Analysis 1 - Journal of Medical Geneticsjmg.bmj.com/content/jmedgenet/35/7/545.full.pdf · D3Sk445/ Exact determination of the breakpoints in ... (II.8 and II.9, fig 1). ... Pro-metaphase

J7Med Genet 1998;35:545-553

Analysis of a familial three way translocationinvolving chromosomes 3q, 6q, and 1 5q by highresolution banding and fluorescent in situhybridisation (FISH) shows two differentunbalanced karyotypes in sibs

Dagmar Wieczorek, Hartmut Engels, Renate Viersbach, Barbara Henke, Gesa Schwanitz,Eberhard Passarge

AbstractWe report on a familial Itranslocation involving chrom6, and 15 identified by pro:banding and fluorescence in silsation (FISH). Two mentally relwith different phenotypic abn4their phenotypically normalmother, and two fetuses of ttypically normal sister wereThe terminal regions ofchrom46q, and 15q were involved in atranslocation, in addition to a pinversion of the derivative ch15. Conventional cytogenetic sthigh resolution GTG bandinresolve this rearrangement. Fwhole chromosome paintsidentified the chromosomal rvolved, except the aberrant relwhich was undetectable wprobes. Investigation of this r4the subtelomeric FISH probeD3S1446 showed a balanced46,XX,t(3;15;6) (q29;q26.1;q26),(q15.1q26.1) in two adult femalfetus. It was unbalanced inshowing two different types of utranslocation resulting in parti3q in combination with partial j

6q in one patient and partial ti

three wayiosomes 3,metaphasetu hybridi-tarded sibsormalities,sister andhe pheno-analysed.

osomes 3q,reciprocaloaracentricromosometudies withg did notISH using

(WCPs)egions in-gion of 3q,rith theseeffion with

with partial monosomy 6q in theother patient and one fetus. These repre-sent apparently new chromosomal pheno-types.(7Med Genet 1998;35:545-553)

Keywords: three way translocation; partial trisomy 3(q29-*qter); partial trisomy 15 (q26.1-*qter); partialmonosomy 6 (q26-qter)

Complex chromosomal rearrangements(CCRs) involve three or more chromosomesand have at least three breakpoints.' 2CCRs with up to seven chromosomesinvolved3 and 10 breakpoints4 have beendescribed. The interpretation of CCRs orcomplex translocations by conventional band-ing techniques alone may be impossible, espe-cially when deletions, insertions, or inversionsare present in addition to the reciprocaltranslocations.5

Exact determination of the breakpoints inD3Sk445/ complex translocations is necessary to estimate

kai r(yype, the risk of unbalanced offspring and theies and one spectrum of phenotypic abnormalities fort-wo sibs genetic counselling. The application of FISHnbalanced with chromosome specific DNA libraries6altrisomy allows the recognition of aberrant chromo-

mnonosomy somal regions and cryptic translocations be-somy 15q yond the previous limits of detection.

We report a familial three way reciprocaltranslocation involving the terminal regions ofthe long arm of chromosomes 3, 6, and 15. Inaddition, a paracentric inversion was present inthe chromosome 15 involved in the transloca-tion. Three derivative chromosomes and four

_ breakpoints were analysed by GTG prometa-phase banding and FISH using whole chromo-some paints and a band specific probe(D3S1445/D3S1446) for the smallest translo-cated segment which was undetectable by thewhole chromosome paint.The proband and her mother are carriers

of a balanced three way translocation, whereasher two mentally retarded adult sibs havedifferent unbalanced karyotypes. Theyrepresent previously undescribed combina-tions of chromosomal imbalances, partialmonosomy 6q with partial trisomy 3q and par-tial monosomy 6q with partial trisomy 15q,respectively.

Institut firHumangenetik,UniversitatsklinikumEssen, Hufelandstrasse55, 45122 Essen,GermanyD WieczorekB HenkeE Passarge

Institut firHumangenetik,UniversitatsklinikumBonn, Wilhehmstrasse31, 53111 Bonn,GermanyH EngelsR ViersbachG Schwanitz

Correspondence to:D Wieczorek or H Engels.

