Prenatal diagnosis of 24 cases of Microduplication 22q11.

2: an

Investigation of Phenotype-Genotype Correlations

Running Head: Prenatal diagnosis of microduplication 22q11.2

Manuscript word count: 3625 number of figures: 2 number of tables: 1

Céline Dupont1, Francesca Romana Grati2, Kwong Wai Choy3, Sylvie Jaillard4, Jérôme Toutain5,
Marie-Laure Maurin6, Jose Antonio Martinez-Conejero7, Claire Beneteau8, Aurélie Coussement9,
Denise Molina Gomes10, Nina Horelli-Kuitunen11, Azzedine Aboura1, Anne-Claude Tabet1, Justine
Besseau10,12, Bettina Bessieres-Grattagliano6, Giuseppe Simoni2, Gustavo Ayala7 , Brigitte
Benzacken1,13 and François Vialard10,12.
1
Unité de Cytogénétique, Département de Génétique, Hôpital Robert Debré-AP-HP, CHU Paris, Paris, France
2
TOMA Advanced Biomedical Assays S.p.A, Busto Arsizio, Varese, Italy
3
Department of Obstetrics and Gynecology, The Chinese University of Hong Kong, Hong Kong
4
Département de Cytogénétique, Hôpital Universitaire de Pontchaillou, Rennes, France.
5
Laboratoire de Cytogénétique, Département de Génétique Médicale, Maternité de Pellegrin, Hôpital
Universitaire de Bordeaux, Bordeaux, France
6
Service d’histologie, embryologie et cytogénétique, Hôpital Necker Enfants Malades, AP-HP, Paris, France
7
Igenomix, Valencia, Spain
8
Département de Génétique, Hôpital Universitaire de Nantes, France
9
Service de Cytogénétique, Hôpital Cochin, AP-HP, Paris, France
10
Département de Cytogénétique, Obstétrique et Gynécologie, Hôpital de Poissy St Germain, Poissy, France
11
United Medix Laboratories Limited, Helsinki, Finland
12
EA2493, Université de Versailles Saint Quentin en Yvelines, Versailles, France
13
CHU Paris, Hôpital Jean Verdier, Unité de Cytogénétique, Bondy, France, Université Paris 13, Sorbonne
Paris Cité, URM 1141

Corresponding author:
Dr Céline Dupont
Unité de Cytogénétique, Département de Génétique, Hôpital Robert Debré (AP-HP), 48 Bd Sérurier, F-75935
Paris, France
E-mail: c.dupont@rdb.aphp.fr/ Tel.: +33 140 034 093/ Fax: +33 140 035 319

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article
as doi: 10.1002/pd.4478

This article is protected by copyright. All rights reserved.

None funding source that supported the work

The authors have no conflict of interest to declare

What’s already known about this topic?

FISH analysis of the 22q11.2 region in a large cohort of children referred for DGS/VCFS led to the

identification of a new chromosomal syndrome:microduplication 22q11.2. This syndrome has a highly variable

clinical phenotype.

What does this study add?

This study is the first prenatal series about the 22q11.2 microduplication syndrome (24 cases). We searched for

possible correlations between fetal phenotypes, indications for invasive prenatal diagnosis, inheritance,

pregnancy outcomes, and presence of others CNVs.

This article is protected by copyright. All rights reserved.

ABSTRACT
Objective

Microduplication 22q11.2 is primarily characterized by a highly variable clinical phenotype,

which ranges from apparently normal or slightly dysmorphic features (in the presence or

absence of learning disorders) to severe malformations with profound mental retardation.

Hence, genetic counseling is particularly challenging when microduplication 22q11.2 is

identified in a prenatal diagnosis. Here, we report on 24 prenatal cases of microduplication

22q11.2.

Methods

Seventeen of the cases were also reanalyzed by microarray analysis, in order to determine

copy number variations (CNVs, which are thought to influence expressivity). We also

searched for possible correlations between fetal phenotypes, indications for invasive prenatal

diagnosis, inheritance and pregnancy outcomes.

Results

Of the 24 cases, 15 were inherited, 6 occurred de novo and 3 were of unknown origin.

Termination of pregnancy occurred in 7 cases and was mainly decided on the basis of

ultrasound findings. Moreover, additional CNVs were found in some patients and we try to

make a genotype-phenotype correlation.

Conclusion

We discuss the complexity of genetic counseling for microduplication 22q11.2 and comment

on possible explanations for the clinical heterogeneity of this syndrome. In particular, we

assessed the co-existence of additional CNVs and their contribution to phenotypic variations

in chromosome 22q11.2 microduplication syndrome.

Keywords: microduplication 22q11.2 syndrome, Prenatal BoBs, prenatal diagnosis,

microarray, CNVs.

This article is protected by copyright. All rights reserved.

