CLINICAL REPORT

Distal 22q11.2 Microduplication Combined WithTypical 22q11.2 Proximal Deletion: A Case ReportMiriam Coelho Molck,1 Tarsis Paiva Vieira,1 Milena Simioni,1 Ilaria Cristina Sgardioli,1

Ana Paula dos Santos,1 Ana Carolina Xavier,2 and Vera Lucia Gil-da-Silva-Lopes1*1Department of Medical Genetics, Faculty of Medical Sciences, University of Campinas (UNICAMP), Sao Paulo, Brazil2Center for Research and Rehabilitation of Lip and Palate Lesions (CRRLPL) Centrinho Prefeito Luiz Gomes, Joinville, Santa Catarina,

Brazil3Assistance Center for Cleft Lip and Palate (CAIF), Curitiba, Parana, Brazil

Manuscript Received: 24 December 2013; Manuscript Accepted: 11 September 2014>

The 22q11 chromosomal region contains low copy repeats (LCRs)

sequences that mediate non-allelic homologous recombination,

which predisposes to copy number variations (CNVs) at this locus.

Hemizygous deletions of the proximal 22q11.2 region result in the

22q11.2 deletion syndrome (22q11.2 DS). In addition, 22q11.2

duplications involving the distal LCR22s have been reported. This

article describes a patient presenting a 2.5-Mb de novo deletion at

proximal 22q11.21 region (between LCRs A-D), combined with a

1.3-Mb maternally inherited duplication at distal 22q11.23 region

(between LCRs F-H). The presence of concomitant chromosomal

imbalances found in this patient has not been reported previously.

Clinical and molecular data were compared with literature, in

order to contribute to genotype-phenotype correlation. These

findings exemplify the complexity and genetic heterogeneity

observed in 22q11.2 deletion syndrome and highlights the diffi-

culty to make genetic counseling and predict phenotypic con-

sequences in these situations. Ó 2014 Wiley Periodicals, Inc.

Key words: 22q11.2 deletion; 22q11.2 distal duplication; copy

number variations; genotype-phenotype correlation

INTRODUCTION

The chromosome 22q11 region contains low copy repeats (LCRs)

sequences that mediate non-allelic homologous recombination and

predispose to copy number variations (CNVs) (i.e., deletions and

duplications) [Edelmann et al., 1999a,b; Shaikh et al., 2001]. Eight

LCRs designated A-H have been characterized at chromosome

22q11 [Shaikh et al., 2000].

Hemizygous deletions of chromosome 22q11 region result in the

22q11.2 deletion syndrome (22q11.2 DS). The phenotype is variable

but commonly includes conotruncal cardiac defects, characteristic

facial appearance, palatal abnormalities, immunodeficiency, endo-

crine dysfunctions, hypocalcemia, urogenital abnormalities, and

learning and behavioral problems [McDonald-McGinn et al., 1999;

Kobrynski and Sullivan, 2007; Monteiro et al., 2013].

Around 90% of cases of 22q11.2 DS results from a 3-Mb deletion

of chromosome 22q11 region spanning LCR22-A to LCR22-D. A

minority of cases present either a common approximately 1.5-Mb

nested deletion spanning LCR22-A to LCR22-B [Edelmann

et al., 1999b]. Deletions distal to 22q11.21 have been reported.

In most cases, the proximal breakpoint lies within LCR22-D, and

the distal breakpoint lies within one of the distal LCR22s [Tan

et al., 2011; Molck et al., 2013].

The 22q11 region can be classified as one of the most unstable of

human genome, containing unusual DNA configuration [Capra

et al., 2013]. Both deletions and duplications are expected to occur

in equal proportions because of reciprocal LCR-mediated events,

although fewer cases of 22q11 microduplications have been

reported so far [Portnoı, 2009]. Clinical manifestations observed

in patients with 22q11 microduplications may range from milder

phenotypes to multiple severe defects and shares features with

How to Cite this Article:Molck MC, Vieira TP, Simioni M, Sgardioli

IC, Santos APd, Xavier AC, Gil-da-Silva-

Lopes VL. 2014. Distal 22q11.2

microduplication combined with typical

22q11.2 proximal deletion: A case report.

Am J Med Genet Part A 9999:1–6.