Received 23 June 1997Revised version accepted forpublication10 December 1997

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Wieczorek, Engels, Viersbach, et al

Table 1 Clinicalfindings in patients with partial monosomy 6q

[13], [14], [17], [18], [19, patient 1], [29, [14],This report This report patient 2], [20, patient2], [21] [30, patient [20, patient 2],(patient 1) (patient 2) [15], [29] 1], [30, patient 2], [31], [42], [43], [44] [21], [22], [45] [46], [47]

MonosomyTrisomy

SexMental retardationSeizuresMotor coordination disturbedFeeding problemsMuscular hypotoniaShort statureTall statureMicrocephalyBrachycephalyAsymmetrical head/faceCoarse facial featuresHypertelorismDeeply set eyesNarrow palpebral fissuresEpicanthic foldsStrabismusPtosisRetinal pigmentary anomalyHigh nasal bridgeBroad nasal bridgeLong nasal septumBulbous tip of noseChoanal atresiaLong philtrumFlat philtrumLarge mouthHigh palateCleft palateMicrognathiaLow set earsDysplastic earsPreauricular pits/tagsShort neckWebbed neckNarrow thoraxScoliosisSitus inversusDiaphragmatic herniaCongenital heart defectHydrocephalusImperforate anusSacral dimpleSpina bifidaGenital anomaliesDeformity of fingersTapering fingersBrachydactylyArachnodactylyDysplastic nailsDysplastic hipsOverriding toes

Not reported. Numbers in square brackets are reference numbers.

MethodsASCERTAINMENT AND FAMILY HISTORY

The consultand (II.2) asked for genetic coun-

selling during the early gestational weeks of herfirst pregnancy because of two sibs with mentalretardation (II.8 and II.9, fig 1). One youngersister with hydrocephalus (II.3) died at the ageof 6 months (further data are not available).One younger brother (II.5) is normal andchromosomal analysis (GTG banding) in an

outside laboratory was reported to be normal(fig 1 ).

In routine chromosomal analysis of the con-

sultand (II.2) using GTG banding at a resolu-tion level of 400 bands per haploid genome, wefound a paracentric inversion of chromosome15 with breakpoints in 15qi5 and 15q26. Pro-metaphase G banding analysis at a resolutionof 650 bands showed a translocation involvingchromosomes 3, 6, and 15. In order to clarifythese findings, the mother (I.2), the twomentally retarded sibs (II.8, II.9), one sponta-

neous abortion (III.2), and the third pregnancy(III.3) of the consultand were studied asdescribed below.

CYTOGENETIC METHODS

Metaphase and prometaphase chromosomepreparations were obtained from PHA stimu-lated lymphocyte cultures from the consultand,her parents, and her mentally retarded sibsusing the MTX/bromodeoxyuridine (BrdU)method of Pai and Thomas.8 Fibroblast andamniotic cell cultures of her two fetuses were

performed using standard methods. Chromo-some banding was performed using thetrypsin-Giemsa technique of Seabright.9

FLUORESCENCE IN SITU HYBRIDISATION

Slides were hybridised with whole chromo-some 3 paint (Angewandte GentechnologieSysteme Heidelberg, Germany) and the probe"telomere 3q" (D3S 1 445/D3S 1446, Oncor-Appligene, Gaithersburgh, VA, USA) accord-ing to the manufacturer's instructions.

