2 Med Genet 1994;31:234-237

Brief papers

Value of chromosome painting in determiningthe chromosomal outcome in offspring of a 12;16translocation carrier

C A Brandt, T Lyngbye, S Pedersen, L Bolund, U Friedrich

AbstractWe currently use direct and reverse chro-mosome painting in prenatal diagnosis. Ina family with a subtle 12;16 translocation,adjacent 1 segregation was diagnosed inthe first child, a boy, in whom symptomscompatible with partial trisomy 16p andpartial monosomy 12q were seen. In thenext pregnancy, a chorionic villus biopsywas tested using chromosome painting.Only by supplementing conventionalcytogenetic methods with molecularcytogenetic techniques could the truekaryotype be unequivocally determined.Reverse painting, using DOP-PCRamplified, flow sorted paternal derivativechromosomes as a DNA library to paintthe chorionic villus cells, was especiallyinformative.

(J Med Genet 1994;31:234-237)

Institute of HumanGenetics, TheBartholin Building,University of Aarhus,DK-8000 Aarhus C,DenmarkC A BrandtS PedersenL BolundU Friedrich

PaediatricDepartment, AarhusMunicipal Hospital,DenmarkT Lyngbye

CytogeneticLaboratory, Instituteof Basic Research inPsychiatry,Psychiatric Hospital,Aarhus, DenmarkU Friedrich

Correspondence toDr Friedrich.

Received 1 September 1993Accepted for publication7 October 1993

Partial trisomy of the short arm of chromo-some 16 has been reported several times (areview of 14 cases is given by Leonard et all).The phenotype varies depending on the size ofthe triplicated segment of the short arm ofchromosome 16 and the concomitant struc-tural abnormalities in the other chromosomesowing to unbalanced inheritance of the trans-location. Leonard et all pointed out that innearly half of the progeny with unbalancedtranslocations from the parents carrying thetranslocation, the breakpoint was in the telo-meric region. When small chromosomal frag-ments are exchanged and the shape of thechromosomes involved are not changed, thediagnosis in an unbalanced translocation car-rier may be difficult by conventional cytogene-tic methods. However, with molecular cytoge-netic methods subtle translocations are easilydiagnosed, even in chorionic villus samples, inspite of the inferior quality of chromosomes.We describe the diagnostic evaluation usingsuch methods in a family where a 12;16 trans-location was segregating.

Case reportThe proband, a boy, was the first child ofhealthy white parents who did not have anyhistory of genetic disease. At the time of his

birth the mother was 25 and the father 26 yearsof age.On routine fetal ultrasound scanning in the

16th week of pregnancy a cyst was found in thenuchal region. Consequently, a chorionic vil-lus biopsy was performed, which showed anormal karyotype; re-examination by ultra-sound in the 28th week showed that the cysthad disappeared. The findings were con-sidered to represent spontaneous regression ofa cystic hygroma. Otherwise, the pregnancywas uneventful.The delivery was spontaneous after 35

weeks and four days of gestation. The amnioticfluid was stained with meconium. Apart fromthis, the birth was normal with an Apgar scoreof 9 at five minutes. The birth weight was2440g. The child was vigorous with a loudcry. He had increased flexor responses in hisarms and hands and extensor tone of the body.The shape of the neurocranium was remark-ably round. He had hypertelorism as well asblepharophimosis and microphthalmia (fig 1),and bilateral cheilognathopalatoschisis al-though the gnathoschisis was not complete onthe left side. The mandible was small. Therewas a "tag" in front of the right ear and bothhelix and tragus of the left ear was smaller thanthe right. The skin was abundant in the gla-bella region and at the back of the neck, and healso had oedema on the back of the feet. Thesethree findings were considered to be suggestiveof hygroma. The hands exhibited flexion con-tractures of all proximal interphalangeal joints,and there was a tendency to overlapping of thefingers. The nails on both hands and feet weredysplastic and the fingernails were also slenderand hyperconvex. The feet showed a "sandalgap" between the first and the second toes. Aclosed sinus was seen over the sacral area.For the first two months he required diur-

etic treatment for uncompensated heart dis-ease, which proved to be the result of bothatrial and ventricular septal defects. When hewas 3 months old he was operated on forbilateral inguinal hernias. He showed muscu-lar hypertonia. From the beginning it wasclear that his rate of development was below50%. His ability to use his sight developeddespite the microphthalmia. At the age of 8months he developed seizures and exhibitedelectroencephalographic hypsarrhythmia.

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Value of chromosome painting in determining the chromosomal outcome in offspring of a 12;16 translocation carrier

)I

i.

Figure 1 The appearance of the proband in the first month after birth.

