CLINICAL REPORT

A De Novo Supernumerary Genomic DiscontinuousRing Chromosome 21 in a Child With MildIntellectual DisabilityNicoletta Villa,1 Angela Bentivegna,2 Adam Ertel,3 Serena Redaelli,2 Carla Colombo,4

Renata Nacinovich,5 Fiorenza Broggi,5 Sara Lissoni,1 Silvia Bungaro,6 Sankar Addya,3 Paolo Fortina,3,7

and Leda Dalpr�a1,2*1Medical Genetics Laboratory, S. Gerardo Hospital, Monza, Italy2Department of Neuroscience and Biomedical Technologies, School of Medicine, University of Milano-Bicocca, Monza, Italy3Kimmel Cancer Center, Department of Cancer Biology, Thomas Jefferson University, Jefferson Medical College, Philadelphia, Pennsylvania4Neonatal Intensive Care Unit, S. Gerardo Hospital, Monza, Italy5Department of Child and Adolescent Neuropsychiatry, S. Gerardo Hospital, Monza, Italy6M. Tettamanti Research Center, Pediatric Clinic, University of Milano Bicocca, S. Gerardo Hospital, Monza, Italy7Department of Molecular Medicine, School of Medicine, University ‘‘La Sapienza’’, Rome, Italy

Received 12 November 2010; Accepted 17 February 2011

Small supernumerary marker chromosomes (sSMCs) are struc-

turally abnormal extra chromosomes that cannot be unambigu-

ously identified or characterized by conventional banding

techniques alone, and they are generally equal in size or smaller

than chromosome 20 of the same metaphase spread. Small

supernumerary ring chromosomes (sSRCs), a smaller class of

marker chromosomes, comprise about 10% of the cases. For

various reasons these marker chromosomes have been the most

difficult to characterize; although specific syndromes have not

yet been defined, 60% of cases are associated with an abnormal

phenotype. The chromosomal material involved, the degree and

tissutal distribution of mosaicism, and the possible presence of

uniparental disomy, are the important factors determining

whether or not the ring chromosome will give rise to symptoms.

Using conventional and molecular cytogenetics approaches we

identified a de novo chromosome 21 sSRC in a child with speech

delay and mild intellectual disability. By using aCGH analysis

and SNP arrays, we report the presence of two discontinuous

regions of chromosome 21 and the paternal origin of the sSRC. A

thorough neuropsychiatric evaluation is also provided. Only few

other cases of complex discontinuous ring chromosomes have

been described in detail. � 2011 Wiley-Liss, Inc.

Key words: ring chromosome 21; fluorescence in situ hybridiza-

tion (FISH); array-CGH

INTRODUCTION

Small supernumerary marker chromosomes (sSMCs) are structur-

ally abnormal extra chromosomes that cannot be unambiguously

identified or characterized by conventional banding techniques

and are generally equal in size or smaller than chromosome 20 on

the same metaphase spread [Liehr et al., 2004]. According to the

data reviewed by Liehr and Weise [2007], sSMCs were present in

0.075% of all analyzed prenatal cases but only in 0.044% of

consecutively studied postnatal ones. In infertile subjects,

0.125% were sSMC carriers (male/female rate 7.5:1). In develop-

mentally retarded patients the sSMC rate was elevated to 0.288%

Additional supporting information may be found in the online version of

this article.

Grant sponsor: Fondazione Cariplo; Grant sponsor: AIRC; Grant sponsor:

MIUR.

Nicoletta Villa and Angela Bentivegna contributed equally to this work.

*Correspondence to:

Leda Dalpr�a, M.D., Dipartimento di Neuroscienze e Tecnologie

Biomediche, School of Medicine, Universit�a degli Studi di Milano-

Bicocca, Via Cadore 48, 20052 Monza, MI, Italy.

E-mail: leda.dalpra@unimib.it

Published online 13 May 2011 in Wiley Online Library

(wileyonlinelibrary.com).