6q26-qter3q29-qter

M+

+

6q26-qter1 5q26. 1-qter

F

+

6q26-qter1 q44-qter99

2M2/21/2

1/21/20/21/21/21/21/22/22/21/22/22/21/21/10/22/20/20/2

2/20/21/21/10/12/21/22/20/22/20/20/21/2

1/21/1

2/22/2

1/1

1/21/2

6q25-qter1 7q25-qter"22ql 1.2-qter185M/8F10/105/5

2/26/75/91/911/115/54/41/105/106/103/119/116/81/92/24/1110/114/104/101/16/103/85/114/54/511/137/1211/113/38/82/2

2/21/11/16/103/4

3/31/14/93/3

1/14/51/1

6q24-qter

2M/2F3/34/4

1/11/13/30/34/41/1

1/12/22/31/22/21/11/1

0/21/11/21/3

3/41/31/12/23/33/34/43/31/13/32/21/2

2/21/11/1

2/32/21/11/2

1/1

2/2

6q23-qter

2M/1F1/1

1/10/21/20/2

1/1

0/12/21/11/11/10/1

0/11/10/11/1

1/10/10/1

1/13/33/32/2

2/22/2

1/1

1/12/21/1

1/11/1

2/20/21/1

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A familial three way translocation involving 3q, 6q, and 15q

Figure 2 Front view ofpatient 1 showing long and narrowface with prominent chin, small eyes, hypoplastic nasal alae,large mouth with prominent lips, widely spaced teeth, andbroad neck. (Photographs reproduced with permission.)

Simultaneous hybridisation and detection ofwhole chromosome 6 paint (personal gift fromJ W Gray)'0 and whole chromosome 15 paint(Angewandte Gentechnologie SystemeHeidelberg, Germany) were performed as fol-lows. The whole chromosome 6 paint waslabelled with digoxigenin in a nick translationreaction using 10 x DIG DNA Labelling Mix-ture (Boehringer Mannheim, Germany) andDNA polymerase 1/DNAseI mixture (GibcoLife Technologies) according to the manufac-turer's instructions, mixed with 2 jg cotl-DNA and 10 gg salmon sperm DNA, and pre-cipitated. The probe was dissolved in 10 gl50% deionised formamide/l x SSC/10% dex-tran sulphate, mixed with 10 ,ul whole

..

W. W~~~~~~~~~~~~~~~~~~W.......Figure 3 (A) Front view of the face ofpatient 2 showing narrow face with smal eyes,strabismus, large mouth with prominent lips, and widely spaced teeth. (B) Patient 2showing thin build, broad neck, and torsion dystonia of the hands.

chromosome 15 paint (Angewandte Gentech-nologie Systeme), and denatured at 750C forfive minutes. Preannealing was performed at370C for 20 minutes. Slide denaturation,hybridisation, and high stringency washeswere performed according to the manufactur-er's instructions. Signals were detected andenhanced simultaneously by consecutive30-45 minute incubations with fluorescein-avidin (Vector)/mouse anti-digoxigenin(Sigma), biotinylated goat anti-avidin-IgG(Vector)/rabbit anti-mouse IgG-TRITC(Sigma), and fluorescein-avidin (Vector)/goat-anti-rabbit IgG-TRITC (Sigma).Chromosomes were counterstained with

DAPI, documented, and analysed with anApplied Imaging Cytovision 3.1 systemequipped with a CCD camera mounted on aLeitz Diaplan epifluorescence microscope.

ResultsCLINICAL DATAPatient 1 (I1.8) is a 26 year old male with severemental retardation and a distinct dysmorphicphenotype (fig 2, table 1). His height is 192 cm(+2.1 SD), weight 95.5 kg, and head circum-ference 56.5 cm (normal). He has a long andnarrow face with a prominent chin, deeply seteyes of normal distance apart, strabismus, anda broad neck. His nose is prominent withhypoplastic nasal alae. His mouth is large, lipsprominent, and teeth widely spaced andnormal in number. The palate is normal. Theears are normally set, but small and dysplastic(fig 2). The distal phalanx of the right thumb isbroader and shorter than the left. Thisdifference in thumb size was also present in hismother and his mentally retarded sister. Whenexcited, he develops torsion dystonia of hisupper extremities. Cranial CT scan showed noanomalies.