CYTOGENETIC STUDIESChromosomes from the proband wereobtained both from cultured amniocytes andfrom peripheral blood lymphocytes after birth.The metaphase chromosome preparationswere Q banded according to standard tech-niques. During routine prenatal diagnosis anormal male karyotype was found. After birth,the clinical findings prompted prometaphaseanalysis. Prometaphase chromosomes werestained with acridine orange and the bandingpattern of chromosome 12 disclosed a surplusband on the long arm of unknown origin.Re-examination of metaphase chromosomesshowed an apparently normal karyotypeexcept that the distal light region of one chro-mosome 12 seemed to be enlarged (fig 2).Chromosome analysis of both parents showeda normal karyotype in the mother and abalanced 12;16 translocation in the father(fig 2). His karyotype was 46,XY,t(12;16)(q24;pl2).Chromosome painting using a DNA library

generated from flow sorted normal chromo-somes 16 by the DOP-PCR technique wasperformed as described by Pedersen et al.2 Atranslocated short segment of chromosome 16could easily be recognised on the long arm ofone chromosome 12 in the father and in theproband. At the same time, one chromosome16 was too short in the father, whereas twonormal chromosomes 16 were observed in theproband. The proband has thus inherited anunbalanced translocation from his father withpartial trisomy 16p and partial monosomy 12q.

Q band R band

Father

Child

Figure 2 Q banded metaphase and R banded prometaphase chromosomes 12 and 16from the father and son. Arrows indicate the translocated segments.

Telomeric staining was performed by thePRINS technique as described elsewhere.5An oligonucleotide (CCCTAA), representingtelomeric sequences was used to stain the telo-meres of the chromosomes. The result showsnormal telomeres on all chromosomes includ-ing the derivative chromosomes der(12) andder(16) from the father and the proband andno signs of telomeres at the breakpoints. Thechromosomes were identified by Q bandingafter telomeric staining (data not shown).

In the next pregnancy, a chorionic villusbiopsy was taken in the ninth week. Chromo-some preparations were made after short termincubation. The conventional cytogenetic ana-lysis disclosed an apparently normal karyo-type. The chromosomes obtained from shortterm incubation were of inferior quality.Nevertheless, direct chromosome paintingwith the DNA library specific for chromo-some 16 showed two apparently normal chro-mosomes 16 and, more importantly, no chro-mosome 16 material on the long arm of chro-mosome 12 (fig 3c), supporting the diagnosisof a normal karyotype. The result was con-firmed by the reverse chromosome paintingtechnique6 using the DNA library generatedby flow sorting of the paternal derivative chro-mosome der(12) applied to metaphasesobtained from the cultured chorionic cells (fig3d). The reverse painting signals on pter ofboth chromosomes 16 unequivocally showedthe presence of terminal chromosome 16material and therefore the absence of theder(16) chromosome. Thus, a normal 46,XYkaryotype could be confirmed in the fetus.

DiscussionDetection of subtle reciprocal translocationsby fluorescence in situ hybridisation (FISH)has recently been described.7 The applicationof whole chromosome libraries for chromo-some in situ suppression (CISS) hybridisationhas great advantages89 in the detection andcharacterisation of chromosomal rearrange-ments. We are currently applying both directand reverse chromosome painting to the prac-tice of prenatal diagnosis.

In the first pregnancy in this family anunbalanced subtle translocation was over-looked, since the size, proportion, and band-ing pattern of the terminal rearrangementappeared almost unchanged compared to thenormal homologous chromosome. Severalclinical signs in the proband after birth indi-cated, however, a chromosomal imbalance.This suspicion was enhanced by the finding ofa slight difference in staining intensity in thefaintly banded chromosome region of the dis-tal long arm of chromosome 12. Thus, morerefined cytogenetic methods were applied.Still, only when the exact nature of thebalanced karyotype in the father was knownwas it possible to perform prenatal diagnosisin this family using molecular cytogeneticmethods. A combination of direct paintingusing a DNA library generated from flowsorted chromosomes 16 (a DNA library fromflow chromosome 12 is not obtainable by flow

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Brandt, Lyngbye, Pedersen, Bolund, Friedrich

Figure 3 Direct chromosome painting with a chromosome 16 library showing (a) one normal chromosome 16, theshorter der(16) chromosome, and the der(12) in the father who has a karyotype 46,XY, t(12;16) (q24;pl2); (b)painting of two normal chromosomes 16 in combination with der(12) indicating an unbalanced karyotype in the son;and (c) painting (short term incubated chorionic villus cells) of two normal chromosomes 16. There are no signals onchromosome 12, indicating a normal karyotype in the fetus. (d) Reverse painting using the DNA library generatedfrom the derivative chromosome der (12) from the father confirmed the normal karyotype in the fetus in chorionicvillus preparations cultured for 10 days. The picture was taken from the computer screen of the confocal scanningequipment.

sorting, because chromosomes 9, 10, 11, and12 cannot be distinguished by conventionalflow fluorometric analysis) and reverse paint-ing using a DNA library generated from flowsorting of the paternal, slightly enlargedder(12) chromosome were used. This strategyproved to be more sensitive in establishing thesegregation of the 12;16 translocation from thefather than conventional banding analysis andtherefore indispensable in this prenatal dia-

gnosis, because of the small size of the translo-cated segment.The fact that Leonard et all found most

cases of trisomy 16p to be caused by inherit-ance of translocation chromosomes makes thediagnosis more feasible in cases of subtle trans-locations. On the other hand, the location ofthe breakpoint in the telomeric region makesthe diagnosis more difficult, especially whentesting cultured tissue as in prenatal diagnosis.