INTRODUCTION

The chromosomal region 22q11.2 is known to be involved in many clinical syndromes,

including velocardiofacial syndrome (VCFS, OMIM#192430), DiGeorge syndrome (DGS,

OMIM#188400) and cat eye syndrome (OMIM# 115470). Fluorescence in situ hybridization

(FISH) analysis of the 22q11.2 region in a large cohort of children referred for DGS/VCFS

led to the identification of a new chromosomal syndrome: microduplication 22q11.21. This

syndrome is associated with a highly variable clinical phenotype, which ranges from

apparently normal or slightly dysmorphic features with very moderate learning disabilities to

severe malformations with profound mental retardation1,2. Low copy repeats (LCR22A to

LCR22H) and segmental duplications in the 22q11.2 region are known to be responsible for

the chromosomal cryptic rearrangements associated with these syndromes, with mediation by

non-allelic homologous recombination mechanisms3. Most reported cases of 22q11.2

microduplication are the reciprocal products of the 3Mbp typically deleted region (TDR)

between LCRA and LCRD, for which a gene dosage effect has been demonstrated1. The main

candidate gene for the microdeletion-associated phenotype is TBX1, which encodes a T-box

transcription factor involved in the regulation of developmental processes. Liao et al. showed

that transgenic mice overexpressing human TBX1 have much the same malformations (cleft

palate, selected outfow tract defects) as patients with VCFS/DGS4. Further research has

demonstrated that missense gain-of-function mutations of TBX1 result in the same phenotype

(a conotruncal heart defect and absence of the thymus) as the 22q11.2 deletion in humans5.

The CNV morbidity map of developmental delay (based on a large study of cohorts of

patients and controls) confirmed the high penetrance of this duplication6. The main feature of

microduplication 22q11.2 is its high phenotypic variability. Familial cases have been

described in which an unaffected or slightly affected parent transmits the duplication to their

affected child. However, some sibs are not affected or may be affected with different

This article is protected by copyright. All rights reserved.

phenotypes. Hence, genetic counseling is very difficult when microduplication 22q11.2 is

diagnosed prenatally (especially in the absence of ultrasound findings); prediction of the

child's phenotype is not reliable. To the best of our knowledge, only four prenatal cases have

been reported7-10 and all occurred in a familial context.

The value of prenatal screening for malformations associated with a low risk of cryptic

imbalance is still subject to debate. A number of array comparative genomic hybridization

(aCGH) studies11-13 , Prenatal BACs-On-Beads (Prenatal BoBs, PerkinElmer, Inc.,

Shelton, CT, USA)14,15 and MLPA16 have demonstrated the utility of these molecular

cytogenetic methods for the diagnosis of microdeletion/microduplication syndromes.

Here, we report on 24 cases in which microduplication 22q11.2 was diagnosed prenatally.

DESCRIPTION OF THE STUDY POPULATION (Table 1)

Between January 2011 and November 2013, a total of 24 prenatal cases of microduplication

22q11.2 (P1 to P24) were diagnosed by 12 cytogenetic laboratories worldwide. The

diagnoses were performed on direct chorionic villus samples (n=11), direct amniotic fluid

(n=11) or fetal tissue (n=2) retrieved at gestational weeks 12 to 36 (mean: 19).

The prenatal diagnosis was prompted by the observation of ultrasound findings (n=19,

including increased nuchal translucency (n=7), multiple congenital abnormalities (n=7),

hydramnios (n=2), isolated bilateral cleft palate (n=1), isolated congenital heart defect (n=1),

and in utero death (n=1)), positive maternal serum screening for Down's syndrome (n=2), a

family history of microduplication 22q11.2 (n=2), and advanced maternal age (n=1).

This article is protected by copyright. All rights reserved.

MATERIAL AND METHODS (Figure 1)

Microduplication 22q11.2 was diagnosed with Prenatal BoBs (n=14), interphase FISH (n=6)

or array-CGH (n=4) (depending on each laboratory's facilities) after a conventional R- and G-

banded cytogenetic analysis had revealed a normal karyotype.

Prenatal BACs on BEADS™ (BoBs™) technology

Prenatal BoBs™ is a multiplexed, microsphere-based suspension array technology used to

detect common aneuploidies and gains/losses of nine frequent microdeletion syndrome

critical regions (Wolf-Hirschhorn, Cri du Chat, Williams-Beuren, Langer-Giedion, Prader-

Willi, Angelmann, Miller-Dieker, Smith-Magenis and DiGeorge-1/2).

DNA was extracted from amniotic fluid or chorionic villus samples and tested with Prenatal

BoBs™ technology15. All positive tests were confirmed by FISH (using commercial probes)

on at least 100 interphase nuclei and 10 metaphases.

The various 22q11 probes in the BoBs Kit used in the present study covered a region

spanning from 19,356 Mb to 19,920 Mb (NCBI37.2/Hg19) on chromosome 22 (including the

TBX1 gene).

Samples with microduplication 22q11.2 gave a ratio outside the expected range (≈+1.2) for

all 22q11.2 probes included in the assay (Figure 1A).

Fluorescence in situ hybridization

Fluorescence in situ hybridization analysis (using the Di George syndrome-specific TBX1

probe) was performed according to the manufacturer’s instructions on at least 100 interphase

nuclei and 10 metaphases. The FISH patterns on interphase nuclei with three separate

fluorescent signals were considered as evidence of duplication (Figure 1B). Parental studies

were also performed with the same FISH probe, in order to investigate the inheritance of the

imbalance and thus better assess the risk of transmission.

This article is protected by copyright. All rights reserved.