This patient was born at term after anuneventful pregnancy with a birth weight of4500 g (+2.3 SD) and a length of 54 cm (+0.1SD). Head circumference at birth is notknown. He was able to walk without help at theage of 18 months and began to speak at the ageof 24 months. His further development wasretarded. Childhood photographs show coarsefacial features with deep set eyes. The similar-ity to his sister's face at that time is remarkable.He attended kindergarten and a school formentally retarded children before he began towork in an institution for handicapped people.He can write by copying, but cannot write hisown text or calculate. His speech developmentis surprisingly good and exceeds his generalintelligence. IQ tests were not done. His motorcontrol does not allow him to ride a bicycle. Helives in a secure institution for the mentallyretarded because he has been convicted ofsexual assault of children.

Patient 2 (II.9) is a 25 year old female withmental retardation and a distinct, very slender("marfanoid") phenotype that differs from herbrother (table 1). Her height is 190 cm (+3.9SD), weight 55 kg, and head circumference 53cm (-1.4 SD). She cannot speak wholesentences, but single words only with a hoarsevoice. Her face is long and narrow with a

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I.

6 der(6) 15 der(15)

Figure 6 GTG banded karyotype ofpatient 2 withpartial monosomy 6q and partial trisomy 15q. The normalchromosome 6 is shown on the left, the derivative on theright.

an abduction device. She could walk alone atthe age of 3 years. Speech development was

severely delayed. IQ tests were not performed.She went to a school and an institution formentally retarded children. She lives with herparents.

3 der(3) 6 der(6) 15 der(15)Figure 4 (A) GTG banded karyotype of the proband showing normal chromosomes 3, 6,and 15 on the left, and the derivative chromosomes on the right, respectively. (B) Ideogramsof the chromosomes: der(3): 3pter-3q29::6q26-qter, der(6): 6pter-6q26::15q26. 1-qter, andder(15): 15pter-15ql5. 1::15q26. 1-15ql5. 1::3q29-3qter.

prominent chin similar to her brother, deeplyset eyes, and strabismus. Her mouth is widewith prominent lips, widely spaced teeth (fig3A), and a high palate with normal vaulting.Her ears are small and normally set and herneck is broad. She has long, slender, dystrophicextremities. Marked arachnodactyly with a

bilateral hand length of 20 cm (+2 SD) ispresent and her thumbs differ in width andlength like her brother's and mother's. She hasmarked thoracolumbar scoliosis and torsiondystonia of all extremities, especially of herarms and hands (fig 3B). Striae distensae are

present on both thighs.Patient 2 was born at term after an unevent-

ful pregnancy with a birth weight of 5000 g(+4.7 SD) and a length of 59 cm (+3.5 SD).Head circumference at birth is not known.Because of hip dysplasia she was treated with

CYTOGENETIC DATA

Prometaphase analysisThe consultand (II.2) and her mother (I.2) hadan abnormal GTG banding pattern in the ter-minal regions of the long arm of three chromo-somes, 3, 6, and 15, interpreted after FISHanalysis as a three way balanced translocationof 3 to 15 to 6, involving breakpoints 3q29,15q26.1, and 6q26. In addition, a paracentricinversion of the rearranged chromosome 15with presumed breakpoints in 15ql5.1 and1 5q26. 1 was present (fig 4). In contrast, patient1 (II.8) had an abnormal banding pattern inthe subtelomeric region of a chromosome 6and a chromosome 15, an inversion in the samechromosome 15, and two normal chromo-somes 3 (fig 5). Patient 2 (II.9) had two normalchromosomes 3 and 15 and no inversion 15.However, chromosome 6 had abnormal bandsin the telomeric region of the long arm (fig 6).No permanent cell lines are available.

FLUORESCENT IN SITU HYBRIDISATION

Consultand (11.2)Using whole chromosome 3 paint (WCP3), theaberrant chromosome 3 showed positive

3 der(3)

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Figure 7 FISH results of the consultand. (A) D3SI445D3Sl446: one chromosome 3and one chromosome 15 show clear hybridisation signals in their long arm terminal regions.(B) Whole chromosome 6 paint (redlTRITC) and whole chromosome 15 paint(green/FITC): with WCP6 (red) one chromosome 6 is painted entirely while the second onelacks hybridisation signals in its long arm terminal region. An additional signal is visible inthe terminal region of one chromosome 3q. Using WCPI5 (green), one chromosome 15 ispainted brightly while the second one shows fainter hybridisation signals in its long armterminal region (arrowhead). An additional signal is visible in the terminal region ofonechromosome 6q. Cross hybridisation can be seen in the short arm regions of acrocentricchromosomes.

