Characterization of a Small Supernumerary RingMarker Derived From Chromosome 2 by Forwardand Reverse Chromosome Painting

Nadezhda V. Ostroverkhova,1 Sergey A. Nazarenko,1* Nikolay B. Rubtsov,2 Ludmila P. Nazarenko,1and Elena N. Bunina3

1Institute of Medical Genetics, Tomsk, Russia2Institute of Cytology and Genetics, Novosibirsk, Russia3Regional Medical Diagnostic Center, Chita, Russia

A small ring-shaped supernumerary markerchromosome (SMC) was detected in 50% ofmetaphase cells in an 18-month-old boy withmental retardation and multiple congenitalanomalies. Conventional cytogenetic meth-ods had failed to identify the origin of themarker. When the patient was age 11.5years, we defined the origin of the SMC byfluorescence in situ hybridization using abattery of centromere-specific DNA probes.The marker was positive with the probe forlocus D2Z. More detailed characterizationwas achieved by using chromosome 2 arm-specific and marker-specific DNA libraries,which were constructed by microdissectionof the two arms chromosome 2 and SMCwith subsequent amplification of the chro-mosomal material by a degenerate oligo-nucleotide-primed polymerase chain reac-tion (DOP-PCR). The marker was identifiedas r(2)(p11.2→q14.1). The propositus haddolichocephaly, coarse hair, low-set ears,exophthalmos, epicanthal folds, strabismus,depressed nasal bridge, high-arched palate,excess of skin on the neck, tapered fingerswith mild clinodactyly, talipes varus on theright, inguinal hernia, hypogenitalism, mus-cular hypotonia, and mental retardation.This is the first case of SMC derived fromchromosome 2 that was characterized byforward and reverse chromosome painting.Am. J. Med. Genet. 87:217–220, 1999.© 1999 Wiley-Liss, Inc.

KEY WORDS: supernumerary marker chro-mosome; microdissection;ring chromosome; proximal

trisomy 2q; phenotype/karyo-type correlations

INTRODUCTION

Supernumerary marker chromosomes (SMCs) arerelatively rare in the general population. They arefound in approximately 0.3–1.2/1,000 newborn infantsand more frequently (3.27/1,000) among mentally re-tarded patients [Nielsen and Rasmussen, 1975; Buck-ton et al., 1985]. SMCs represent a heterogeneousgroup and are described in patients with different phe-notypes. The range of phenotypic expression is relatedto the size of a marker, its euchromatic content, chro-mosomal origin, and level of mosaicism.

The chromosomal origin of small markers is oftendifficult to determine by conventional cytogeneticmethods. To date approximately 150 cases with SMCswere identified with molecular cytogenetic techniques[Plattner et al., 1993b; Blennow et al., 1995; Crolla,1998; Crolla et al., 1998; Viersbach et al., 1998]. Mostwere studied by using centromere-specific DNA probes,which allow identification of only the chromosomal ori-gin of the markers. Recently developed techniques ofmicrodissection of banded human chromosomes in con-junction with fluorescence in situ hybridization (FISH)(reverse and forward chromosome painting) providemore precise characterization of SMCs and conse-quently of their correlation with the clinical phenotype[Blennow et al., 1992; Viersbach et al., 1994].

Chromosome 2 is not often involved in the formationof marker chromosomes [Callen et al., 1992; Crolla,1998; Crolla et al., 1998; Viersbach et al., 1998]. Onlytwo cases with a very small SMC derived from of chro-mosome 2 have been detected to date by FISH withcentromere-specific DNA probes [Plattner et al., 1993b;Daniel et al., 1994]. We describe a new case of small-ring SMC derived from chromosome 2 that was char-acterized by forward and reverse chromosome paintingwith chromosome 2 arm-specific and marker-specificDNA libraries.

*Correspondence to: Prof. Sergey A. Nazarenko, Institute ofMedical Genetics, Ushaika 10, Tomsk, 634050, Russia. E-mail:snaz@img.tsu.ru

Received 5 March 1999; Accepted 13 July 1999

American Journal of Medical Genetics 87:217–220 (1999)

© 1999 Wiley-Liss, Inc.