Array CGH

A high-resolution, genome-wide aCGH assay was performed using various technology

platforms (Agilent 180K, Agilent 105K, Agilent 60K, Agilent targeted custom design 44K,

PerkinElmer-CGX12 135K and Illumina 300K) according to the manufacturers’ standard

protocols, in order to (i) diagnose microduplication 22q11.2, (ii) confirm cases identified with

targeted approaches (Prenatal BoBsTM or FISH) and (iii) identify additive CNVs.

RESULTS

Analysis of R- and G-banded chromosomes from prenatal samples revealed a normal

karyotype in all cases. A metaphase FISH analysis confirmed the chromosomal mechanism

by discriminating between intra- and interchromosomal duplications (Figure 1C).

Inheritance:

The microduplication was inherited from a healthy parent in 15 patients (65%) (from the

father in 9 cases and from the mother in 6 cases). We found 6 de novo cases. In the remaining

3 cases, the inheritance could not be established because the parents were not available for (or

did not agree to) testing (Table 1).

Pregnancy outcomes (Table 1):

In 15 cases, pregnancy is either still on-going (n=3) or led to the delivery of a healthy baby

(despite the detection of an ultrasound marker at the time of the invasive procedure in 10 of

these 15 cases). In seven cases, the couple opted for termination of pregnancy (TOP). Lastly,

in utero death occurred in one case and the diagnosis was performed on a sample of fetal skin.

In the de novo group, the TOP and on-going pregnancy rates did not differ greatly (2 out of 7

(28%) and 4 out of 15 (26%), respectively). Conversely, the prevalence of ultrasound markers

associated with a poor prognosis (multiple fetal anomalies and severe visceral malformations

such as severe heart defect) was significantly higher in terminated pregnancies (6 out of 7)

This article is protected by copyright. All rights reserved.

than in on-going pregnancies (2 out of 15).

Array CGH analysis (Figure 2)

Seventeen cases were analyzed (n=4) or reflexed (n=13) by genome-wide aCGH analysis, in

order to determine concurrent copy gains/losses or size differences that could explain the

phenotypic variability of microduplication 22q11.2. The microduplication sizes are reported

in Table 1. For inherited cases (n=3), parental DNA was also analyzed (when available) in

order to compare the duplication size. No differences in breakpoint locations or the copy gain

size were detected when comparing the two generations. The majority of the

microduplications (n=14) corresponded to the common 3 Mbp critical region. In one case, we

observed a de novo 4 Mbp copy gain associated with non-specific hydramnios (P6). In one

family, we observed an inherited 0.9 Mbp gain associated with a severe congenital heart

defect (P3). The paternal gain did not differ from the fetal gain and the same

microduplication 22q11.2 was found in another pregnancy for the same couple (P4); the

pregnancy led to the delivery of a healthy baby. In between these two pregnancies in the

same couple, another pregnancy was terminated because of maternal inherited Duchenne

muscular dystrophy in a male fetus. It was phenotypically normal with the same

microduplication 22q11.2 but is not reported in Table 1 because of the presence of the

Mendelian disease.

Along with the microduplication 22q11.2, common CNVs ≥100 kbp elsewhere in the genome

were found in 11 of the 17 cases analyzed with aCGH (Table 2 (sup file) and Figure 2). No

rare recurrent CNVs were found in our series. Six chromosomal regions other than 22q11.2

were found to be duplicated or deleted in more than one case: 3q26.1, 6p25.3, 8p11.22,

10q26.3, 14q11.2 and 15q11.1q11.2.

This article is protected by copyright. All rights reserved.

DISCUSSION

At present, several molecular cytogenetic technologies are available for the rapid, accurate

prenatal diagnosis of chromosome abnormalities in general and common aneuploidies in

particular. In France, the current gold standard is interphase FISH on primary amniocytes.

More recently, aCGH has been applied to pregnancies with a high risk of pathogenic

submicroscopic chromosomal imbalance. In low-risk pregnancies, the risk of finding CNVs

with uncertain clinical significance has prompted some laboratories to target a small group of

chromosome regions with clear clinical significance. Prenatal BoBs is one of newest of

these assay technologies and has been validated in the diagnosis of aneuploidy and other

microdeletion syndromes14,15,17. These new technologies have also enabled the diagnosis of

reciprocal microduplications (such as 22q11.2) that may be considered as variants of

unknown significance. The reciprocal microduplications are easier to miss than targeted

microdeletions themselves, since (i) a mild clinical phenotype is expected and (ii) it is

difficult to resolve two nearby FISH signals on interphase nuclei. This means that

microduplications are likely to be underdiagnosed, even when they are associated with

clinically recognizable syndromes (such as Potocki-Lupski)18. Some microduplication

syndromes are characterized by high phenotypic variability and are diagnosed in both

probands with congenital malformations and/or developmental delay and unaffected or

slightly affected subjects or relatives. Microduplication 22q11.2 is a good example of this.

Here, we reported on 24 new prenatally diagnosed cases of microduplication 22q11.2.

Concerning the BoBs analysis, our overall detection rate for the 22q11.2 microduplication

was 0.18% (11 out of 6125 fetuses analyzed by BoBs), which is similar to the value found

in the literature (2 out of 1599 fetuses)15.