hybridisation signals along its entire lengthexcept for a diminished signal intensity over thetelomeric region ofthe long arm. No additionalsignals were detected (data not shown). Thesubtelomeric FISH probe telomere 3q(D3S 1445, D3S 1446) yielded distinct hybridi-sation signals in the telomeric regions of thenormal chromosome 3 and one chromosome15 (fig 7A). Whole chromosome 6 paint(WCP6) gave positive hybridisation signalsalong the entire euchromatic length of theaberrant chromosome 6 except for the distalpart of the long arm (fig 7B). Additional signalswere detected over the terminal region of onechromosome 3. Whole chromosome 15 paint(WCP15) showed fainter hybridisation signalsin the terminal portion of the long arm of theaberrant chromosome 15 than in the proximal

region of the long arm, which was painted nor-mally. An additional signal was present in theterminal long arm region of the aberrant chro-mosome 6 (fig 7B).Based on these findings we determined the

karyotype to be 46,XX,t(3;15;6)(q29;q26.1;q26) inv der(15)(q15.1q26.1).ish t(3;15;6)(wcp3+, wcp6+, D3S1445/D3S 1446-; wcp 15+,D3S 1445/D3S 1446+; wcp6+, wcp 15+). 1

Analysis of fibroblast cultures from thesecond fetus (III.2) showed a balanced karyo-type as in the mother, the consultand (II.2), byall three WCPs and the probe "telomere 3q".Owing to limited quality of banding (320 bandstage) and signal production, this conclusion isnot entirely certain. Maternal origin of thesefibroblast cultures was excluded by Q polymor-phism analysis using quinacrine/actinomycin Dstaining" based on the existence of double sat-ellites of chromosomes 15 and 22 in the fetus,which were clearly absent in the mother.

Analysis of amniotic cell cultures of the thirdpregnancy (III.3) of the consultand showed thesame karyotype as in patient 2 with partial tri-somy 15q and partial monosomy 6q, asmentioned below. Because the consultand'sfamily moved to Cambridge, UK, the amnio-centesis was performed there. The analysisfrom amniotic cells was done in parallel atAddenbrooke's Hospital, Cambridge (Drs A JGreen and L Willatt) and in our laboratorywith identical results. Based on these findingsthe third pregnancy was terminated.

Consultand's mother (I. 2)The karyotype ofthe mother (I.2) was identicalto that of her daughter.

Patient 1 (11.8)After hybridisation with WCP3, both chromo-somes 3 were painted entirely, without addi-tional signals present (data not shown). Usingtelomere 3q (D3S1445, D3S1446), both chro-mosomes 3 and one chromosome 15 showedpositive hybridisation signals in the telomericregion of the long arm (fig 8A). After hybridi-sation with WCP6, one chromosome 6 did notshow hybridisation signals in the terminalregion of the long arm, whereas the rest ofchromosome 6 was painted normally (fig 8B).No other signals were detected. With WCP1 5the findings were identical to the mother (I.2)and the consultand (II.2) (fig 8B).According to FISH analysis the karyotype of

II.8 is 46,XY,der(6),der(15)t(3; 15;6) (q29;q26.1;q26) inv(15)(q15.1q26.1).ish t(3;15;6)(wcp3+, D3S1445/D3S1446+, wcp6-; wcpl5+,D3S1445/D3S1446+; wcp6+,wcpl5+) givingrise to partial trisomy of chromosome 3q29 to3qter and partial monosomy 6q26 to 6qter, asshown in fig 5.

Patient 2 (1. 9)After hybridisation with WCP3, both chromo-somes 3 were painted entirely (data notshown). No additional signals were found.Using the FISH probe telomere 3q (D3S 1445,D3S 1446), both chromosomes 3 showedsignals (fig 9A).