DOI 10.1002/ajmg.a.34010

How to Cite this Article:Villa N, Bentivegna A, Ertel A, Redaelli S,

Colombo C, Nacinovich R, Broggi F, Lissoni

S, Bungaro S, Addya S, Fortina P, Dalpr�a L.

2011. A de novo supernumerary genomic

discontinuous ring chromosome 21 in a child

with mild intellectual disability.

Am J Med Genet Part A 155:1425–1431.

� 2011 Wiley-Liss, Inc. 1425

[Liehr and Weise, 2007]. The small size of sSMCs prohibits

characterization by traditional banding techniques, and instead

requires molecular cytogenetic techniques, such as FISH or array-

based comparative genomic hybridization (aCGH), to determine

the chromosomal origin.

sSMC are scientifically interesting due to an incomplete under-

standing of their formation, karyotypic evolution, and the fact

that their presence may lead to chromatin imbalances (partial

tri-, tetra-, or hexasomies) without detectable clinical consequences

[Liehr et al., 2008]. Subjects with a supernumerary marker

chromosome have duplication and, in some cases, a triplication

of the material comprising the sSMC. The risk of an abnormal

phenotype associated with a randomly ascertained de novo sSMC

derived from acrocentric autosomes (excluding chromosome 15) is

approximately 7%, compared with approximately 28% for sSMC

derived from non-acrocentric autosomes [Crolla, 1998]. A collab-

orative study of 19 Italian laboratories involving 241 sSMCs

[Dalpr�a et al., 2005] has allowed definitive conclusions concerning

karyotype–phenotype correlations. These studies emphasize that

molecular characterization of sSMCs and their associated clinical

phenotypes can provide karyotype–phenotype correlations useful

for genetic counseling.

We report on a de novo small supernumerary ring chromosome

(sSRC) of paternal origin, derived from different regions of chro-

mosome 21, in a child with speech delay and mild intellectual

disability.

MATERIALS AND METHODS

CytogeneticsMetaphase chromosome preparations were obtained from PHA-

stimulated lymphocyte cultures of peripheral blood according to

standard procedures. Chromosome analysis was carried out apply-

ing QFQ banding according to routine procedures, and karyotypes

for the patient (50 metaphases) and for both parents (100 meta-

phases, each) were reconstructed following the guidelines of ISCN

2009 [Shaffer et al., 2009].

Fluorescence In Situ Hybridization (FISH)FISH was performed as previously reported [Lissoni et al., 2009]. To

characterize the sSMC, the following probes were applied: Pan-

centromeric, Pan-telomeric, D13Z1/D21Z1, beta-satellite, and

WCP for chromosome 21 (Oncor, Inc., Gaithersburg, MD). To

define the chromosome 21-derived euchromatic sequences, BAC

probes were applied: RP11-91N21, RP11-106K13, RP11-22D1,

RP11-61A21, RP11-97K13, RP11-78J18, RP11-242C13, RP11-

141K11, and RP11-89M24 (Wellcome Trust Sanger Institute,

Hinxton, Cambridge, UK).

Array-CGHGenomic DNA was extracted from patients’ lymphoblastoid

cultures by phenol–chloroform standard protocols and DNA con-

centration was determined on a NanoDrop ND-1000 spectropho-

tometer (NanoDrop Technologies, Berlin, Germany). Gene copy

number analysis was performed by aCGH using Agilent Human

Genome CGH Microarray 44K kit (Agilent Technologies�, Wall-

dbron, Germany) following manufacturer’s recommendations.

Genomic DNA (Promega�, Mannheim, Germany) was used as

reference in sex-matched hybridizations which were analyzed using

Feature Extraction v10.5 and DNA Analytics v4.0 software (Agilent

Technologies�) applying ADM2 algorithm with a threshold of 5,

minimum absolute average log 2 ratio in called intervals of 0.30 and

a minimum of three probes. Putative chromosome copy number

changes were defined by intervals of three or more adjacent probes

and were considered as duplicated or deleted when results exceeded

the �0.30 range.

High-Resolution Cytogenetics AnalysisDNA samples were hybridized to the Affymetrix Cytogenetics

whole-genome 2.7 M array and processed according to the man-

ufacturer’s recommended protocol (http://www.affymetrix.com).