CLINICAL REPORT

The propositus was referred for investigation at age18 months because of developmental delay and physi-cal anomalies. He was born as the third child of a 28-year-old woman and a 33-year-old man. The older sibsare normal. The pregnancy was unremarkable, and thechild was delivered at 34 weeks of gestation. Birthweight was 2,800 g, length 50 cm, and head circumfer-ence 32 cm. He had feeding problems in early infancy.At age 12 months, in association with a viral respira-tory tract infection, seizures occurred, which continuedfollowing recovery from infection. He sat at age 10months and walked with support at age 18 months. Atthat age he had not yet developed speech. His weight(9.8 kg), length (76 cm), and head circumference (45.5cm) were all below the 10th centile. In addition, he haddolichocephaly, coarse hair, low-set ears, exophthal-mos, epicanthal folds, strabismus, depressed nasalbridge, high-arched palate, excess of skin on the neck,tapered fingers with mild clinodactyly, wide space be-tween first and second toes, talipes varus on the right(Fig. 1). He had also muscular hypotonia and inguinalhernia.

When seen at 11.5 years (Fig. 2), the child had aweight of 34 kg (40th centile), a height of 140 cm (25thcentile), and a head circumference of 51 cm (10th cen-tile). He had moderate mental retardation and hypo-genitalism.

RESULTS

G-banded chromosome analysis on peripheral bloodshowed a mosaic karyotype: 46,XY/47,XY,+r. The ringchromosome was small and present in 50% of the cells.The marker was C-positive, distamicin A/DAPI-negative, and did not contain NOR-positive satellites.The parents’ chromosomes were normal.

In order to determine the origin of SMC, FISH wasperformed with a battery of centromere-specific DNAprobes either for individual chromosomes or groups ofchromosomes (Oncor, Gaithersburg, MD). A clear hy-bridization signal (Fig. 3A) was obtained with an al-phoid DNA probe for the centromeric region of chromo-some 2 (D2Z).

The need for a precise characterization of the markerprompted us to apply an additional molecular cytoge-netics approach. Chromosome 2 and SMC-specific DNAlibraries were constructed using microdissection ofboth arms of chromosome 2 and of SMC with subse-quent amplification of the chromosomal material by adegenerate oligonucleotide-primed polymerase chainreaction (DOP-PCR). The microdissection was carriedout as described by Senger et al. [1990]. Altogether thep arms of seven chromosome 2 were excised using ex-tended glass needles under microscopical view (in-

Fig. 1. Patient at age 18 months. Fig. 2. Patient at age 11.5 years.

218 Ostroverkhova et al.

verted microscope IM 135, Zeiss, Oberkochen, Ger-many). In the same manner seven q arms of chromo-some 2 and of SMC were dissected and prepared forDNA-amplification. After excision of the chromosomesthe fragments were transferred into a 10-nl collectiondrop, containing 10 mM TRIS-HCl, pH 7.5, 10 mMNaCl, 0.1% sodium dodecyl sulfate, and 0.5 mg/ml pro-teinase K, which was overlaid with mineral oil andplaced inside a small moist chamber. Protein digestionwas carried out for 2 hours at 60°C. Then the collectiondrop was transferred to a microreaction tube contain-ing 2.5 ml of the reaction mixture (0.2 × Sequenasebuffer, 400 mM each dNTP). Prior to DNA amplificationthe degenerate oligonucleotide primers [Telenius et al.,1992] were pretreated with topoisomerase I, which sig-nificantly reduces PCR artifacts that are caused by thehigh primer concentration used in the present protocol[Rubtsov et al., 1996]. The PCR conditions and fluores-cence in situ hybridization were essentially as de-scribed by Rubtsov et al. [1996].

Forward chromosome painting using 2p arm-specificpainting probe showed a very weak signal on themarker chromosome (Fig. 3B). However FISH with 2qarm-specific painting probe gave a stronger signal (Fig.3C). This result suggests that the marker chromosomecontains mainly material from proximal 2q.

In order to determine precisely the breakpoints onchromosome 2, reverse chromosome painting withSMC-specific DNA library was performed on normalmetaphases. A clear hybridization signal was obtainedmainly on the proximal part of the long arm of chro-mosome 2 (Fig. 3D) and a weak hybridization signal onthe proximal 2p. The reverse chromosome painting us-ing the marker chromosome library was performed onmetaphases from the patient, which gave wholemarker labeling, i.e., confirming that the microdissec-tion and DOP-PCR reaction were correctly performed(data are not shown). The karyotype of our patientwas interpreted as 46,XY/47,XY,+mar.rev ishr(2)(p11.2→q14.1).