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Table 2 Clinicalfindings in patients with partial trisomy ISq

[24, patient 1], [25, patient 1], [25, patient 2],This report [23, patient 1], [23, [28, patient 1,2,4,5], [51], [52, patient 1],(patient 2) [27], [48] patient 2], [49] [24, patient 2], [50] [53], [54, patient 1],

TrisomyMonosomy

1 5q26. 1-qter 1 5q26-qter6q26-qter 2q37-qter27

4p 1 6-pter48

SexMental retardationSeizuresMotor coordination disturbedFeeding problemsMuscular hypotoniaShort statureTall statureMicrocephalyBrachycephalyAsymmetrical head/faceCoarse facial featuresHypertelorismDeeply set eyesNarrow palpebral fissuresEpicanthic foldsStrabismusPtosisRetinal pigmentary anomalyHigh nasal bridgeBroad nasal bridgeLong nasal septumBulbous tip of noseChoanal atresiaLong philtrumFlat philtrumLarge mouthHigh palateCleft palateMicrognathiaLow set earsDysplastic earsPreauricular pits/tagsShort neckWebbed neckNarrow thoraxScoliosisSitus inversusDiaphragmatic herniaCongenital heart defectHydrocephalusImperforate anusSacral dimpleSpina bifidaGenital anomaliesDeformity of fingersTapering fingersBrachydactylyArachnodactylyDysplastic nailsDysplastic hipsOverriding toes

F

+

2M2/21/1

1/11/20/10/10/10/10/10/10/11/10/10/1

1/11/2

0/10/11/21/10/11/20/12/2

1/20/10/11/1

0/10/1

1/11/1

0/11/1

1 5q25-qter 1 5q23-qter12p I 3-pter49 6q27-qters4

7p22-pter5°

3M3/3

2/3

2/2

0/22/20/10/10/10/10/11/10/1

0/10/10/10/1

3/31/10/13/3

3/33/31/1

2/2

1/13/3

2/22/20/11/1

6M/4F9/93/8

1/15/76/80/86/9

8/87/91/11/14/101/11/15/9

0/18/91/10/1

6/90/90/18/8

4/71/11/12/66/100/10/18/8

5/9

7/71/1

1/10/1

1 5q22-qter21 q22-qter24'1, 8p23.3-pter251 1q25-qter28, 13q32.3-qter2810q26-qter5, 12q24.33-qter531 4q32-qter546M/6F9/94/7

1/15/64/72/96/103/64/101/30/41/36/94/53/43/4

3/57/91/33/5

9/90/62/44/4

9/96/87/71/53/83/5

2/2

7/91/11/4

3/58/92/21/24/4

With WCP6 the aberrant chromosome 6did not show hybridisation signals in theterminal region of its long arm, whereas therest of this chromosome was painted (fig 9B).No other signals were detected. Using WCP1 5,both chromosomes 15 were painted entirelyand an additional hybridisation signal wasdetected in the terminal region of onechromosome 6 (fig 9B). These results are con-sistent with monosomy 6q26 to qter andtrisomy 15q26.1 to qter (46,XX,der(6)t(6;15)(q26; q26.1) .ish t(3; 15;6) (wcp3+,D3S 1445/D3S 1446+, wcp6-; wcp 15+,D3S 1445/D3S1446-; wcp6+,wcpl5+), as shown in fig 6.

DiscussionIn a sequential approach using cytogenetictechniques with increasing power of resolutionand chromosomal specificity, we recognised afamilial complex translocation involving threechromosomes with breakpoints in 3q29, 6q26,and 15q26.1 and a paracentric inversion