Grant sponsor: Fundacao de Amparo a Pesquisa do Estado de Sao Paulo;

Grant numbers: 2008/50421-4, 2009/08756-1, 2008/10596-0, 2011/

23794-7; Grant sponsor: Conselho Nacional de Desenvolvimento

Cientıfico e Tecnologico (CNPq); Grant numbers: 149600/2010-0,

471422/2011-8; Grant sponsor: CNPq; Grant number: 304455/2012-1.�Correspondence to:

Vera Lucia Gil-da-Silva-Lopes, Department of Medical Genetics,

University of Campinas, Tessalia Vieira de Camargo Street, 126 - CEP

13083-887 – Campinas, Sao Paulo, Brazil.

E-mail: vlopes@fcm.unicamp.br

Article first published online in Wiley Online Library

(wileyonlinelibrary.com): 00 Month 2014

DOI 10.1002/ajmg.a.36809

Ó 2014 Wiley Periodicals, Inc. 1

22q11.2 DS, including heart defects, velopharyngeal insufficiency

with or without cleft palate or hypernasal speech, and urogenital

abnormalities [Wentzel et al., 2008].

Duplications of 22q11.2 involving the distal LCR22s have also

been reported [Descartes et al., 2008; Ou et al., 2008; Coppinger

et al., 2009; Wincent et al., 2010; Tan et al., 2011; Ribeiro-Bicudo

et al., 2013]. Similar to the proximal duplications, they appear to

have a high rate of familial transmission and phenotypes vary

among family members carrying this chromosomal abnormality

[Portnoı et al., 2009; Ou et al., 2008; Coppinger et al., 2009; Wincent

et al., 2010]. In addition, no correlation could be recognized

between the severity of clinical features and the size or location

of duplications [Coppinger et al., 2009; Wincent et al., 2010].

In this study, we report on a patient with clinical features of 22q11.2

DS, carrying a de novo typical deletion of 2.5-Mb in the 22q11.2

proximal region (LCRs A-D), combined with a maternally inherited

1.3-Mb duplication in the 22q11.2 distal region (LCRs F-H).

METHODS

This study was approved by the Ethics Committee Board of

University of Campinas (Number 487/2009). Written informed

consent and written permission to use images in this study were

obtained from patient’s parents.

The patient was evaluated by clinical geneticists in a multicenter

study about 22q11.2 DS in Brazil, using a standardized form by

Cranio-Face Brazil Project for clinical data and complementary

examinations. These data are presented on Table I.

G-banding karyotype at the level of 550 bands was performed for

the patient and his parents. Multiplex ligation-dependent probe

amplification (MLPA) analysis was performed for the patient and

his parents using SALSA MLPA kit P250 Di George (A1 and B1

versions, MRC Holland1). Data analysis was made against up to

five control samples using an in-house Excel spreadsheet available

in National Genetics Reference Laboratory, Manchester, UK

[http://www.ngrl.org.uk/Manchester/publications/MLPA%

20Spreadsheets.]

Array genomic hybridization (aGH) analysis for the patient was

performed using the Genome-Wide Human SNP Array 6.0 (Affy-

metrix1) and analyzed by GeneChip1 Operating Software

(GCOS) (Affymetrix1). aGH analysis for patient’s mother and

father was performed using the CytoScan HD chip (Affymetrix1)

and analyzed by Chromosome Analysis Suite (ChAS) Software

(Affymetrix1).

Analysis of polymorphic microsatellite markers was carried

out to determine the parental origin of the deletion, using four

microsatellite markers (D22S264, D22S941, D22S1623, and

D22S1638) mapped within deleted region (NCBI build 36).

Genotyping was performed by polymerase chain reaction and

its products were subsequently separated by capillary electropho-

resis on ABI3500 genetic analyzer and analyzed with the Gene-

Marker1 software.

TABLE I. Description of Clinical Findings Detected on Present Case and Those With Typical 22q11.2 Deletion Syndrome

Patient 22q11.2 DSa

Age (at last evaluation) 9 56% � 5 yearsGender Male Evenly distributedDeletion/duplication interval del LCR22-A to D/dup LCR22-F to H del LCR22-A to B/DDeletion size del 2.5-Mb/dup 1.3-Mb 1.5–3.0-MbBird weigth centile 10th NRWeight centile 90th 41%< 5th centileHeight centile 50th NRHead circumference centile 50th–98th NRCongenital heart disease � 74%Palatal abnormalities þ 69%Characteristic face þ CommonRecurrent infections � CommonDevelopmental delay þ 70–90%Feeding problems � 30%Hearing loss � 39%Eye abnormalities þ �69%Neurological abnormalities � Intermediate frequencyUrinary tract anomalies � 37%Gastrointestinal anomalies � Low frequencySkeletal abnormalities þ Intermediate frequencyGenital anomalies � Low frequencyHypocalcemiab � 50%

del, deletion; dup, duplication; þ, presence; � , absence; NR, Not Reported.aMcDonald-McGinn et al. (1999).bRetrospective data.

