Mirror-Symmetric Duplicated Chromosome 21qWith Minor Proximal Deletion, andWith Neocentromere in a Child Without theClassical Down Syndrome Phenotype

G. Barbi,1* I. Kennerknecht,1 G. Wohr,1 D. Avramopoulos,2 G. Karadima,2 and M.B. Petersen2

1Abteilung Medizinische Genetik der Universitat, Ulm, Germany2Department of Genetics, Institute of Child Health, Aghia Sofia Children’s Hospital, Athens, Greece

We report on a mentally retarded child withmultiple minor anomalies and an unusuallyrearranged chromosome 21. This der(21)chromosome has a deletion of 21p and ofproximal 21q, whereas the main portion of21q is duplicated leading to a mirror-symmetric appearance with the mirror axisat the breakpoint. The centromere is onlycharacterized by a secondary constriction(with a centromeric index of a G chromo-some) at an unexpected distal position, butfluorescence in situ hybridization (FISH)with either chromosome specific or with allhuman centromeres alpha satellite DNAshows no cross hybridization. Thus, themarker chromosome represents a furtherexample of an “analphoid marker with neo-centromere.” Molecular analysis using poly-morphic markers on chromosome 21 veri-fied a very small monosomic segment of theproximal long arm of chromosome 21, andadditionally trisomy of the remaining distalsegment. Although trisomic for almost theentire 21q arm, our patient shows no classi-cal Down syndrome phenotype, but only afew minor anomalies found in trisomy 21and in monosomy of proximal 21q, respec-tively. Am. J. Med. Genet. 91:116–122, 2000.© 2000 Wiley-Liss, Inc.

KEY WORDS: trisomy 21; monosomy 21;analphoid marker chromo-

some; neocentromere; micro-dissection

INTRODUCTION

Down syndrome is thought to depend on the dupli-cation of only a small segment of chromosome 21q, yeteven in “complete” trisomy 21 a broad spectrum of clini-cal variability has been established [Jackson et al.,1976; Avramopoulos et al., 1997], thus making karyo-type/phenotype correlations rather difficult. We addthe observation of a further case with trisomy of almostall of 21q accompanied by a small proximal deletion.However, the phenotype of our patient was dominatedby the findings of monosomy. The chromosomal imbal-ance described is the consequence of a structurally al-tered chromosome 21 with an unexpected centromereposition, which adds a further contribution to the re-cently defined class of analphoid marker chromosomeswith neocentromere [Choo, 1997].

MATERIALS AND METHODSCytogenetic Analysis

Chromosome analysis was performed on lympho-cytes cultured in medium RPMI 1640 plus 16% fetalcalf serum (FCS) after phytohemagglutinin (PHA)stimulation. Chromosomes were prepared and stained[GTG-, QFQ-, CBG-banding, silver staining of nucleo-lus organizer regions (Ag-NOR), RBG replication pat-terns] according to standard techniques. Microdissec-tion was done as described by Senger et al. [1990] usingan inverted microscope type IM (Zeiss, Oberkochen,Germany) equipped with objectives of 5×, 10×, and 63×magnification, and with eyepieces of 16× magnifica-tion, respectively, and a rotatory Zeiss stage gliding onan oil film. Microneedles with a tip of about 0.5 mmdiameter were drawn from borosilicate glass bars of 2mm diameter (HWS Labortechnik, Mainz, Germany)using a vertical pipette puller model 700C (David KopfInstruments, USA), and moved by a motorized me-chanical micromanipulator AM 3 DC-K (Bachofer, Re-

Grant sponsor: E.C. BIOMED; Grant numbers: GENE-CT93-0015, BMH4-CT96-0554; Grant sponsor: Jacob Madsens & Hus-tru Olga Madens Fond, Wedellsborg Fond, Hartmanns Fond,Kirstine Fonden, Kong Christian den Tiendes Fond, Lily Ben-thine Lunds Fond.