15ql 5.1-26.1. This allowed the identificationof two different unbalanced karyotypes in twoadult sibs with distinct phenotypes. Patient 1(partial monosomy 6q/partial trisomy 3q) hadprofound mental retardation, tall stature,strabismus, macrostomia, and webbed neck inaddition to other features (table 1). Patient 2(partial monosomy 6q/partial trisomy 15q) hadmicrocephaly, scoliosis, arachnodactyly, anddysplastic hips among others features (tables 1and 2).Owing to the complex chromosomal imbal-

ances present in both patients, it is difficult torelate particular signs to specific chromosomalregions, for example, monosomy 6q26-qterand trisomy 15q26. 1-qter. Both patients are asseverely retarded as all other patients withmonosomy 6q (listed in table 1) and all patientswith partial trisomy 15q (listed in table 2).The tall stature in both patients is notewor-

thy. This may have been influenced by the gen-eral height in this family, both the consultand

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A familial three way translocation involving 3q, 6q, and IlSq

Figure 8 FISH results ofpatient 1. (A) D3S1445/D3S1446: both chromosomes 3 as wellas one chromosome 15 show clear hybridisation signals in their long arm terminal regions.(B) Whole chromosome 6 paint (red/TRITC) and whole chromosome 15 paint(green/FITC): WCP6 (red) shows lack of hybridisation signals in the long arm terminalregion of one chromosome 6, whereas the second one is painted entirely. No additionalsignals are visible in 3qter. WCP15 (green) gives the same hybridisation result as in theconsultand.

and her mother being 180 cm tall. Neither ofthe carriers of the balanced translocationsshowed any dysmorphic signs. Reviewing pub-lished cases with partial monosomy 6q, twoother patients also had tall stature,"3 14 whereasnine patients had short stature.'5 22 Tall staturehas also been described in four patients withpartial trisomy 15q.'3-25 Therefore, height maybe an inconsistent feature of both monosomy

6q and trisomy 15q, and does not help indistinguishing these chromosomal aberrations.Tallness is also a feature of a further 33chromosomal aberrations described in theCytogenetic Database,26 not including partialtrisomy 3q. In addition, patient 2 had a marfa-noid habitus. A connection to the fibrillin genemapped to 1 5q2 1.1 can be ruled out since nei-ther the translocation nor the inversion break-points are near this chromosomal region.

Strabismus was also noted in both patients.This is a very common clinical feature of both

monosomy 6q (12/14 patients) and trisomy15q (6/7 patients).Macrostomia, another feature of both pa-

tients, cannot be used to distinguish partialmonosomy 6q from partial trisomy 15q,because it is present in both chromosomalaberrations with similar frequencies (9/17 inmonosomy 6q and 4/9 in trisomy 15q). Bothpatients had a broad and webbed neck, whichhas been reported in monosomy 6q (8/10patients) and in trisomy 15q (4/8 patients).

Clinical anomalies present only in patient 2are microcephaly, scoliosis, arachnodactyly,and dysplastic hips. Microcephaly and scoliosisare common features of many chromosomalaberrations, and also of monosomy 6q and tri-somy 15q (tables 1 and 2). In contrast, arachno-dactyly has not been reported in patients withmonosomy 6q, but in patients with trisomy15q. 23 27 28 Thus, the arachnodactyly is morelikely to be related to the partial trisomy 15qthan to monosomy 6q in patient 2. In contrast,dysplastic hips have been described only inpatients with partial monosomy 6q.16 18 29 31This clinical feature in patient 2 may thereforeresult from monosomy 6q.We found no report of patients with partial

trisomy 3q29-qter who could be compared topatient 1. All other aberrations of the long armof chromosome 3 have breakpoints far moreproximal than we have observed in patient 1.Clinical findings in patients with dup(3)(q26-*qter), the most distal duplication 3qpublished,32 36 differed from patient 1. Someshowed a phenotype resembling Cornelia deLange syndrome, features definitely absent inour patient.We tried to find chromosomal differential