Classically, microduplication 22q11.2 patients display slightly dysmorphic features, with

hypertelorism, a broad nasal bridge, down-slanting palpebral fissures, a flat philtrum,

This article is protected by copyright. All rights reserved.

abnormally large ears, micrognathia, midface hypoplasia and microcephaly1. Although the

dysmorphic features in microduplication 22q11.2 differ from those described in 22q11.2

deletion1, they share some clinical features (such as congenital/conotruncal heart defects and

an abnormal oral-facial cleft palate). In the present series, the indications for prenatal

diagnosis varied. We found some overlapping features with microdeletion 22q11.2 syndrome

(congenital heart defects, cleft palate, and thymic hypoplasia). Remarkably, nine patients

(37%) presented with increased nuchal translucency and five patients (21%) displayed a

congenital heart defect.

Moreover, the diagnosis led to seven TOPs. We observed one in utero death and three

pregnancies with a low risk of chromosome abnormalities (with isolated maternal biological

markers or advanced maternal age) which were all continued - even though the

microduplication was de novo in one case (P13).

Our prenatal series confirmed the extreme phenotypic variability of this new chromosomal

syndrome. Patients with microduplication 22q11.2 and normal or near-normal phenotypes

have been reported in a large number of families7,19,20 . In our cohort, the fetal phenotype did

not match the parental phenotype in four of the 15 inherited microduplications. All four cases

resulted in TOP. In one case, the microduplication was associated with monosomy X.

Phenotypic variability is well illustrated by cases P3 and P4. Initially, a prenatal diagnosis

was made for P3 because of Duchenne muscular dystrophy in the mother’s family (for which

the test was negative). Postmortem findings in P3 included a congenital heart defect (with

transposition of the great vessels, ventricular septal defect and abnormal tricuspid valve),

cleft palate, facial dysmorphism, a single umbilical artery and delayed bone maturation.

Microduplication 22q11.2 was diagnosed by BoBs in this fetus because a second-trimester

ultrasound examination had revealed a complex heart defect with cleft palate, a single

umbilical artery and delayed bone maturation. The microduplication 22q11.2 was found to be

This article is protected by copyright. All rights reserved.

inherited from the fetus' healthy father. The parents decided to terminate the pregnancy

because of the severe ultrasound findings. The couple's next two pregnancies were also found

to have this small microduplication. Although the first pregnancy was terminated because of

the presence of Duchenne muscular dystrophy in a male fetus, the latter was phenotypically

normal (but is not reported in Table 1 because of the presence of this Mendelian disease). The

third pregnancy (P4) led to the delivery of a healthy baby.

Our observation agrees with previous reports7,8 in which the diagnosis of 22q11.2

duplications in fetuses with severe congenital heart defects led to TOP - even when the

microduplication was inherited from healthy parents. Nevertheless, the vast majority of

familial cases in our cohort (11 out of 15) were not terminated and resulted in uneventful

pregnancies. In four cases, TOP was chosen on the basis of severe ultrasound findings. Hence,

genetic counseling for this new syndrome is often challenging.

There are several possible explanations for the incomplete penetrance and variable

expressivity observed in this syndrome6: (i) the variable size of the imbalance (as we saw

above), (ii) the influence of several interfering genes or epigenetic factors5,21 and (iii) the co-

existence of additional CNVs20,22.

In terms of the size of the microduplication, non-allelic homologous recombination is known

to be responsible for microduplications and microdeletions of the 22q11.2 region, with

mediation by the number of LCRs (LCR22A to LCR22H). The most common

microduplications occur between LCRA and LCRD and yield an approximately 3 Mbp gain,

which corresponds to the reciprocal event of the TDR observed in DGS/VCFS1.

Microduplications of 22q11.21-q11.23 between LCRD and LCRH (distal to the region

involved in the classical 22q11.2 microduplication syndrome) have also been described23.

Our findings are consistent with previous reports: 13 of the 17 molecularly characterized

cases featured the common 22q11.2 duplication. In one case, a larger (4 Mbp) gain in size

This article is protected by copyright. All rights reserved.

(P6) had already been described19. In contrast, P3 and P4 (from the same family) had a

smaller, atypical microduplication (0.9 Mbp: 18,919,942-19,838,159/hg19). In agreement

with previous studies, the phenotype's severity was not correlated with the size of the

microduplication1,19; for example, P6 (with the larger microduplication and de novo) is a

healthy child with isolated, prenatally diagnosed hydramnios, whereas P3 (with the smaller

microduplication, inherited from an healthy father) showed a complex heart defect with cleft

palate, a single umbilical artery and delayed bone maturation. The smallest yet (437 kbp)

microduplication 22q11.2 was recently identified by Weisfeld-Adams et al. (using an

Affymetrix Genome-Wide Human SNP array 6.0) in a child with microcephaly,

cryptorchidism and patent ductus arteriosus20. This microduplication was maternally inherited

and the child's sister (who presented with mild hypotonia and motor delay) had the same

microduplication. As with the siblings P3 and P4, the breakpoints did not coincide with

known LCR regions. These cases again show that microduplication size is not a good

predictor of the clinical phenotype - even in inherited cases.

