I2 Med Genet 1992; 29: 542-546

Minute Y chromosome derived marker in a childwith gonadoblastoma: cytogenetic and DNAstudies

V Petrovic, S Nasioulas, C W Chow, L Voullaire, M Schmidt, H Dahl

AbstractA 12 year old girl referred for chromo-some analysis because of short staturewas found to have karyotype mos 45,X/46,X, + mar. The marker chromosomewas observed in 58% of her blood lym-phocytes. It was a small, pale staining,spherical fragment with GTL bandingand showed faint differentiation along itslength with CBG banding. DNA analysisusing Y specific probes showed the ab-sence of the testicular determining re-gion and the presence of some short armand centromeric Y chromosomal mater-ial. In situ hybridisation confirmed thatthe Y chromosomal material was associ-ated with the marker chromosome. Atlaparotomy the patient was found to havestreak gonads. Gonadectomy was subse-quently performed and histological ex-amination showed dysgenetic gonadswith a dysgerminoma arising from agonadoblastoma in the left gonad. Thiscase shows that even very small Y derivedmarker chromosomes with pericentricmaterial can predispose the phenotypicfemale to gonadal neoplasia.

Murdoch Institute,Royal Children'sHospital, FlemingtonRoad, Parkville,Melbourne, Victoria3052, Australia.V PetrovicS NasioulasL VoullaireM SchmidtH Dahl

Department ofAnatomicalPathology, RoyalChildren's Hospital,Melbourne, Australia.C W Chow

Correspondence toMs Petrovic.

Received 10 June 1991.Revised version accepted3 January 1992.

An increased risk for gonadoblastoma in phe-notypic females with dysgenetic gonads andthe presence ofY chromosome material is wellknown.' The Y chromosome material can bepresent as a low level Y chromosome mosai-cism or as a cytogenetically indefinablemarker, in which case its identification usingDNA methods is important in determining thepatient's risk for developing gonadoblastoma.

Page2 has postulated a gene (GBY) on the Ychromosome with an undefined physiologicalfunction in normal males, but which predis-poses dysgenetic gonads to develop malig-nancy when present in females. On the basis oftwo reported cases'-5 of females with dysgene-tic gonads, deleted Y chromosomes, and gona-doblastoma, Page2 has argued that the GBYgene is located either near the centromere or

on the long arm of the Y chromosome and isdistinct from the testis determining factor.Identification of the minimal regions of the Ychromosome that are present in patients withgonadoblastoma will clarify those females whoare at risk and may lead to an understanding ofthe oncogenic mechanism.The presence of a very small Y derived

marker chromosome in a girl who was subse-quently found to have gonadal neoplasia has

allowed us, using DNA techniques, to relinethe region of the Y chromosome that maycontain the hypothetical GBY gene.

Case reportThe patient was a 12 year old girl who wasreferred for chromosome analysis because ofshort stature. She was born at term with abirth weight of 2183 g to a 26 year old motherand a 28 year old father. At birth she was notedto have swollen ankles and feet. At 3 years ofage she had bilateral inguinal hernias repairedand at 9 years she had an eosinophilic granu-loma removed from her right clavicle. At 12-9years her weight was 34-5 kg (< 10th centile)and her height 130-5 cm (<3rd centile) withnormal body proportion and a bone age inkeeping with her chronological age. Therewere other features of Turner's syndromeincluding increased carrying angle, lack ofpubertal development, breasts, and pubic hair,and mild lymphoedema of the left ankle. Web-bing of the neck and ear deformities wereabsent. Ultrasound showed a markedly atro-phic uterus, normal cervix and vagina, and theovaries were not clearly visualised. There wasa mild degree of pectus excavatum which wasfamilial. At 13 years of age, after cytogeneticstudies, a laparotomy was carried out whichshowed bilateral streak gonads and gonadec-tomy was performed.

HISTOPATHOLOGYThe right gonad was a streak 23 x 3 x 1-5 mmwith ovarian stroma but no germ cells. The leftgonad was 16 mm in length with a 5 mmnodule on one side. Section of this noduleshowed infiltration by clusters and trabeculaeof a dysgerminoma associated with manylymphoid follicles, focal calcification, and anoccasional nest of gonadoblastoma was present(fig 1).