2 AMERICAN JOURNAL OF MEDICAL GENETICS PART A

CLINICAL REPORT

Patient is the third male child of a healthy and non-consanguineous

couple (mother 30 years, father 32 years). Family history was

uneventful. Pregnancy was complicated by maternal bronchopneu-

monia. The mother was hospitalized for seven days during the

second month. She also used non-specified medications for weight

loss during the first month until she discovered the pregnancy.

Delivery was at term and anthropometric data were: weight 3 kg

(10th centile), length 45 cm (below 3rd centile) and OFC 35 cm (50th

centile). Motor development was normal, but he had speech delay.

Dysmorphologic examination at 9 years old: weight 34 kg (90th

centile), length 132 cm (50th centile), OFC 55 cm (between 50th

and 98th percentiles); characteristic face: low-set ears, mild epi-

canthal folds, typical thin nose with hypoplastic alae nasi (Fig. 1a;

Supplemental Fig. 1a in supporting information online); accessory

nipple (4 cm below the left nipple), prepucial adhesion, ligament

laxity and mild scoliosis. He also presented hypernasal voice,

language impairment and learning difficulties. Nasofibroscopy

exam showed velopharyngeal insufficiency. Doppler echocardio-

gram revealed minimum tricuspid regurgitation. Ophthalmologi-

cal examination showed astigmatism. Odontological evaluation

presented multiple carious teeth. Hearing screening examination

was normal. Intra-abdominal ultrasound and cranioencephalic

magnetic resonance imaging were normal.

G-banding karyotype of the patient and his parents were normal.

MLPA analysis of the patient revealed a de novo typical deletion of

approximately 3-Mb between LCR22-A and LCR22-D (22q11.21)

and an atypical duplication at distal region (22q11.23) between

LCR22-F and LCR22-H (Supplemental Fig. 2a in supporting

information online). MLPA analysis of the mother detected the

same duplication. The aGH analysis of the patient confirmed these

alterations and determined a deletion of 2.5-Mb deletion at

22q11.21 [17,256,416–19,795,835 - hg18] and 1.3-Mb duplication

at 22q11.23 [22,020,300–23,394,964 - hg18] (Supplemental

Fig. 2b). aGH analysis of the patient’s mother by, showed a

duplication of approximately 1.3-Mb at 22q11.23 [23,690,387–

25,041,592 - hg19] (Supplemental Fig. 2c). MLPA and aGH analy-

ses of the father were normal. No other rare CNVs were observed in

the patient and his parents. Analysis of the polymorphic microsat-

ellite markers D22S264 and D22S941 was fully informative and

showed that the deletion was present in the paternal inherited

chromosome.

Patient’s mother was shown by MLPA and aGH to carry the same

duplication of patient. Her dysmorphologic examination at 42 years

old displayed: weight 98.5 kg (above 97th centile), height 158,5 cm

(between 25th and 50th centiles), OFC 55 cm (50th centile), no major

facial dysmorphisms: only a hyperchromic spot in face with 5 cm of

diameter (Fig. 1b; Supplemental Fig. 1b), mild brachydactyly of

hands and feet, mild cutaneous syndactyly between 2nd and 3rd toes,

proximal implantation of 5th toes. Nasofibroscopy exam showed

normal result. Doppler echocardiogram revealed a slight increase in

left atrium. Intra-abdominal ultrasound showed a nodular forma-

tion in the right ovary (possibly dermoid tumor/teratoma); she was

referred for specialized investigation for both alterations.

The father was examined at the age of 45 years old and does not

show any phenotypic abnormality. He denies delay in motor

development. He sat, crawled and walked on the correct marks.

Initiation of speech was normal. He denies delay or changes in

speech. He never showed language impairment, learning difficulties

and behavioral problems.

DISCUSSION

The high frequency of de novo genomic rearrangement on 22q11

chromosome region is attributed to the presence of several large

paralogous low copy repeats (LCRs). Each LCR contain a complex

modular structure and a high degree of sequence identity (>96%)

over large stretches of the repeat [Shaikh et al., 2000]. The LCRs

apparently mediate aberrant intrachromosomal or interchromo-

somal exchanges during meiosis [Saitta et al., 2004], and 22q11

deletions are among the most frequent constitutional rearrange-

ments [Shaikh et al., 2007].