In terms of genetic factors, the main candidate gene for the phenotype modulation of

microduplication 22q11.2 is TBX1 (as in DGS/VCFS). Transgenic mice overexpressing

human TBX1 with a gain-of-function mutation had the same phenotype as with 22q11.2

deletion4,5 . Genetic studies in mice have suggested the presence of modifier genes both in the

critical region and elsewhere in the genome21. Most of these candidate genes are likely be

involved in Tbx1–related pathways in the development of the pharyngeal germ layers. For

example, Tbx1 regulates or is modified by fibroblast growth factors 8 and 10 (encoded by

Fgf8 and Fgf10) 21, Shh24, Crkl and retinoic acid25. These studies suggest that many different

genetic factors can modify the expression of Tbx1 or, more generally, influence the

expressivity of 22q11.2 region disorders.

With regard to the CNVs' potential involvement in modulation of the phenotypic

This article is protected by copyright. All rights reserved.

manifestation of DGS/VCFS critical region CNVs, Li et al. observed the co-existence of

additional CNVs26: a 605 kbp duplication of the 15q13.3 region encompassing CHRNA7 gene

and a 209 kbp deletion involving the RBFOX1 gene in 16p13.2. Li et al. suggested that these

two genes (which have already been implicated in autism and schizophrenia) might modify

the phenotype26. On the same lines, Girirajan et al., recently analyzed the genomes of 2312

children known to carry a CNV associated with intellectual disability and congenital

abnormalities27. The researchers found that 10.1% of the children carried a second, large

CNV in addition to the primary genetic lesion27. On this basis, we screened for the presence

of other CNVs ≥100 kbp elsewhere in the genome (Table 2 (sup file), Figure 2). Six

chromosomal regions were found to be duplicated or deleted in more than one patient (3q26.1

[n=3 patients], 6p25.3 [n=2], 8p11.22 [n=5], 10q26.3 [n=3], 14q11.2 [n=3], and

15q11.1q11.2 [n=4]). Even though the tests were not all performed in the same country or

same laboratory or even by the same technician, it is known that the polymorphisms detected

by each type of array may depend on the chip and not only on the patient DNA. Two of the

six common regions appear to be particularly relevant. The first is the 8p11.2 region (starting

at nt:39,237,438/hg19). It must be borne in mind that recurrent CNVs in 8p11.22 are

frequently found by Agilent arrays because the region can be detected in reference DNA.

However, the 8p11.22 CNV is also a common variation in the population and was not found

in all our patients. Five fetuses (P1, P3, P15, P16 and P22) had a 120-150 kbp rearrangement

of this region; four of these cases presented a severe congenital heart defect or multiple

malformations and the only healthy fetus (P1) had a microdeletion in this region.

Interestingly, in the two families in which more than one fetus had the microduplication

22q11.2, the malformed fetuses had an 8p11.2 microrearrangement (P3 and P22) and the

healthy fetuses did not have any CNVs in this region (P4 and P23). Unfortunately, array-

based testing could not be performed in all parents. However, in one French center in which

This article is protected by copyright. All rights reserved.

the parents were systematically screened, it was found that the father of P3 and P4 (who had

the microduplication 22q11.2 associated with a normal phenotype) did not have a

rearrangement in 8p11.22. In fact, the 8p11.2 region is known to contain common nucleotide

polymorphisms in healthy populations. One can legitimately hypothesize that the

combination of an 8p11.2 microrearrangement and a 22q11.2 microduplication results in the

most severe phenotype. Furthermore, the 8p11.2 region contains a binding site for the CTCF

zinc-finger transcription factor (known to be an enhancer-blocking transcriptional insulator).

Along with laminin and the cohesins, CTCF is one of the main organizers of the genomic

architecture within the interphase nucleus28. Several observations indicate that CTCF and

cohesins may control gene expression by mediating local changes in the chromatin

conformation - thereby determining which promoters can directly interact with enhancers or

other regulatory sequences28. However, before postulating correlations between this CNV and

microduplication 22q11.2, we would have to check whether this CNV is less frequent in the

rest of the analyzed cases than in our 22q11.2 cohort. At present, we do not have enough

cases to assess the reliability of this analysis. However, detection of other CNVs that might

contribute to the phenotype may be used for counseling (even though the data on common

CNVs must be interpreted with care).

On the same lines, two microdeletions of the 3q26.1 region (starting at nt: 162,514,534/hg19

and spanning 104 kbp) were found in healthy babies in our series, whereas microduplication

of the exact same region was combined with microduplication 22q11.2 in a fetus with

multiple defects. Interestingly, this region is also a binding site for CTCF. We did not find

any genotype-phenotype correlations for the four other common areas (6p25.3, 10q26.3,

14q11.2, and 15q11.1q11.2).

