RESEARCH LETTER

De Novo Duplication of 18p11.21–18q12.1 ina Female With Anorectal MalformationCharlotte Schramm,1,2 Markus Draaken,1,2 Enrika Bartels,1 Thomas M. Boemers,3 Eberhard Schmiedeke,4

Sabine Grasshoff-Derr,5 Stefanie M€arzheuser,6 Stuart Hosie,7 Stefan Holland-Cunz,8

Friederike Baudisch,1,9 Lutz Priebe,1,2 Per Hoffmann,1,2 Alexander M. Zink,1 Hartmut Engels,1

Felix F. Brockschmidt,1,2 Stefan Aretz,1 Markus M. N€othen,1,2 Michael Ludwig,9 and Heiko Reutter1,10*1Institute of Human Genetics, University of Bonn, Bonn, Germany2Department of Genomics, Life and Brain Center, University of Bonn, Bonn, Germany3Department of Pediatric Surgery and Pediatric Urology, Children’s Hospital, Cologne, Germany4Department of Pediatric Surgery and Urology, Center for Child and Adolescent Health, Hospital Bremen-Mitte, Bremen, Germany5Department of Pediatric Surgery, University Hospital W€urzburg, W€urzburg, Germany6Department of Pediatric Surgery, Campus Virchow Clinic, Charit�e University Hospital Berlin, Berlin, Germany7Department of Pediatric Surgery, Klinikum Schwabing, Technische Universit€at M€unchen, M€unchen, Germany8Department of Pediatric Surgery, University of Heidelberg, Heidelberg, Germany9Department of Clinical Chemistry and Clinical Pharmacology, University of Bonn, Bonn, Germany10Department of Neonatology, Children’s Hospital, University of Bonn, Bonn, Germany

Received 15 September 2010; Accepted 1 November 2010

TO THE EDITOR:

Anorectal malformations (ARM) occur in about 1 in 2,500

live births [Cuschieri, 2001]. ARM can be classified into different

groups regarding their diagnostic, therapeutic, and prognostic

features [Levitt and Pe~na, 2007], ranging from ARM without fistula

(e.g., imperforate anus without fistula), with fistula (e.g., recto-

perineal malformation) to complex ARM including cloacal

malformations. Similar to most congenital defects, ARM can occur

as an isolated malformation or associated with other defects or

syndromes. Among the defined syndromic forms are chromosomal

abnormalities (e.g., trisomy 13, 18, and 21; cat eye syndrome),

monogenic syndromes (e.g., Townes–Brocks, Klippel–Feil, or

Pallister–Hall), sequences (Pierre Robin) or associations

(VATER/VACTERL) [Levitt and Pe~na, 2007; Stoll et al., 2007].

Other than the molecular genetic elucidation of a few syndromic

forms, the current knowledge of genetic factors underlying most

ARM is very limited. Since many ARM phenotypes are associated

with reduced reproductive fitness, it is reasonable to assume

that a significant proportion of patients carry new (de novo)

mutations. On this basis we performed a systematic screening

of 13 ARM patients (Table I) and their unaffected parents using

a SNP-array-based genome-wide approach to search for mutations

characterized by a loss or gain of genomic material. The parallel

investigation of the patients’ parents allowed for identification of de

Charlotte Schramm and Markus Draaken contributed equally to this work.

Grant sponsor: German Federal Ministry of Education and Research

(Bundesministerium f€ur Bildung und Forschung, BMBF); Grant

number: 01GM08107; Grant sponsor: University of Bonn; Grant

number: O-149.0093.

*Correspondence to:

Heiko Reutter, Department of Neonatology, Institute of Human Genetics,

University of Bonn, Sigmund-Freud-Str. 25, D-53127 Bonn, Germany.

E-mail: reutter@uni-bonn.de

Published online 11 January 2011 in Wiley Online Library

(wileyonlinelibrary.com).

DOI 10.1002/ajmg.a.33820

How to Cite this Article:Schramm C, Draaken M, Bartels E, Boemers

TM, Schmiedeke E, Grasshoff-Derr S,

M€arzheuser S, Hosie S, Holland-Cunz S,

Baudisch F, Priebe L, Hoffmann P, Zink AM,

Engels H, Brockschmidt FF, Aretz S, N€othen

MM, Ludwig M, Reutter H. 2011. De novo

duplication of 18p11.21–18q12.1 in a female

with anorectal malformation.

Am J Med Genet Part A 155:445–449.

� 2011 Wiley-Liss, Inc. 445

novo mutations. For the present analysis, patients presenting with

distinct clinical syndromes, for example, Currarino syndrome,

Townes–Brocks syndrome, or VACTERL/VATER association, or

with a positive family history were excluded from the analysis.