Several “non-standard” deletions/duplications with variable

breakpoints were reported in addition to the commonly deleted/

duplicated region (3-Mb, LCRs A through D) flanked at LCR22s

at both proximal and distal breakpoints. These are caused by

different recombination mediated by the LCR22-A to LCR22-H.

Beyond LCRs-mediated NAHR mechanism, some other mech-

anisms were proposed to explain the formation of CNVs on

chromosome 22, such as non-homologous end joining, micro-

homology-mediated break-induced replication and variable

number of tandem repeats-mediated genomic rearrangements

[Yu et al., 2012].

In this article, we present a patient with concomitant chromo-

somal imbalances not described before in the 22q11 region: a de

novo typical deletion of 2.5-Mb in the proximal region combined

with a maternally inherited 1.3-Mb duplication in the distal 22q11.2

region. The analysis of polymorphic microsatellite markers showed

that the deletion was present in the paternal inherited chromosome.

The identification of a 1.3-Mb duplication between LCR22-F

and LCR22-H in this patient and his mother reinforces the hy-

FIG. 1. Facial features of affected individuals. a: Patient at age

9 years, showing low-set ears, mild epicanthal folds and typical

thin nose with hypoplastic alae nasi and round nasal tip. b:

Patient’s mother at age 42 years, showing no major facial

dysmorphisms.

MOLCK ET AL. 3

pothesis by Shaikh et al. [2007] that “the orientation of the BCRL

(BCR-related loci) module may predict the substrates for NAHR”.

Further, the LCRs in direct orientation mediate deletions and

duplications [Shaffer and Lupski, 2000]. Based on this orientation,

BCRL-D1, BCRL-E, BCRL-F and BCRL-H are in one group and

BCRL-D2 and BCRL-G are in the second group [Shaikh

et al., 2007].

Coppinger et al. [2009] identified several duplications between

LCRs E-H and F-H in patients with idiopathic mental retardation

and/or congenital anomalies. Among them, there were six cases

presenting F-H mediated duplications. Four of them had inherited

duplications, similar to the patient herein reported. These results

reinforce the hypothesis that duplications seem to be more com-

monly inherited than deletions [Ou et al., 2008]. It is also interesting

to point that, generally, individuals with inherited duplications had

a minimally affected or phenotypically normal parent [Descartes

et al., 2008; Ou et al., 2008; Coppinger et al., 2009; Wincent

et al., 2010], consistent with our patient. Several possibilities

may account for phenotypic variability including variation in

genetic background, epigenetic phenomena, gene expression

and/or regulatory variation among genes in rearranged regions

[Ou et al., 2008; Fernandez et al., 2009].

Previous reports emphasize phenotypic variability and lack of

common facial gestalt among patients with distal 22q11.2 duplica-

tion [Ou et al., 2008; Descartes et al., 2008; Coppinger et al., 2009

Wincent et al., 2010; Ribeiro-Bicudo et al., 2013]. At Database of

Genomic Variants (DGV) [http://dgv.tcag.ca/dgv/app/home],

there are CNVs of different sizes in the 22q11.23 region. However,

none of them overlaps more than 50% in size compared to 1.3-Mb

segment duplicated in our patient. At Database of Chromosomal

Imbalance and Phenotype in Humans (DECIPHER) [http://deci-

pher.sanger.ac.uk], there are seven cases reported that overlaps part

of segment duplicated. Phenotypes were not reported in two of

them. Three patients (255591, 2727, and 251448) share a phenotype

of intellectual disability and other (255653) of cognitive im-

pairment with our patient. In two of them (255591 and 2727)

duplications were inherited from normal parents. It is worth to

highlight that cases 255591 and 2727 showed additional CNVs,

including an 8.4-Mb deletion in chromosome 1 and a 440 Kb

duplication in chromosome 3, respectively. Moreover, none of

seven cases reported at DECIPHER showed the 22q11.21 deleted

region presented in our case.

The overlapped 1.3-Mb duplicated segment at 22q11.23 region

comprises 38 genes. Some of them were selected, considering

function and biological process involved, and are listed in

Table II. One of these genes is the SMARCB1. Mutations that

alters the function of this gene could result in several forms of

cognitive deficits due to a mechanism involving epigenetic

regulation [Day and Sweatt, 2011; Kleefstra et al., 2012]. In

fact, this mechanism was also pointed in the 22q11.23 dele-

tion/duplication by Ribeiro-Bicudo et al. [2013]. Other candidate

genes are: SPECC1L that has a critical role in actin-cytoskeletal

reorganization during facial morphogenesis [Saadi et al., 2011],

and SNRPD3 that was related to aberrant splicing events, a

mechanism established as direct cause of several human disorders

[Khan et al., 2012].