Lastly, epigenetic factors could potentially modify the expressivity of this syndrome. For

example, it is known that TBX1 expression is inhibited by retinoic acid. During development,

This article is protected by copyright. All rights reserved.

environmental factors (of maternal origin) that interfere with retinoic acid or vitamin A

metabolism (e.g. alcohol) might therefore change the expression of TBX1 and thus the

expressivity of this syndrome5. Moreover, a recent paper29 reported that the histone

acetyltransferase MOZ (MYST3/KAT6A) regulates the transcriptional activity of the Tbx1

locus. The researchers suggested the presence of a molecular mechanism for specific

modification of the chromatin at the Tbx1 locus, which might explain the phenotype

variability in DGS. Similarly, one can argue that the same epigenetic mechanism can also

modulate phenotype variability in the microduplication 22q11.2 syndrome.

In contrast to inheritance of the 22q11.2 deletion (which was recently reported as being of

maternal origin in 76.1% of cases30), the duplication 22q11.2 appears to be inherited from the

father or mother with the same frequency.

In conclusion, we have reported on the first prenatal series of microduplication 22q11.2

syndrome. Our data are consistent with the extreme phenotype variability of this condition.

Novel molecular cytogenetics technologies (such as Prenatal BoBs and routine prenatal

microarray analysis) will probably increase the detection rate for this and other cryptic

microduplication rearrangements. Further studies using aCGH or SNP arrays in these patients

might lead to the description of different "molecular profiles" by characterizing CNVs that

may modulate the phenotype of microduplication 22q11.2 syndrome. Furthermore, it may

soon become possible to detect microdeletion/microduplication syndromes with non-invasive

prenatal testing of cfDNA and thus increase the detection rate.

Moreover, detailed clinical follow-up of children with prenatally diagnosed microduplication

22q11.2 syndrome is likely to provide us with a better understanding of the underlying

disease mechanism. Although these findings need to be confirmed in a larger series, it

appears that the parents' decision is driven more by the presence of poor prognostic

ultrasound findings (such as a severe heart defect and multiple congenital malformations)

This article is protected by copyright. All rights reserved.

than by inheritance of the imbalance. In contrast, it appears to be difficult to completely

reassure parents during prenatal genetic counseling when a detailed prenatal ultrasound

examination has not evidenced any structural signs. We need to consider that in patients with

a developmental delay (which is not always associated with congenital structural

abnormalities), the typical microduplication 22q11.2 shows a penetrance close to 1 (0.91)

with a high degree of statistical significance (p=1.26E-05)6.

REFERENCES

1 Portnoi MF.. Microduplication 22q11.2: a new chromosomal syndrome. Eur J Med

Genet 2009; 52:88-93.

2 Ou Z, Berg JS, Yonath H, et al. Microduplications of 22q11.2 are frequently inherited

and are associated with variable phenotypes. Genet Med 2008; 10:267-77.

3 Edelmann L, Pandita RK, Spiteri E, et al. A common molecular basis for rearrangement

disorders on chromosome 22q11. Hum Mol Genet 1999; 8:1157-67.

4 Liao J, Kochilas L, Nowotschin S, et al. Full spectrum of malformations in velo-cardio-

facial syndrome/DiGeorge syndrome mouse models by altering Tbx1 dosage. Hum Mol

Genet 2004; 13:1577-85.

5 Torres-Juan L, Rosell J, Morla M, et al. Mutations in TBX1 genocopy the 22q11.2

deletion and duplication syndromes: a new susceptibility factor for mental retardation.

Eur J Hum Genet 2007; 15:658-63.

This article is protected by copyright. All rights reserved.

6 Cooper GM, Coe BP, Girirajan S, et al. A copy number variation morbidity map of

developmental delay. Nat Genet 2011; 43:838-46.

7 de La Rochebrochard C, Joly-Hélas G, Goldenberg A, et al. The intrafamilial variability

of the 22q11.2 microduplication encompasses a spectrum from minor cognitive deficits to

severe congenital anomalies. Am J Med Genet A 2006; 140:1608-13.

8 Hu P, Ji X, Yang C, et al. 22q11.2 microduplication in a family with recurrent fetal

congenital heart disease. Eur J Med Genet 2011; 54:e433-6.

9 Christopoulou G, Sismani C, Sakellariou M, et al. Clinical and molecular description of

the prenatal diagnosis of a fetus with a maternally inherited microduplication 22q11.2 of

2.5 Mb. Gene 2013; 527:694-7.

10 Brady PD, Delle Chiaie B, Christenhusz G, et al. A prosepctive study of the clinical

utility of prenatal chromosomal microarray analysis in fetuses with ultrasound

abnormalities and an exploration of a framework for reporting unclassified variants and

risk factors. Get Med 2014; 16:469-76.

11 Vialard F, Molina Gomes D, Leroy B, et al. Array comparative genomic hybridization

in prenatal diagnosis: another experience. Fetal Diagn Ther 2009; 25:277-84.

12 Leung TY, Vogel I, Lau TK, et al. Identification of submicroscopic chromosomal

aberrations in fetuses with increased nuchal translucency and apparently normal

karyotype. Ultrasound Obstet Gynecol 2011; 38:314-9.

This article is protected by copyright. All rights reserved.

13 Shaffer LG, Dabell MP, Fisher AJ, et al. Experience with microarray-based

comparative genomic hybridization for prenatal diagnosis in over 5000 pregnancies.

Prenat Diagn 2012; 32:976-85.