*Correspondence to: G. Barbi, Abteilung Medizinische Genetik,Universitat Ulm, Parkstr. 11, D-89073 Ulm, Germany.E-mail: gotthold.barbi@medizin.uni-ulm.de

Received 7 June 1999; Accepted 30 November 1999

American Journal of Medical Genetics 91:116–122 (2000)

© 2000 Wiley-Liss, Inc.

utlingen, Germany). Briefly, using fresh metaphasespreads dropped under sterile conditions on a pre-cleaned coverglass, and G-banded immediately prior tomicrodissection, a certain chromosome or segment wasdissected, attached to the needle, and collected in acollection drop of between 10 and 100 nl volume (1volume: 10 mM Tris/HCl, pH 7.5; 10 mM NaCl; 0.1%sodium dodecyl sulfate (SDS); 0.5 mg/ml Proteinase K+ 1 volume: glycerol). After collection of at least 10copies, amplification of the selected DNA by polymer-ase chain reaction (PCR) with degenerate oligonucleo-tide primers (DOP-PCR) [Telenius et al., 1992], andsubsequent labeling with biotinylated dUTP (Boe-hringer, Mannheim, Germany) was performed as de-scribed [Chudoba et al., 1996].

A whole chromosome painting library specific forchromosome 21 (AGS, Heidelberg, Germany) was hy-bridized according to the supplier’s protocol. Plasmidprobe L1.26 (ATTC, Rockville, MD) containing alphasatellite DNA specific for the centromeres of chromo-somes 13 and 21 (D13Z1/21Z1) was used as a fluores-cence in situ hybridization (FISH) probe after labelingwith biotinylated dUTP by nick translation. Biotinyl-ated alpha satellite DNA used for the detection of allhuman centromeres, and digoxigenin-labeled cosmidDNA representing locus D21S65 (ONCOR, Gaithers-burg, MD), were hybridized according to the supplier’sprotocol. For detection of biotinylated probes Fluores-cein isothiocyanate (FITC)-avidin was used; digoxi-genin-labeled probes were detected by mouse anti-digoxigenin antibody followed by incubation withFITC-conjugated anti-mouse antibody from sheep andsubsequently with FITC-anti-sheep antibody.

FISH experiments were recorded at a Zeiss Axioplanmicroscope equipped with appropriate filter sets for thefluorochromes FITC, 48, 6-diamidino-2-phenylindoledihydrochloride (DAPI), and propidium iodide, anddocumented with the digital imaging capture systemISIS (Metasystems, Altlussheim, Germany) using amonochrome camera.

Molecular Analysis

Molecular analysis was done using DNA from bloodlymphocytes of the proband and his parents using stan-dard extraction procedures [Miller et al., 1988]. TheDNA was used for PCR amplification of polymorphicshort sequence repeat sequences (microsatellites) local-ized along the entire length of 21q. The polymorphicalleles were visualized after end labeling of primer,electrophoresis of the radiolabeled PCR productsthrough denaturing acrylamide gels, and autoradiog-raphy, as previously described [Economou et al., 1990;Petersen et al., 1990]. The scoring of polymorphic al-leles was by visual comparison of the intensity of al-leles as described [Petersen et al., 1991]. The order ofmarkers used was known from the linkage and physi-cal maps of human chromosome 21 [Korenberg et al.,1995; Shimizu et al., 1995].

RESULTS

Our patient is the first child of a 30-year-old primi-gravida and her non-consanguineous 31-year-old hus-

band. During pregnancy, at 12 weeks of gestation, anuchal edema had been detected by ultrasonic surveil-lance. After amniocentesis, a “normal” karyotype(46,XY) had been reported in another laboratory. Atgestational age of 37 weeks, premature rupture of themembranes was reported which resulted in spontane-ous delivery at 38 weeks.