Materials and methodsCYTOGENETICSMetaphase cells were obtained from PHA sti-mulated blood lymphocytes using standardtechniques. Chromosomes were examinedusing various staining methods includingGTL banding, CBG banding, Ag-NOR band-ing, and GBG banding. Three separate cellcultures were established from the gonadaltissue and chromosomes from the dividingfibroblasts were examined.

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Minute Y chromosome derived marker in a child with gonadoblastoma: cytogenetic and DNA studies

* W.

:J. - . , ; *1

Figure 1 Section through left gonad of the patientshowing infiltration by dysgerminoma with lymphoidfollicle and focal calcification (arrowed) (H&E).

IN SITU HYBRIDISATIONFluorescent in situ hybridisation to thepatient's chromosomes using the biotinlabelled Y centromeric probe pY846 was car-ried out according to published methods.'Chromosomes were photographed usingEktachrome ASA 400 slide film and printeddirectly onto black and white paper. The slideswere subsequently stained with Giemsa andrephotographed.

DNA STUDIESDNA was extracted from white blood cellsfrom the proband and from a normal male anda normal female as described by Miller et al.7Genomic DNA, digested with restrictionendonucleases EcoRI, TaqI, HindIII, orHaeIII, was electrophoresed on a 07% agar-ose gel, blotted onto a Gene Screen Plus mem-brane (New England Du Pont), hybridised,and washed at high stringency(0p 1 S.SC, 01i%SDS, 70'C) following manufacturers' recom-mendations. To determine gene dosage, equalamounts of DNA were loaded onto the gels.The following probes which were used have

been assigned to a deletion interval map of thehuman Y chromosome.' A schematic represen-tationIof this map is shown in fig 2.Probe pDP1007 detects Y chromosome spe-

cific sequences which map to deletion interval1 at Yp 1 1 and cross hybridises to the short armof the X chromosome. On TaqI digests itdetects a 2-8 kb Y specific fragment and an Xlinked fragment of 2-3 kb. This probe recog-nises the ZFY locus.10

p

1

2

3

4a

4b

5

6

7

A

Icen

B C

I

I

q

Figure 2 Interval map of the human Y chromosome.On the right the bars indicate the regions remaining inthe deleted Y chromosomes in patients described by(A) Disteche et al,3 (B) Magenis et al,45 and (C) thepresent case.

Probe pDP61 is a subclone derived fromplasmid p115."1 It detects homologous X andY chromosome sequences (DXYS8) of 1 8 and2-8 kb. On TaqI digests, it detects a Y linkedfragment of 2 1 or 2-6 kb (Y linked RFLP)mapping to deletion interval 2.3Probe pDP105(A/B) defines a family of Y

specific repetitive sequences. On TaqI digestsit detects Y linked fragments of 8-0, 5-2, and2-5 kb. The 2-5 kb fragment A maps to dele-tion interval 3 (Yp), while the 5-2 kb fragmentB maps to deletion interval 6 (Yq)." 12Probe pDP34 detects highly homologous

sequences on the X and Y chromosome. OnTaqI digests it detects a Y linked fragment of15 kb specific to deletion interval 4A. It alsodetects an X linked allele at 11 kb and 12 kbfragments. 13

Probe pDP97 is a subclone derived fromcosmid Y97. It detects an alphoid repeatedsequence at the centromere of the Y chromo-some. On EcoRI digests it detects a Y linkedfragment of 5-5 kb specific for deletion interval4b."1Probe pDP527 on TaqI digests detects a

7 kb Y specific fragment which maps to dele-tion interval 5 or 6 on Yq, and two X specificfragments of 4-2 and 3-3 kb (D C Page, per-sonal communication).Probe 116/21 is a subclone derived from an

independent cosmid 116. On HindIII digestsit detects 5-5 and 9 0 kb Y specific sequencesmapping to the boundary of intervals 6 and 7.6Probe pY431 -HinfA detects highly repeated

