SR) the external genitaliawere hypoplastic and glandular hypospadiaswas noted. The basal levels of FSH and LHwere described as being at the lowest limits ofthe normal range. The other subject (TM) hadhypoplastic external genitalia with cryp-torchidism. FSH and LH levels were not indi-cated. The proximal breakpoint in both caseswas located between DXS43 and DXS41, dis-tal to both DSS and DXS28, similar to thebreakpoint described in our patient. Thesedata, together with the patient described here,suggest that there might be a gene in this posi-tion implicated in the physiology of thegonadotrophins.

Thirdly, it cannot be excluded, in the casepresented here, that the duplication may have asilencing effect on a DSS gene located atXp21.3. The DAX-1 gene has been mappedwithin the DSS critical interval. Loss offunction mutations in DAX-1 are responsiblefor the HHG and AHC in a 46,XY subject.'2The subject described here had a wild typeDAX-1 sequence. In addition Southern analy-sis failed to detect any abnormality within theDSS interval. It is formally possible that HHGwas caused by a long range position effect ofthe duplication on DAX-1 expression or othergenes in the DSS critical region.

The authors wish to thank Chantal Binet, Sylvie Chataignier,and Nicole Souleyreau for excellent technical assistance.

1 2 3

14 kb)

2

10Kbki: '.

D782 pd i(:5i6DXS 1 43

1 2 3-)

7 5 kb un- ;_ ;

... .....

C7DXS28

..:

4.2 kb) - i-

DXS3 9

Figure 5 Southern blot dosage analysis of the Xp duplication. 7

chromosomal position are indicated. The size of the fragment is shpanel lane 1 corresponds to the proband, lane 2 a normalfemale,male. Genomic DNA was digested with the restriction enzymes AHindIII (pY53.3), EcoRV (C7), BclI (pdic56), or PstI (p99.6).

1 Bernstein R, Jenkins T, Dawson B, et al. Female phenotypeand multiple abnormalities in sibs with a Y chromosomeand partial X chromosome duplication: H-Y antigen andXg blood group findings. JMed Genet 1980;17:291-300.

2 Scherer G, Schempp W, Baccichetti C, et al. Duplication ofan Xp segment that includes the ZFX locus causes sex

k bt- inversion in man. Hum Genet 1989;81:291-4.1D)\ J *- ='"'@3 Stern HJ, Garrity AM, Seal HM, Wangsa D, Disteche CM.Duplication Xp21 and sex reversal: insight into the mech-anism of sex determination. Am J Hum Genet 1990;47(suppl):A41.

4 Ogata T, Hawkins JR, Taylor A, Matsuo N, Hata J, Goodfel-low PN. Sex reversal in a child with a 46,X,Yp +karyotype: support for the existence of gene(s), located indistal Xp, involved in testis formation. J Med Genet 1992;

mb.3 6 ~ 29:226-30.XS4-, 5 Narahara K, Kodama Y, Kimura S, Kimoto H. Probable

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- .1 1982;19:222-4.- - 7 May KM, Grizald KA, Blackson RD. Sex reversal and mul-

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