Fig. 3. Molecular cytogenetics characterization of SMC. A: FISH to metaphase chromosome from the patient using chromosome 2 centromere-specificDNA probe D2Z. Signal is present on both normal chromosome 2 centromeres and marker chromosome (arrow). Forward chromosome painting using 2p(B) and 2q (C) arm-specific probes. Signal is present on both arms of chromosome 2 and on the marker chromosome (arrow). Note weak signal on makerwith 2p arm-specific probe and strong signal with 2q arm-specific probe. D: Reverse chromosome painting using marker-specific probe. Note signal mainlyon the proximal part of the long arm of chromosome 2 (arrows).

Small Supernumerary Marker Chromosome 2 219

DISCUSSION

Some carriers of a SMC have a normal phenotype,but certain nonsatellite markers can be associated withmental retardation and malformations [Callen et al.,1992; Blennow et al., 1995; Plattner et al., 1993b].Chromosome 2 is not often involved in the formation ofmarker chromosomes [Crolla et al., 1998; Crolla, 1998;Viersbach et al., 1998]. To date, only two cases with avery small ring SMC derived from chromosome 2 andidentified by FISH using a-satellite DNA-probes havebeen reported [Plattner et al., 1993b; Daniel et al.,1994]. In the first case the SMC was detected in a 7.5-year-old boy with developmental delay and autism butwithout physical anomalies [Plattner et al., 1993a].This marker arose de novo and was found in 30% ofperipheral leukocytes. In the second case the SMC wasidentified in a 30-year-old phenotypically normal malein 100% of the cells examined and had familial occur-rence [Daniel et al., 1994]. Absence of significant clini-cal manifestations suggests that this SMC probablyconsisted of repetitive DNA sequences.

Most reported cases of 2q trisomy are de novo distaltrisomies. Proximal 2q trisomy is a rare event. Apartfrom two above-mentioned cases with a SMC arisingfrom chromosome 2 there is only one reported case withpartial trisomy of proximal 2q in which the breakpointswere identified [Mu et al., 1984]. The proposita, a 3.5-year-old girl with physical anomalies and mental re-tardation had a de novo direct tandem duplication ofthe region 2(q11.2→q14.2). Her abnormalities includedshort stature, microcephaly, brachycephaly, depressednasal bridge, prominent philtrum, mild clinodactyly,congenital glaucoma, muscular hypotonia, and mentalretardation. Although the phenotype of our patientshowed some similar clinical findings the delineation ofa proximal 2q trisomy syndrome may be possible onlywhen further cases with similar chromosomal abnor-malities will be detected.

There are other mechanisms that may have causedthe manifestations in patients with an SMC. Individu-als bearing SMSs might have an increased risk of beinguniparental disomic for the structurally normal homo-logues of the SMC [Robinson et al., 1993; James et al.,1995]. The parental origin of the ring chromosome aswell as of the chromosome 2 homologs was not inves-tigated because the patient’s parents are inaccessibleto follow-up. Uniparental disomy for chromosome 2 is apossible, though unlikely, explanation for the abnor-mal phenotypic findings in our patient, because in onereported case there is no evidence of imprinting effectsassociated with maternal uniparental disomy 2 [Ber-nasconi et el., 1996]. Phenotypical abnormalities in thesecond case of maternal isodisomy for chromosome 2(such as severe intrauterine growth retardation,marked oligohydroamnios, bronchopulmonary dyspla-sia, perineal hypospadias, preauricular ear pits, andsignificant pectus carinatum [Shaffer et al., 1997]) aswell as in some other reported cases of maternal di-somy with confined placental mosaicism for chromo-some 2, which are discussed by Shaffer et al. [1997], arenot common with multiple congenital anomalies of ourpatient. Therefore, the symptoms in our patient are

caused by partial trisomy mainly of the proximal partof the long arm of chromosome 2. A combined approachusing microdissection and FISH with chromosomearm-specific and marker-specific DNA libraries pro-vided an efficient identification of marker chromo-somes and delineation of new chromosomal syndromes.

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