This platform interrogates approximately 2.7 million markers,

providing high-density coverage across the entire genome. Cyto-

genetics data was processed using Chromosome Analysis Suite

(ChAS) software from Affymetrix (Santa Clara, CA). ChAS soft-

ware was used to process the raw data (.CEL files) and determine

regions of copy number segment gains/losses as well as copy-neutral

loss of heterozygosity (cn-LOH). Copy number segments were

filtered to include only gains represented by at least 250 markers

extending over 500 kb, and losses represented by at least 100

markers over a 200 kb region. Confidence levels were restricted to

at least 85%. Following copy number analysis on the Affymetrix

Cytogenetics platform, the source of the chromosomal duplication

was determined from trisomic genotype calls in the chromosome 21

ring region, which were estimated using the Standardized Centered

Allelic Ratio (SCAR). The SCAR value was computed from

Affymetrix software, where positive values map to the homozygous

‘‘A’’ allele, negative values to the homozygous ‘‘B’’ allele, and values

close to 0 indicate heterozygosity. In duplicated regions, positive

SCAR values indicate duplication of the ‘‘A’’ allele and negative

SCAR values indicate duplication of the ‘‘B’’ allele. Heterozygous

alleles in the proband where one parent carried the homozygous

‘‘A’’ allele and the other parent carried the homozygous ‘‘B’’ allele

provided evidence to determine the source of the extra copy.

Sequence In Silico AnalysisIn silico sequence analysis was performed using the following

database and bioinformatic tools: Entrez Nucleotides Database,

http://www.ncbi.nlm.nih.gov/Entrez/query.fcgi?db¼nucleotide;

NCBI BLAST, http://www.ncbi.nlm.nih.gov/Blast/; UCSC

Genome Bioinformatics, http://www.genome.ucsc.edu/ (for

sequence homology analyses for identification of gene content and

for BAC/PAC clones used as probes in the FISH experiments).

CLINICAL REPORT

The female patient was born at 36 weeks of gestation after an

uneventful pregnancy, as the second child of healthy, unrelated

parents. The mother and father were 17 and 25 years old, respec-

tively, and had a healthy older female child. Birth weight of the

1426 AMERICAN JOURNAL OF MEDICAL GENETICS PART A

patient was 2,370 g with an Apgar score of 7 at 1 min. She was

cytogenetically studied because of mild facial dysmorphisms and

hypotonia. The family pedigree shows a history of Down syndrome

(DS) (Supplemental Fig. 1). At 2 months, she was hospitalized for a

suspected non-febrile seizure; weight was 5,250 g (90th to 97th

centile), length of 55 cm (25th to 50th centile). Clinical evaluation

confirmed a general muscular hypotonia, malformed ears, umbili-

cal hernia, and gastroesophageal reflux, but neuropsychiatric eval-

uation, EEG, and MRI of the brain were normal. At 5 months her

weight was 11,700 g (>97th centile). She was later referred because

of slight delay in psychomotor development, especially speech

delay. She could sit without support at the age of 7 months, and

walk without support at the age of 20 months.

Neuropsychiatric EvaluationThe patient was referred for clinical assessment at the local Child

Neuropsychiatry Service at the age of 29 months. The first psycho-

motor assessment (Brunet Lezine Test) showed a disharmonic

psychomotor retardation: 20.8 months of developmental quotient

(DQ) compared to 29 months of chronological age, with posture

control (DQ 24 months), coordination (DQ 24 months), sociali-

zation (DQ 21 months), and significant impairments in the lan-

guage domain (DQ 10 months). At the time of the psychomotor

development check up at 39 months of chronological age the

disharmonic psychomotor retardation was confirmed (DQ 24.9

months), with small improvement in all developmental domains,

but with an increased retardation compared to the standardized

average development. The differences between chronological and

psychomotor age shifted from 8.4 months at the first psychomotor

assessment (29 months of chronological age) to 14.3 months at the

second check up (39 months of chronological age).