The newborn infant [birth weight (BW) 3,660 g,birth length (BL) 54 cm, occipitofrontal circumference(OFC) 36 cm] presented with general muscular hypo-tonia, nuchal fold, epicanthus. Cardiologic examinationshowed a complete atrioventricular septum defect(mixed type/type A according to Rastelli) , and a fibro-muscular subaortic stenosis. Banding of pulmonary ar-tery and ductus ligation were performed at age 1month. Seventeen months after birth, our patient hadan ileus with surgical removal of a Meckel diverticu-lum, and resection of the aganglionic segment of thecolon 1 year later.

Fig. 1. Our patient at 3.5 years.

Mirror-Symmetric Duplicated Chromosome 21q 117

On clinical reexamination at 3.5 years (Fig.1; TablesI and II) the patient had severe statomotoric and men-tal retardation: he could not stand freely nor speak asingle word. He had a peculiar face with prominenceof the metoptic suture, epicanthus, low nasal bridge,anteverted nostrils, long smooth philtrum, invertedupper lip, retrognathia, high-arched palate, a deeplyfurrowed tongue, a low-set left ear, nuchal cutis laxa,inguinal testes, atypical single palmar creases andclinodactyly of the fifth fingers of both hands, andoverlapping toes bilaterally. Fontanelles had closed onlyat the age of 3 years. Chronic enteritis was diagnosed.

Because of the obvious anomalies of the infant, asecond chromosome analysis was performed using cul-tured lymphocytes after PHA stimulation, which led tothe detection of a structurally altered G chromosomeinstead of a normal chromosome 21 as the only detect-able aberration in each of more than 50 cells analyzed(Fig. 2). This marker chromosome had a broader Gband than the normal chromosome 21 suggesting a du-plication of 21q chromatin (Fig. 2a,b). The marker hada subtelomeric primary constriction as the only evi-dence of an active centromere (Fig. 2c). However, C-banding did not stain the pericentromeric chromatinproperly (Fig. 2d). The der(21) had no satellites eitherby QFQ-banding or by homogeneous Giemsa staining(Fig. 2b,c). Silver-staining of Ag-NOR was negative onthe marker (Fig. 2e), whereas all 4 parental homolo-gous chromosomes 21 had Ag-positive NORs and sat-ellites (not shown). Irrespective of the position of thecentromere, banding-patterns (GTG, QFQ, RBG) sug-gested a mirror-symmetric appearance of the der(21)

with presumed mirror axis in the proximal long arm(Fig. 2a,b,f).

After FISH study with a whole chromosome 21 spe-cific library, the marker chromosome was completelypainted (not shown) indicating a duplication of 21qchromatin. However, neither with chromosomes 13/21specific alpha satellite DNA (D13Z1/D21Z1) (notshown) nor with all human centromere specific DNA(Fig. 3) a specific hybridization signal could be demon-strated on the der(21) chromosome.

The mirror-symmetric appearance of the der(21)chromosome, suggested by standard banding, couldalso be visualized by a symmetric signal after hybrid-ization with a cosmid from locus D21S65 in 21q22 (Fig.4), and with a microdissection library of distal 21q froma normal individual. Painting with this library gavesignals of anteverted polarity at both distal ends of theder(21) chromosome (Fig. 5). Together with the resultsobtained by standard cytogenetics, this experimentalso demonstrates, that the “short” and “proximal longarm” of the marker chromosome consists of chromatinoriginating from distal 21q.

Reverse painting with a microdissection library ofthe whole der(21) chromosome showed cross hybridiza-tion only with the marker and the long arm of the nor-mal chromosome 21 (not shown). Thus, 21p chromatinseemed to be deleted in the marker. Furthermore,within the limits of the sensitivity of the methods used,a complex translocation involving a chromosome differ-ent from 21 can be excluded.