diagnoses in patients 1 and 2 using the HumanCytogenetic Database.26 We found two chro-mosomal aberrations when matching tall stat-ure, macrostomia, broad neck, and strabismusas differential diagnoses for patient 1 (trisomy8 mosaicism and del(18)(q12.2-*q21.19)). Wecombined tall stature, microcephaly, long face,and arachnodactyly for patient 2 and found sixchromosomal aberrations: del (3)(pter-*p25)and dup(20)(ql3-*qter), trisomy 8 mosaicism,del(9) (pter--p22), dup(13) (q22-*qter), dup(15) (q22-*qter), and trip(1 8p). Interestingly,duplication 15q, with more distal breakpointspresent in patient 2, was mentioned. Thus, thephenotype in patient 2 may be representative ofa terminal duplication of 15q.Our investigation was preceded by routine

genetic counselling during the first pregnancyof the proband because of sibs with mentalretardation of unknown cause. When prometa-phase analysis suggested that three chromo-somes were involved, detailed investigations bymolecular cytogenetic techniques were carriedout. First, the exact nature of the balancedtranslocation was shown by FISH to be a threeway terminal translocation, 3q29-qter to15q26.1, 15q26.1-qter to 6q26, and 6q26-qterto 3q29. The translocation involved the termi-nal regions of the long arms of the threechromosomes, including a small translocatedsegment which was unrecognisable by conven-tional chromosome painting. The presence of a

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Figure 9 FISH results ofpatient 2. (A) D3Sl445/D3S1446: both chromosomes 3 showclear hybridisation signals in their long arm terminal regions, no signal is present in 15qter.(B) Whole chromosome 6 paint (red/TRITC) and whole chromosome 15 paint

(green/FITC): the results with WCP6 (red) are identical to the ones found in patient 1

while hybridisation with WCP15 (green) paints both chromosomes 15 entirely as well as

the long arm terminal region of one chromosome 6.

paracentric inversion of the chromosome 15involved in the translocation posed a particu-larly vexing problem. In contrast to theproximal breakpoint of the inversion (1 5ql 5.1),the distal breakpoint (15q26.1) was the same inthe inversion and the translocation. In patient 2and the balanced carriers, a subtelomeric FISHprobe specific for 3q29 (D3S1445, D3S1446)showed that chromosome 3 material was trans-

located to chromosome 15 in patient 2 and thebalanced carriers. The size of this translocationwas below the resolution limit of the wholechromosome paint.37 Failure to detect thecorrect chromosomal recipient would haveresulted in false results of the prenatal diagnosisperformed in the second and third pregnancy ofthe consultand.

This finding stresses the importance of cryp-tic terminal translocations in clinical cytoge-

netics. As Flint et at15 pointed out for patientswith so-called idiopathic mental retardation,

subtle translocations, among them terminalones, may be present in persons whosechromosomes appear normal in high resolutionbanded preparations and should be checkedwith appropriate subtelomeric FISH probes.Judging from our findings the same is true ifchromosomes give normal results after FISHwith whole chromosome painting probes.The risk for offspring of a carrier of a

balanced CCR is difficult to estimate." Batistaet at° gave a risk of miscarriage of about 50%based on an extended review of 30 familieswith CCRs. Gorski et at' analysed 25 CCRfamilies with 67 informative pregnancies andfound a risk of abnormalities of 18.4% for livebirths. Nearly 50% of all liveborn offspringwere also CCR carriers. The overall risk of anabnormal outcome of pregnancy seems to be ofthe order of about 50%. The consultand'smother gave birth to two healthy children (II.2and I1.5), one of them a balanced translocationcarrier, and two mentally retarded sibs withdifferent unbalanced karyotypes. Two furtherpregnancies resulted in abortions which havenot been investigated. One daughter died dur-ing the first weeks of life because of hydro-cephalus. One might speculate that she alsomay have had an unbalanced karyotype,because hydrocephalus is frequently found inpartial trisomy 6q and in partial monosomy15q. Both in the consultand and her mother,abnormal outcome of pregnancy was higherthan 50%.

The first two authors contributed equally to this work. Wewould like to thank the patients and their family for their coop-eration, Dr Gabriele Gillessen-Kaesbach for helpful discussions,and Dr Albert Schinzel and Dr Olivier Cohen for reviewingtheir databases. In addition, we thank Drs A J Green and L Wil-latt for providing amniotic cell cultures from the thirdpregnancy.

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