14 Shaffer LG, Coppinger J, Morton SA, et al. The development of a rapid assay for

prenatal testing of common aneuploidies and microdeletion syndromes. Prenat Diagn

2011; 31:778-87.

15 Vialard F, Simoni G, Gomes DM, et al. Prenatal BACs-on-Beads: the prospective

experience of five prenatal diagnosis laboratories. Prenat Diagn 2012; 32:329-35.

16 MLPA: a prenatal diagnostic tool for the study of congenital heart defects?

Mademont-Soler I, Morales C, Soler A,et al. Gene 2012; 500:151-4

17 Gross SJ, Bajaj K, Garry D, et al. Rapid and novel prenatal molecular assay for

detecting aneuploidies and microdeletion syndromes. Prenat Diagn 2011; 31:259-66.

18 Popowski T, Molina-Gomes D, Loeuillet L, et al. Prenatal diagnosis of the

duplication 17p11.2 associated with Potocki-Lupski syndrome in a foetus presenting with

mildly dysmorphic features. Eur J Med Genet 2012; 55:723-6.

19 Ensenauer RE, Adeyinka A, Flynn HC, et al. Microduplication 22q11.2, an emerging

syndrome: clinical, cytogenetic, and molecular analysis of thirteen patients. Am J Hum

Genet 2003; 73:1027-40.

This article is protected by copyright. All rights reserved.

20 Weisfeld-Adams JD, Edelmann L, Gadi IK, Mehta L. Phenotypic heterogeneity in a

family with a small atypical microduplication of chromosome 22q11.2 involving TBX1.

Eur J Med Genet 2012; 55:732-6.

21 Bi W, Probst FJ, Wiszniewska J, et al. Co-occurrence of recurrent duplications of the

DiGeorge syndrome region on both chromosome 22 homologues due to inherited and de

novo events. J Med Genet 2012; 49:681-8.

22 Aggarwal VS, Morrow BE. Genetic modifiers of the physical malformations in velo-

cardio-facial syndrome/DiGeorge syndrome. Dev Disabil Res Rev 2008; 14:19-25.

23 Coppinger J, McDonald-McGinn D, Zackai E, et al. Identification of familial and de

novo microduplications of 22q11.21-q11.23 distal to the 22q11.21 microdeletion

syndrome region. Human molecular genetics 2009; 18:1377-83

24 Garg V, Yamagishi C, Hu T, et al. Tbx1, a DiGeorge syndrome candidate gene, is

regulated by sonic hedgehog during pharyngeal arch development. Dev Biol 2001;

235:62-73.

25 Guris DL, Duester G, Papaioannou VE, Imamoto A. Dose-dependent interaction of

Tbx1 and Crkl and locally aberrant RA signaling in a model of del22q11 syndrome. Dev

Cell 2006; 10:81-92.

26 Li D, Tekin M, Buch M, Fan YS. 2012. Co-existence of other copy number variations

This article is protected by copyright. All rights reserved.

with 22q11.2 deletion or duplication: a modifier for variable phenotypes of the syndrome?

Mol Cytogenet 5:18

27 Girirajan S, Rosenfeld JA, Coe BP, et al. Phenotypic heterogeneity of genomic

disorders and rare copy-number variants. The New England journal of medicine . 2012;

367:1321-31.

28 Wendt KS, Peters JM. How cohesin and CTCF cooperate in regulating gene

expression. Chromosome Res 2009; 17:201-14.

29 Voss AK, Vanyai HK, Collin C, et al. MOZ regulates the Tbx1 locus, and Moz

mutation partially phenocopies DiGeorge syndrome. Dev Cell 2012; 23:652-63.

30 Besseau-Ayasse J, Violle-Poirsier C, Bazin A, et al.. A French collaborative survey of

272 fetuses with 22q11.2 deletion: ultrasound findings, fetal autopsies and pregnancy

outcomes. Prenat diagn 2014 oi: 10.1002/pd.4321.

This article is protected by copyright. All rights reserved.

 PATIENT INDICATION OF PRENATAL DIAGNOSIS Detailled TISSUE WG METHOD ISSUE INHERITED ARRAY START (hg19) END (hg19) LENGH

P1 Ultrasound findings minor heart defect (right aortic arch) Amniotic fluid 22 BoBS born, healthy pat Agilent 180K 19023824 21464119 2440295

P2 Ultrasound findings increased nuchal translucency chorionic villus sample 13 BoBS born, healthy mat Agilent 180K 18909044 21464119 2555075

Familial study (Duchenne myopathy in mother family) then severe congenital heart defect (TGA,VSD)+ bilateral cleft palate+facial
P3 chorionic villus sample 13 BoBS TOP pat Agilent 180K 18919942 19838159 918217
Ultrasound findings dysmorphism+delayed bone maturation+single umbilical artery

Familial study (father with dup22, Duchenne myopathy in

P4 absence of ultrasound finding chorionic villus sample 13 BoBS born, healthy pat Agilent 180K 18919942 19838159 918217
mother family)

P5 Ultrasound findings isolated bilateral cleft palate Amniotic fluid 29 BoBS TOP de novo PerkinElmer CGX12 18919469 21811314 2891845

P6 Ultrasound findings isolated hydramnios Amniotic fluid 30 BoBS born, healthy de novo PerkinElmer CGX12 18209486 22129322 3919836