The patient underwent psychomotor therapy from age 4 to

6 years and speech therapy from age 6 to 8 years. When the speech

therapy began, she showed difficulties with attention, language

comprehension, and critical reasoning, and she was able to read by

syllabicating. At the end of the speech therapy, at the age of 8, she

was able to perform syllabic fusion, though decoding was very slow.

The language, which had shown expressive delay, was simply

structured and phonologically adequate, but lexical and semantic

limits were more evident. Comprehension was limited to concrete

contexts or related to personal experiences.

Results of the Leiter-R and speech assessment confirmed a mild

intellectual disability with difficulties with logical, communicative

and language abilities, attention, spatial memory, and short- and

long-term memory (see a full report on Supplemental Material:

Complete Neuropsychiatric Evaluation).

METHODS AND RESULTS

Chromosome analyses from the patient’s blood lymphocyte cul-

tures showed a sSMC, which was DA/DAPI negative, in all 50

metaphases investigated (Fig. 1a), while analysis of both parents

showed normal karyotypes (100 metaphases investigated). FISH

analysis using the Pan-centromeric probe and the Pan-telomeric

probe (Oncor, Inc.) revealed a small monocentric sSRC, positive for

the alphoid probe D13Z1/D21Z1, but negative for the beta-satellite

probe (Fig. 1b–d). Finally, the WCP probe for chromosome 21 was

positive demonstrating chromosome 21 origin and suggesting

additional euchromatic sequences in the r(21) (Fig. 1e). Using

ISCN 2009 recommendation, the aberration was characterized as:

47,XX,þmar de novo.ish r(21) (wcp21þ,D13Z1/D21Z1þ,bsat-).

In order to define the chromosome 21-derived euchromatic

sequences and attempt to identify the breakpoint region, FISH

analysis with a battery of BAC clones was performed on EBV-

transformed lymphoblastoid cell line of the patient (Fig. 1f and

Supplemental Fig. 2). The r(21) showed hybridization signals for

RP11-97K13 and RP11-78J18, but none for RP11-141K11, while for

RP11-242C13 there is no clear interpretation, since it showed a

weak hybridization signal or no signals. From these results we were

able to define a partial trisomy of about 3 Mb extending from cen to

21q21.1, with a breakpoint region of 158 kb by the size of RP11-

242C13 (from nt 15,546,175 to 15,704,232). By aCGH analysis, two

different regions of copy gain on chromosome 21 were evidenced

(all the positions on chromosome 21 were referred to the old

Assembly Mar. 2006 hg18 of UCSC): (a) the proximal one’s, from

21q11.2 to 21q21.1 (nt 13,548,935–15,510,501), extending for

1.96 Mb; (b) the distal one’s, from 21q21.3 to 21q22.11 (nt

29,278,776–31,123,552), extending for 1.84 Mb (Fig. 2). The

two regions of copy gain were confirmed during subsequent

whole genome analysis on the Affymetrix Cytogenetics array

(Supplemental Fig. 3). Copy number segment analysis using the

higher resolution cytogenetics array identified the amplified

regions as 21q11.2–21q21.1 (1,066 markers; nt 13,527,258–15,552, 772) and 21q21.3–21q22.11 (2,613 markers; nt

29,265,617–31,950,671).

Uniparental disomy (UPD) was excluded using microsatellite

analysis (data not shown). These results were confirmed by the

Affymetrix Cytogenetics array, which determined this region was

free from long contiguous stretches of homozygosity (LCSH),

which are characteristic of UPD. The paternal origin of the ring

was assessed using allele signal intensities from the Affymetrix

Cytogenetics Array. Trisomic genotypes were determined for the

proband in regions 21q11.2 and 21q21.3–q22.11 and compared to

the parental genotypes at locations where one parent carried the

homozygous ‘‘A’’ allele and the other parent carried the homo-

zygous ‘‘B’’ allele. The amplified allele in 24 out of 29 valid trisomic

genotypes in this region matched the homozygous allele of the

father (P< 0.001) (Supplementary Table I).