The 2.5-Mb deletion at 22q11.21 reported in this patient is

common to 22q11.2 DS. Our patient shares some clinical features

with this syndrome, including facial dysmorphic features, palatal

abnormalities and developmental delay. This region contains more

TABLE II. Candidate Genes at 22q11.23 Region

Gene OMIM Functions Process

ZNF70 194544 DNA binding, transcription factor activity,zinc ion binding

Regulation of transcription, DNA-dependent transcription

MMP11 185261 Metalloendopeptidase activity, calcium/zincion binding

Collagen catabolic process, extracellular matrixdisassembly/organization, multicellular organismaldevelopment

SMARCB1 601607 Protein binding, transcription coactivatoractivity

Chromatin remodeling, DNA integration/repair, celldifferentiation, mitotic cell cycle phase transition,nervous system development

MIF 153620 Chemoattractant activity, cytokine activity,protein/receptor binding

DNA damage response, carboxylic acid metabolic process,cell aging/proliferation, cell surface receptor signalingpathway

CABIN1 604251 Protein phosphatase, inhibitor activity Cell surface receptor signaling pathway, chromatinmodification

SPECC1L 614140 Actin-cytoskeletal reorganization duringfacial morphogenesis

Actin cytoskeleton organization, cell cycle/division/migration, regulation of microtubule depolymerization

ADORA2A 102776 G-protein coupled adenosine receptoractivity, alpha-actinin binding, enzymebinding, protein binding

Apoptotic process, blood circulation/coagulation, cell-cellsignaling, central nervous system development, eatingbehavior

SNRPD3 601062 Enzyme binding, histone pre-mRNA DCPbinding, protein binding

RNA metabolic process, RNA splicing gene expression,histone mRNA metabolic process

OMIM, Online Mendelian Inheritance In Man, available in http://www.ncbi.nlm.nih.gov/omim

4 AMERICAN JOURNAL OF MEDICAL GENETICS PART A

than 40 known genes and several candidate genes, such as TBX1 and

COMT have been postulated for the behavioral phenotype in

22q11.2 DS. TBX1 also has been show to play an important

role in congenital heart disease, craniofacial anomalies and other

anomalies related to pharyngeal apparatus development; and

COMT has been related to psychiatric or behavioral problems

[Prasad et al., 2008].

The distal 22q11.2 microduplication presented by our patient

may act as a susceptibility factor, requiring a modifying factor for a

clinical phenotype to emerge [Ribeiro-Bicudo et al., 2013]. The

putative modifiers are likely to be different in each system, thus

producing interindividual variability [Tan et al., 2011], while

regulation of neuronal function through epigenetic control of

gene transcription seems to be an important mechanism related

to intellectual deficiency [van Bokhoven Kramer, 2010].

In the patient reported here, the cognitive and behavioral findings

as well as physical signs were those usually observed in both 22q11.2

deletions and duplications. In this way, it is not possible to conclude

which features are related to deletion or duplication. However, since

most of clinical signs found in the patient are common in the

22q11.2DS, we believe that the deletion could have a major role

in the phenotype. Additional similar reports would be necessary to

further elucidate genotype/phenotype correlations. Maternal phe-

notype is very mild and there is no evidence that features described

are related to the 1.3-Mb duplication at 22q11.23.

To our knowledge, the combined deletion of 2.5-Mb in the

22q11.2 proximal region with a 1.3-Mb duplication in the 22q11.2

distal region has not been reported previously. In conclusion, these

findings exemplify the complexity and genetic heterogeneity ob-

served in 22q11.2 DS and corroborate the idea that genetic modi-

fiers contribute to the phenotypic variability found in this

syndrome. In addition, this study reinforces the importance to

perform parental investigations after the identification of a dele-

tion/duplication in a proband.

ACKNOWLEDGMENTS

We acknowledge the patients and their parents for their coopera-

tion and the Microarray Laboratory at Brazilian Biosciences Na-

tional Laboratory (LNBio), CNPEM, Campinas, Brazil for their

support with the use of equipment, fluidics station and scanner

GeneChip.

This study was supported by Fapesp - Fundacao de Amparo a

Pesquisa do Estado de Sao Paulo (2008/50421-4, 2009/08756-1,

2008/10596-0 and 2011/23794-7) and CNPq - Conselho Nacional

de Desenvolvimento Cientıfico e Tecnologico (149600/2010-0

and 471422/2011-8). VLGSL is supported by CNPq (304455/

2012-1).

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