As shown in Table III, most of 21q molecular mark-ers (consecutive loci from D21S11 to D21S1575) are

TABLE I. Signs of Down Syndrome in the Propositus, in Patient VK of Bradley et al. [1986]/Van Keuren et al. [1989], and inSome Patients With Comparable Partial Trisomies 21 Alone [Korenberg et al., 1994]*

Patients:proximal breakpoint of duplication:

Pro-bandq21.1

VKq21

DSq21.1

SOSq21.1

1413q21.3

SOLq22.1

JSB NA NO BA SM

q22.12/q22.13

Phenotypic findingShort stature + ± − ± − + + +Microcephaly − + − + − − − −Brachycephaly − − − + + − − − −Flat facies + + + + + + + +Upslanting palpebral fissures − + + + + + + + + + +Epicanthic folds − + + + + + + + +Brushfield spots ? + − + − − −Flat nasal bridge + + + − + + + + +Open mouth − + + + − + + + −Furrowed tongue + + + − + − −High-arched palate + + − + − − +Narrow palate +Malpositioned ears + + − + + − − − +Small/dysmorphic ears − + + − + + + − − +Short neck − + + + − − − +Loose skin of neck +Short and broad hands − + + + + + + + +Brachydactyly/short 5th finger − + + − + + + + + −Incurved 5th finger − + + − + + + −Transverse palmar crease (+) + − + − + + +Gap between toes 1 and 2 + + + + − + −Cardiac anomaly + − + − + − + − + +Duodenal stenosis − − + − − − − −Joint hyperflexibility − + − + + + −Muscular hypotonia + − − + − − + −

*Presence (+)/absence (−) of signs from the list of Jackson et al. [1976] modified according to Korenberg et al. [1994].

118 Barbi et al.

represented by one maternal and two paternal alleles,whereas a small pericentric segment (at least betweenthe loci D21S258 and D21S1228, more proximal lociwere uninformative) is only represented by single ma-ternal alleles, respectively. These data demonstratethat the marker is of paternal origin with a deletion inproximal 21q, and a duplication of the remainder of21q, with no evidence for a disomic segment at thejunction. Duplication of identical paternal alleles sug-gests a “mitotic origin,” e.g., inverted duplication byfusion of sister chromatids after breakage in a G2 phasecell. The molecular genetic findings support the cyto-genetic interpretation of a mirror-symmetric derivativechromosome with loss of its original centromere andacquisition of a neocentromere.

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Fig. 2. Homologous chromosomes 21 of our patient (normal chromo-some left, marker chromosome right) after (a) GTG-banding, (b) QFQ-banding, (c) homogeneous Giemsa staining, (d) CBG-banding, (e) silver-staining of nucleolus-organizer-regions (Ag-NOR), (f) RBG replicationpattern, (g) representative ideogram.

Mirror-Symmetric Duplicated Chromosome 21q 119

DISCUSSION

We report on a patient with both proximal monosomy21 and distal trisomy 21 with no hint of a disomic seg-ment between. The combination of monosomy and tri-somy makes a comparison with other patients forkaryotype/phenotype correlations difficult. Cases withmere duplication of distal 21q represent the variabilityof full trisomy 21 in their clinical picture, as shown bycomparison with a few selected cases (Table I). Al-though our patient presents a comparable score ofsigns common with Down syndrome as other patientswho have a smaller duplicated segment [Jackson et al.,1976; Korenberg et al., 1994] (Table I), these signs aredominated by the manifestations of monosomy 21, sothat his aspect would never suggest a clinical diagnosisof Down syndrome without knowledge of the trisomy.

Only a few cases with a comparable deletion of theproximal 21q arm have been characterized with mo-lecular methods (Tables II and III). Among these ourpatient and the one described by Huret et al. [1995]have the smallest deletions. These two patients presentmore severe psychomotor retardation, which is also nottypical for Down syndrome. In contrast, patient GMU,who has a larger deletion extending from pter to locusD21S11 in q21.1, is reported to have normal intelli-gence [Chettouh et al., 1995].