In Silico Sequence AnalysisIn order to clarify the discontinuity of this ring chromosome, an in

silico sequence analysis to delineate the genomic structural features

of the two genomic regions involved was performed. The two

regions of trisomy lie in the same contig (NT_011512: Homo

sapiens chromosome 21 genomic contig, GRCh37.p2 reference

primary assembly). BLAST analysis using the proximal region

(as query) versus the distal one’s (as subject), evidenced more

than 30,000 blast hits on the query sequence, with a query

coverage of 13% (see Supplemental Material: In silico Results,

A). Furthermore, variation and repeats analysis by UCSC

Genome Browser on Human Mar. 2006 (NCBI36/hg18) assembly

was conducted for both regions. In the proximal region

VILLA ET AL. 1427

(chr21:13,548,935–15,510,501) several duplications of >1,000

bases of non-repeatmasked sequence were evidenced; three of them

(chr21:28204081, chr21:28321445, and chr21:28335703) have a

genomic size of 94,550 bases, 9,791 and 20,456 bases, respectively,

and a similarity>96% with sequences belonging to the distal region

of trisomy (chr21:29,278,776–31,123,552) (see Supplemental

Material: In silico Results, B). Finally, again for the proximal region,

The database of chromosomal imbalance and phenotype in humans

using ensembl resources (DECIPHER) (decipher@sanger.ac.uk)

reported one patient (249224) with a deletion of 6.37 Mb

(chr21:14320039–20690911) affected by speech delay.

DISCUSSION

Here we report on the first case of a supernumerary discontinuous

ring chromosome 21. However, this is the first instance where the

sSRC was characterized in detail by FISH, array-CGH and then

confirmed by the Affymetrix Cytogenetics array, as a de novo ring

from chromosome 21. Previously, one case of ring chromosome

originating from three discontinuous regions of chromosome 4

[Fang et al., 1995], and a second from different parts of the q arm of

chromosome 20 [Anderlid et al., 2001] were described. Both

reported the ring chromosomes were formed during repeated

breakage and reunion cycles as a result of the formation of inter-

locked rings during cell division [Fang et al., 1995; Anderlid et al.,

2001]. Two similar cases of r(1)s were identified in the work of

Callen et al. [1999], but the discontinuous regions of chromosome 1

were not well characterized. R€othlisberger et al. [2009], report a

discontinous sSMC(18), while Starke et al. [2001] described an

acquired one from chromosome 11. Similar cases can be found

at http://www.med.uni-jena.de/fish/sSMC/00START.htm, like

08-W-p23.3/1-1 for chromosome 8 and 14-W-q13.3/1-1 for chro-

mosome 14. Although several discontinuous sSMCs have been

reported, few were the sSRCs accurately studied.

In our case, we evidenced two different regions of partial trisomy

on the q arm of chromosome 21 by array-CGH and also confirmed

by the Affymetrix Cytogenetics array. Starting from Affymetrix

Cytogenetics array data and microsatellite analysis it is possible to

postulate a mechanism of formation for r(21) as initiating from

a complete trisomy 21, originated more likely through a non-

disjunction event at the second meiotic paternal division, or, less

likely, at an early post-zygotic division. Then, in early stages of

mitotic division, the supernumerary chromosome was reduced to a

ring. The loss of genomic material from r(21) occurred by the

FIG. 1. Results of cytogenetic and molecular-cytogenetic investigations showing a de novo supernumerary ring chromosome 21 in the proband. a: QFQ

banding; (b) alphoid probe D13Z1/D21Z1; (c) beta-satellite probe; (d) Pan-telomeric probe; (e) WCP probe for chromosome 21; (f) RP11-106K13 BAC

clone. The white arrows indicate the r(21) while the red signals indicate presence of the various probes.