P7 Down syndrom maternal serum screening unknown Amniotic fluid BoBS born, healthy pat

advance maternal age+previous fetus died for isolated

P8 unknown Amniotic fluid 17 BoBS born, healthy pat
diaphragmatic hernia

Ultrasound findings+ previous child/pregnancy with increased nuchal translucency+ nasal bone defect+ single umbilical artery+ Agilent Target designed
P9 chorionic villus sample 12 BoBS born,healthy de novo 18909032 21801661 2892629
retinoblastoma stomach invisible. 44K

P10 Ultrasound findings increased nuchal translucency (7.9mm) chorionic villus sample 13 BoBS TOP (+ 45,X) pat

P11 in utero death at 36+5 WG absence of dysmorphic features skin 36 BoBS IUD ? Illumina 300K 18656495 21452237 2795742

Ultrasound findings+advance maternal age+ down syndrom positive Down screening risk: 1:164; US: increased nuchal translucency Agilent Target designed
P12 Amniotic fluid 20 BoBS born, healthy mat 18909032 21357982 2448951
maternal serum screening (7.8mm)+ prominent stomach 44K
Agilent Target designed
P13 Down syndrom maternal serum screening positive Down screening risk 1:16 Amniotic fluid 18 BoBS born, healthy de novo 18909032 21357982 2448951
44K

positive Down screening risk: 1:2; US: increased nuchal translucency Agilent Target designed
P14 Ultrasound findings+Down syndrome maternal serum screening chorionic villus sample 13 BoBS TOP de novo 18909032 21357952 2448951
(4.3mm) 44K

polymalformed fetus: left labial cleft+ hypertelorism+ retrognathism+

P15 Ultrasound findings Amniotic fluid 23 FISH TOP pat Agilent 60K 18919942 21801661 2881719
single umbilical artery+thymic hypoplasia+clubfeet

polymalformed fetus : macrosomia+facial dysmorphism (short neck,

anteverted nostrils, long philtrum)+ bilateral brachymesophalangia of the
P16 Ultrasound findings Amniotic fluid 33 FISH TOP ? Agilent 105K 18894835 21561514 2666679
5th+ complex congenital heart defect (double aortic and mitral stenosis,
small left ventricle with hypertrophy)

P17 Ultrasound findings increased nuchal translucency (6mm) chorionic villus sample 13 FISH born, healthy mat

P18 Ultrasound findings increased nuchal translucency (4mm) chorionic villus sample 13 FISH born, healthy mat

P19 Ultrasound findings isolated hydramnios Amniotic fluid 29 FISH born,healthy de novo

P20 Ultrasound findings increased nuchal translucency (3.6mm) chorionic villus sample 13 FISH continue pat

P21 Ultrasound findings increased nuchal translucency (3.5mm) chorionic villus sample 13 CGH array continue pat Agilent 60K 18919942 21440514 2520572

polymalformed fetus: complex congenital heart defect+ facial

P22 Ultrasound findings muscle 28 CGH array TOP mat Agilent 180K 18661524 21812179 3150655
dysmorphism+ thymic hypoplasia+ left pyelectasia

P23 Familial study (mother with dup22) minor external features: dysplastic left ear chorionic villus sample 12 CGH array born, healthy mat Agilent 180K 18894635 21995624 3100989

amniotic fluid in excess, ventricular septal defect, pulmonary stenosis,

P24 Ultrasound findings Amniotic fluid 25 CGH array continue ? Agilent 60K 18570312 21466514 2896202
eosophageal atresia suspicion and crisped hands

Table 1: The main characteristics of study participants with prenatally detected microduplication 22q11.2:
Description of each case of microduplication 22q11.2 (Indication and term for prenatal diagnosis, ultrasound findings, methods of diagnosis, issues of the pregnancies,
inherited status, time and results of array when done).
WG: weeks of gestation; BoBs: BACs-on-BeadsTM; aCGH: array CGH; TOP: termination of pregnancy; pat: paternally inherited; mat: maternally inherite

This article is protected by copyright. All rights reserved.

Figure 1

1A/ Partial Prenatal BoBs profile of Patient 5: focus on beads from the 22q11.2 region.
The amount of target bound to each bead type is then compared to the amount of target bound
in reference samples (male: blue line and female: red line). The results are showed as ratio
plots indicating where a gain or a loss of genomic material has occurred. Here, the profile is
deflected to the right (≈ 1.2), indicating microduplication.

1B/ Interphase FISH of Patient 5: the three red fluorescent signals correspond to the TBX1
probe (control probe: ARSA, green, 22q13.3).

1C/ Metaphase FISH of Patient 5: showing the intrachromosomal nature of the 22q11.2
microduplication with a large TBX1 fluorescent signal.

This article is protected by copyright. All rights reserved.

Figure 2

Schematic representation of the location of CNVs

Schematic representation of the location of CNVs that co-exist with the 22q11.2
microduplication in patients having undergone an aCGH analysis (P1, P2, P3, P5, P9, P12,
P14, P15, P16, P22 and P23). Microduplications are shown in blue and microdeletions are
shown in red.

This article is protected by copyright. All rights reserved.