Y specific sequences which map to deletioninterval 7 (the heterochromatic region of Yq).Using HaeIII digests an intense 2 1 kb Ylinked fragment is detected (probe made by KSmith, D C Page, personal communication).The probes were labelled with 32p using the

random oligonucleotide method.'4

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Petrovic, Nasioulas, Chow, Voullaire, Schmidt, Dahl

Presence or absence of the SRY gene was

determined by the polymerase chain reaction.'5The SRY specific primers were 5'-AGTGTG-AAACGGGAGAAAAC-3' (nucleotides 544-56316) and 5'-TACAACCTGTTGTCCAG-TTG-3' (nucleotides 882-90116).

ResultsCYTOGENETIC STUDIESFifty G banded metaphase cells obtained fromperipheral blood lymphocyte culture showed a

mosaic karyotype, mos 45,X/46,X, + mar. Themarker was present in 58% of cells (29/50 ofcells counted). With GTL banding the markerappeared to be a small pale staining spherical

~.VI0%

W. ...--

Figure 3 Marker chromosome seen in the patient after (A) GTL banding,(B) CBG banding.

-A B

j

Figure 4 In situ hybridisation using the biotin labelled

probe p Y84 (A) showing signal on marker chromosome

and (B) showing the same cell without signal.

fragment (fig 3A). With CBG banding therewas faint differentiation along the marker (fig3B). It stained negatively with Ag-NOR band-ing and showed late replication with GBGbanding (data not shown). Parental chromo-somes were normal.Chromosome analysis of fibroblast cells

from gonadal tissue showed the same mosaickaryotype seen in the blood lymphocytes butthe proportions of the two cell lines variedslightly. The marker chromosome was presentin 32% of cells (16/50 of cells counted) fromthe fibroblast culture of the right gonad, in82% of fibroblast cells (41/50 of cells counted)from the left gonad, and in 62% of fibroblastcells (31/46 of cells counted) from the nodulein the left gonad.

IN SITU HYBRIDISATIONThe Y centromeric probe pY84 was found tohybridise to the marker seen in the patient'skaryotype (fig 4).

DNA ANALYSISDNA from the patient was examined for thepresence of Y chromosome material using theY specific probes listed in Materials and meth-ods. These probes were chosen to cover theseven intervals of the Y chromosome.9 Theresults of Southern blot analysis using theeight probes described are shown in fig 5.Probes pDP105A (2-5 kb fragment) andpDP97 hybridised to the patient's DNA.Probes pDP1007, pDP61, pDP105B, pDP34,pDP527, 116/21, and pY431 gave no Y specificsignals in the patient. DNA from the normalmale control showed hybridisation with all theprobes used. These results are summarised infig 6. The SRY gene was not detected in thepatient by use of specific primers in the PCRreaction (fig 7). Thus, as summarised in fig 6,the marker chromosome in the patient isdeleted for intervals 1, 2, and 4a of the shortarm, and, ofthe long arm, intervals 6 and 7 andpossibly some of 5 has been deleted. Thepatient's marker chromosome, then, carriessome Y short arm and centromeric materialand possibly some long arm material.

DiscussionA very small marker chromosome was seen in agirl with features of Turner's syndrome whowas found to have gonadoblastoma and dys-germinoma. DNA studies showed that thepatient's karyotype contained Y short arm andcentromeric material and possibly some proxi-mal long arm material. The SRY gene was notdetected. In situ hybridisation confirmed thatthe marker chromosome carried the Y centro-meric material. We therefore interpret themarker chromosome as a Y derived ring chro-mosome where the breakpoints have occurredat p11.2 and qll.2.On the deletion interval map of the human Y

chromosome (fig 6) our patient is deleted for

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Minute Y chromosome derived marker in a child with gonadoblastoma: cytogenetic and DNA studies

pDP1 G5

(Taq IPM F

mW 80

pDP34(Taq!)PM F

15(Y)

126

5 2(Y,B)