A similar mirror-symmetric duplicated der(21) chro-mosome has been reported [Bradley et al., 1986; VanKeuren et al., 1989], which, however, had retained itscentromere and short arm material. It is interesting tocompare our patient with this one, who has a largerproximal deletion, and a smaller distal trisomic seg-ment, respectively (Table III), but was reported to pre-sent facial signs typical of Down syndrome.

The structural chromosome aberration detected inour patient is an inverted duplication of a terminalchromosome arm. This marker chromosome was pres-ent in each of the cells investigated, and it has an un-characterized centromere at an unexpected position.Cytogenetic techniques including FISH with alpha sat-ellite DNA, or reverse painting with total marker DNA,failed to detect the regular characteristics of a humancentromere in the der(21), except a mere primary con-striction. During recent years several reports of similarmitotically stable structurally aberrant marker chro-mosomes have been published, which obviously lackalpha satellite DNA sequences at their active centro-meres [Voullaire et al., 1993; Blennow et al., 1994;Ohashi et al., 1994; Bukvic et al., 1996; Sacchi et al.,1996; Depinet et al., 1997; Vance et al., 1997]. Theder(21) chromosome described here most probably rep-resents a further example of such an “analphoidmarker chromosome with a neocentromere.” This newclassification of marker chromosomes was recentlytermed and summarized, and is mainly represented bymirror-symmetric duplicated terminal chromosomearms with a functional centromere at an unexpected

Fig. 3. FISH with “all centromeres” alpha satellite DNA (arrowheadpoints at the der(21) chromosome).

Fig. 4. Hybridization signals (arrows) at locus D21S65 on the normaland the derivative hromosome 21.

Fig. 5. Painting with a microdissection library from terminal 21q on theder(21) (anteverted arrows) and the normal chromosome 21 (arrow).

120 Barbi et al.

site [Choo, 1997]. As already expected by Choo [1997],our example also contributes a further “putative latent-centromeric site” within distal 21q according to the hy-pothesis, that latent DNA different from alpha satel-lites and with sequences dispersed over the genomemay be reactivated by a hitherto unknown mechanismto bind centromeric proteins and to become an activecentromere [Voullaire et al., 1993]. The duplication ofidentical paternal alleles at polymorphic loci in themarker presented here suggests a “mitotic origin” suchas breakage and fusion of sister chromatids in a G2phase cell, as already suggested for other examples ofanalphoid marker chromosomes with neocentromeres[Choo, 1997]. Further investigations such as trappingthe DNA of such neocentromeres by immunohisto-chemical binding studies with centromeric proteinsand subsequent characterization of the associatedDNA sequences in as many cases as possible mighthelp to elucidate the essential role of DNA sequenceand structure with respect to centromere function.

ACKNOWLEDGMENTS

This work was supported by E.C. BIOMED grantsGENE-CT93-0015 and BMH4-CT96-0554 to the Euro-pean Chromosome 21 Consortium, Fru C. HermansensMindelegat, Direktør Jacob Madsens & Hustru OlgaMadsens Fond, Else og Mogens Wedell-WedellsborgsFond, Smedemester Niels Hansen og hustru Johanne f.Frederiksen’s Legat, Brødrene Hartmanns FondKirstine Fonden, Kong Christian den Tiendes Fond,and Lily Benthine Lunds Fond (MBP). The authorsthank Antje Kollak and Ingrid Peter for skillful tech-nical assistance.

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TABLE III. Molecular Genetic Data of the Proband Compared With Literature Data on Partial Monosomy 21q

Band Locus

Genotypea Cases from the Literatureb–d

Fa Prob Mo VK BM CO HE JC RO RE

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D21S13 − − − ? − −21.1 D21S192 11 2 22 ? ?

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21.3 D21S217 11 112 22D21S218 12 122 13D21S54 + −D21S226 + ?

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22.2 HMG14 11 111 12 S17+D21S39 +D21S53 +

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22.3 D21S171 11 111 11 + ?D21S1575 12 122 13

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