1428 AMERICAN JOURNAL OF MEDICAL GENETICS PART A

mechanism proposed by Fang et al. [1995], or through non-

allelic homologous recombination (NAHR), Indeed, NAHR can

be mediated by the presence of low-copy repeats (LCRs, also called

segmental duplications, SD) [Stankiewicz and Lupski, 2002; Shaw

and Lupski, 2004]. LCRs are region-specific DNA blocks usually of

10–300 kb in size and of >95–97% similarity to each other

[Stankiewicz and Lupski, 2002; Bailey and Eichler, 2006]. We

evidenced the presence of at least three SDs in the two regions of

trisomy, that may have triggered the mechanism NAHR, function-

ing as substrates (see Fig. 3).

Finally, as the paternal lineage shows two relatives with DS, one

could assume that there is a tendency to non-disjunction of the two

chromosome 21s at meiosis in this family.

It is the distal trisomy region of r(21), not identified by FISH

analysis and with higher gene content, to fall in the critical area

associated with severe clinical problems (nt 18,100,000-q-tel)

[http://www.med.uni-jena.de/fish/sSMC/21.htm#Start21]; this is

in accordance with the model proposed by Liehr, based on the

interpretation of the presently available data on sSMC and on the

hypothesis that a genetic imbalance induced by sSMC presence, is

the major reason for clinical symptoms in sSMC carriers. Supple-

mentary Table II and Supplemental Figure 6 show an attempt of

genotype–phenotype correlation in sSMC cases derived from chro-

mosome 21 reported in the literature, in addition to the one

presented here.

Our case emphasizes the difficulty of a genotype–phenotype

correlation for cases with partial trisomy 21, as already discussed by

others [Kondo et al., 2006; Lyle et al., 2009]. Kondo and coworkers

reported two cases of partial trisomy 21 (pter-q22.12 and pter-

q22.11) without the major features of DS. Both patients had a

similarly sized extra chromosome 21, lacked all of DS critical region

(DSCR) on 21q22, but they presented with moderate intellectual

disability, delayed motor development, and speech delay, as our

patient. Although the two cases described by Kondo et al. was

associated with a distal partial trisomy 3p and 14q, respectively, the

authors conclude that the moderate intellectual disability most

likely results from partial trisomy 21pter-q22.1. On the other hand,

Lyle and coworkers reported on genotype–phenotype correlations

in DS in 30 cases of partial trisomy 21. Patients with milder

phenotype are those in which the partial trisomy overlaps with

one of the two regions of trisomy identified in our ring. From all this

evidence one might speculate that a critical region, associated with

psychomotor and intellectual disability and speech delay, maps to

21q22.11.

In conclusion, we were able, using aCGH analysis and SNP array,

to identify the real genomic content of the r(21) in our patient, to

determine its paternal origin (with important implications for

counseling), to correlate with the phenotype and to hypothesize

a possible mechanism of formation. It is increasingly evident that,

given the rarity of these partial trisomies of chromosome 21, there is

a need for a web-based collection of cases with both phenotypic and

genotypic characterization, as suggested by Lyle et al.; in addition

the use of oligonucleotide platforms for array-CGH may reveal not

only additional cases that could be undiagnosed or better map the

breakpoints, but also may help to define which regions contribute to

phenotypic features.

FIG. 2. Array-CGH profile of chromosome 21 showing the two regions of trisomy present in r(21): the centromeric region covers 1.96 Mb, encompassing

from 21q11.2 to 21q21.1 (nt 13,548,935–15,510,501) while the distal region covers 1.84 Mb, encompassing from 21q21.3 to 21q22.11 (nt

29,278,776–31,123,552). The colored boxes show magnification of the two regions with their known gene content.

VILLA ET AL. 1429

ACKNOWLEDGMENTS

We are grateful to the family for agreeing to participate in this study.

We wish to thank the Galliera Genetic Bank for EBV-transformed

lymphoblastoid cell line; Galbiati M. and Lettieri A. (M. Tettamanti

Research Center, Pediatric Clinic, University of Milano, Bicocca,

San Gerardo Hospital), Colombo D. (Istituto Auxologico Italiano)

and Surrey S. (Thomas Jefferson University, Philadelphia, PA,

USA) for technical support and editorial assistance. This work was

supported in part by Fondazione Cariplo, AIRC, and MIUR (S.B.,

M. Tettamanti Research Center).

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