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Value of chromosome painting in determining the chromosomal outcome in offspring of a 12;16 translocation carrier

Direct painting has proven its value in charac-terising the constitution of the derivative chro-mosome in cases of terminal rearrangements.Furthermore, in cases of 3: 1 segregation,which Leonard et all found in eight out of 14cases with dup(16p), reverse chromosomepainting using a DNA library generated from aflow sorted preparation of the supernumerarychromosome can establish the origin and com-position of the extra chromosome.The presence of interstitial telomeric se-

quences has been suggested in several reportsinvolving terminal rearrangements.'0 Thehexanucleotide sequence, a human telomereconsensus (TTAGGG)., identified by Moyziset al," was used as a priming oligonucleotide((CCCTAA)7) to visualise the telomeres byPRINS of the derivative chromosomesinvolved in our case. The presence of thesequence only at the terminus of the chromo-somes indicates that the breakpoint in thereciprocal translocation 12;16 was proximal tothe region where the telomeric sequences areplaced.

Molecular cytogenetics has become an inte-grated part of our cytogenetic procedures.Time and economy preclude the use of thesetechniques as routine tools as yet. However,molecular cytogenetics should be used as asupplement to routine cytogenetic studies, if akaryotype appears to be normal under circum-stances where there is particular suspicion ofchromosomal aberration. This is so in caseslike the one presented here, when fetal ultra-sound or clinical investigation raise suspicion.We have, in the present case, significantly

increased the diagnostic sensitivity with theintroduction of direct and reverse chromosomepainting to prenatal diagnosis of terminal re-arrangements.

The expert technical assistance of Monna Caprani and HelleStr0mkjwr is appreciated. Kim Keilberg and Kirsten Millgaardare thanked for the photographic work, Anette Sorensen fortyping the manuscript, and Tracey Flint for language correc-tion. This work was supported by grant 5.18.10.03 from theDanish Human Genome Research Programme (CAB, LB).

1 Leonard C, Huret JL, Imbert M-C, et al. Trisomy 16p in aliveborn offspring due to matemal translocationt(16;21)(ql 1;pl 1) and review of the literature. AmJ7 MedGenet 1992;43:621-5.

2 Pedersen S, Hindkjoer J, Brandt CA, Bolund L, Kolvraa S.Reverse chromosome painting. In: Choo KHA, ed.Methods in molecular biology. In situ hybridization proto-cols. New Jersey: The Humana Press 99-102.

3 Hindkjer J, Koch J, Mogensen J, et al. Primed in situlabelling of nucleic acids. Int 7 Biotech 1991;12:752-6.

4 Brandt CA, Kierkegaard 0, Hindkjxr J, et al. Ring chromo-some 20 with loss of telomeric sequences detected bymulticolour PRINS. Clin Genet 1993;44:26-31.

5 Brandt CA, Djernes B, StromkjTr H, et al. Pseudodicentricchromosome 18 diagnosed by chromosome painting andprimed in situ labelling (PRINS). 7 Med Genet1994;31:99-102.

6 Telenius H, Palmear AH, Tunnacliffe A, et al. Cytogeneticanalysis by chromosome painting using DOP-PCR am-plified flow-sorted chromosomes. Genes Chrom Cancer1992;4:257-63.

7 Speleman F, van Roy N, Wiegant J, et al. Detection ofsubtle reciprocal translocations by fluorescence in situhybridization. Clin Genet 1992;41:169-74.

8 Pinkel D, Landegent J, Collins C, et al. Fluorescence in situhybridization with human chromosome-specific libraries:detection of trisomy 21 and translocation of chromosome4. Proc Natl Acad Sci USA 1988;85:9138-42.

9 Jauch A, Daumer C, Lichter P, et al. Chromosomal in situsuppression hybridization of human gonosomes and auto-somes and its use in clinical cytogenetics. Hum Genet1990;85: 145-50.

10 Park VM, Gustashaw KM, Wathen TM. The presence ofinterstitial telomeric sequences in constitutional chromo-some abnormalities. AmJ Hum Genet 1992;50:914-23.

11 Moyzis RK, Buckingham JM, Cram LS, et al. A highlyconserved repetitive DNA sequence, (TTAGGG)n, pre-sent at the telomeres of human chromosomes. Proc NatlAcad Sci USA 1988;85:6622-6.

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