- 2 8(Y) m - 2 8-2 3 Om --2 6(Y)

pDP527

(TaqI)PM F

W -- 70(Y)55(Y)

2 5(Y,A)

p1 16/21

(Hindl II )PM F

0

pY431

(Haelli)PM F

-- 90(Y)

_- 55(Y)- - <- 4 2

3.3

#F -21(Y)

Figure 5 Detection of Y chromosome DNA in the patient by Southern blot analysis.DNA from the patient (P), a normal control male (M), and a normal control female(F) was digested with restriction endonucleases TaqI, EcoRI, HaeIII, or HindIII asindicated. The filters were hybridised to the eight DNA probes as shown. The sizes ofthe detected fragments, the Y specific fragments (Y), and two intervals identifying Yspecific fragments detected by probe pDPO05 (A and B) are also indicated.

intervals 1, 2, and 4a of the short arm, and, ofthe long arm, intervals 6 and 7 and possiblysome of interval 5 has been deleted. The dele-tion of interval 4a, with the presence of interval3 and 4b has also occurred in an XY female

(patient 2) described by Disteche et aP (fig 2).This has been interpreted by Page'7 to be aresult of the rearrangement occurring in a Ychromosome that has an inversion of intervals3 and 4a. Our report of another case supportsthe hypothesis that this inversion is a popula-tion dimorphism.'7

It is known that the testis determining gene(TDF) is located towards the terminal end ofthe Y chromosome short arm in interval 1L8The gene SRY, which has recently been iso-lated and proposed as TDF,'6 is located withinthis region. The loss of interval 1 and theabsence of SRY in the proband's marker Ychromosome implies the absence of TDF inthe patient and is consistent with the lack oftesticular development. The presence of gona-doblastoma in our patient without testis de-velopment and in the absence of the testisdetermining region of the Y chromosome givessupport to the view that the development ofthis tumour is not dependent on either thepresence of testicular tissue or the TDF gene.

If the GBY gene postulated by Page2 exists,then it must have been present on the markerchromosome in our patient. The marker chro-mosome seen in our patient is smaller thanthose previously investigated with DNA tech-niques in patients with similar clinicalfeatures35 (summarised in fig 2). In particular,interval 7, which is the heterochromatic regionof the Y chromosome, and interval 6 are absentin our patient. The DNA studies on previouslypublished cases together with those on thepresent case suggest that the postulated GBYgene must map close to the centromere. Thus,we conclude that currently a Y centromericprobe is the most suitable probe for screeningfor malignancy risk in females with dysgeneticgonads and small marker chromosomes of un-certain origin. However, a centromeric probecould fail to indicate the presence of the GBYgene in the event of this gene being translo-cated onto another chromosome.

Y chromosomedeletion intervals

p

2

3

4a

4b

5

6

7

Probe

pDP1 007

pDP61

pDP1 05/A

pDP34

pDP97cen

pDP527pDP1 05/B116/21

pY431 -Hinf-A

Locus

ZFY, ZFX

DXYS8

DYZ4/A

DXYS1

DYZ3

STSPDYZ4/B

DYZ2

Hybridisation

Normal Normal

P M F

+

+

+ +

+

+ + _

+

+

_ + _

q

Figure 6 DNA probes used to test for the presence ( + ) or absence (-) of Y specific restriction fragments (see fig2) in the patient, normal male, and normal female controls. The loci have been assigned to a deletion interval map ofthe Y chromosome as shown.

pDP1 007

(Taql)PM F

pDP61

(Taq I)PM F

pDP97

(Eco RI)

PM F

.9

-I - ..

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1230-

653 ..u:s.' ......':,',

..........

394298

| I | . I , , . .V'O-n

Figure 7 SRY gene analysis. Using the SIspecific primers in the polymerase chain reac,presence of the SRY gene is indicated by a 3amplified band. Lane 1 is the amplification pthe patient, lane 2 from a control male, anda control female.

A lymphoblast cell line (MICY8800lished from this patient is availableTissue Culture Laboratory, Murd4tute. We thank Dr D C Page and Drfor the Y specific probes.

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