The study was approved by the local Ethics Committee and

the families provided informed consent. EDTA blood samples

were obtained from each family and isolation of genomic

DNA was carried out using the QIAmp DNA Blood Kit (Qiagen,

Hilden, Germany). Molecular karyotyping was performed using

SNP-based genome-wide arrays (Human660-Quad-v1 BeadChip

and Illumina HumanOmni1-Quad BeadChip [Illumina, San Die-

go, CA] containing 657,366 and 1,140,419 markers, respectively).

All analyses were performed according to the manufacturer’s

protocol. An analysis was considered to have failed if less than

95% of the loci were informative on the BeadChip. To identify

potential copy number variants (CNVs), the SNP fluorescence

intensity data of each individual were analyzed with QuantiSNP

(v2.1, www.well.ox.ac.uk/QuantiSNP/) using an Objective-Bayes

Hidden-Markov model for calling putative CNVs [Colella et al.,

2007]. CNVs with a log Bayes factor below 30 were disregarded, as

recommended. The results were also checked visually using the

GenomeStudio (v2009.2, www.illumina.com/) genotyping module

(see legend for Fig. 1). Filtering of CNVs for de novo events, known

variable regions and gene content was carried out using the

Cartagenia Bench� software (Cartagenia, Leuven, Belgium). Sex

chromosomes were excluded from further analysis.

Molecular karyotyping of the entire sample of 13 cases defined

a duplication of chromosomal region 18p11.21–18q12.1 in a female

patient (case 1; Table I) with anal atresia and rectovesical fistula,

patent ductus arteriosus (PDA) and myopia. No further de novo

chromosomal microaberration containing known genes which

did not correspond to a known variable region listed in the Database

of Genomic Variants (DGV, http://projects.tcag.ca/variation/)

were found in the remaining 12 ARM patients.

The patient (case 1) was born after 41 weeks of gestation as the

first child to a 35-year-old woman (gravida 1, para 1). Pregnancy

was uneventful. The healthy, nonconsanguineous parents were

of Caucasian origin with no family history of malformations. The

patient has a healthy younger sister who was 5 years of age.

At birth, the patient presented with anal atresia and rectovesical

fistula, PDA, slight facial dysmorphism and hypotonia. Because

of the anal atresia, a colostomy was performed. The anal atresia

was surgically corrected at the age of 6 months, and the colostomy

was closed. At the age of 3 years myopia (�7/�4 dpt) was diag-

nosed. Motor development was slightly delayed during infancy, but

treatment was not considered necessary. Cognitive functioning was

evaluated with the Bayley Scales of Infant Development at the age

of 3 years and 3 months [Bayley, 1993] with normal results. In

accordance with this, the girl showed normal development without

any signs of cognitive, language, or social impairments over the

following years. Likewise her gross motor skills were normal with

only slight deficits in her fine motor skills. At the time of assessment

the patient was 7 years of age and successfully attending primary

school, showing no signs of physical illness. Furthermore her slight

facial dysmorphism described in infancy was no longer apparent.

Because of her congenital anomalies, a karyotype (G-banding)

had been performed when she was a neonate. This revealed a 46,XX

karyotype with the presence of a small duplication on chromosome

TABLE I. Phenotypes of the Investigated ARM Patients

Case Chip array Sex ARM Other major malformations/deformations1a 660 F þ Slight facial dysmorphism in infancy, myopia, PDA2 660 M þ Myelomeningocele, Arnold–Chiari malformation II,

glandular hypospadias, right-sided renal agenesis3 660 M þ Craniosynostosis (coronal and lambdal synostosis),

bilateral pre-auricular tags, hemifacial microsomia4 660 M þ Left-sided renal dysplasia, bilateral VUR IV�,

cryptorchidism, persistent urachus5 660 M þ Prune-belly syndrome, right-sided cystic renal dysplasia,

caudal regression syndrome, costal bone deformity6 660 F þ Hyperopia, oral cutaneous fistula, left-sided pre-auricular fistula,

left-sided UPJ obstruction, left-sidedhydronephrosis, Hypomelanosis Ito

7 Omni F þ Right-sided VUR II–III�, left-sided VUR I�, atrial septal defect type II8 Omni M þ Left-sided renal agenesis, right-sided UPJ obstruction,

right-sided hydronephrosis, glandular hypospadias9 Omni F þ PFO, right-sided UPJ obstruction,

right-sided hydronephrosis, bicornuate uterus10 Omni F þ Vaginal atresia, bicornuate uterus, sacral dysplasia11 Omni F þ gibbus, missing teeth12 Omni M þ Phimosis, cryptorchidism, left-sided VUR II–III�

13 Omni M þ Left-sided renal agenesis, glandular hypospadias

VUR, vesico-ureteral reflux; PDA, patent ductus arteriosus; UPJ obstruction, ureteropelvic junction obstruction; PFO, persistent foramen ovale.aPatient with de novo duplication of chromosomal region 18p11.21–18q12.1.

446 AMERICAN JOURNAL OF MEDICAL GENETICS PART A

18p, without involvement of 18q. However, molecular karyotyping

of the patient in our study showed duplication of chromosomal

region 18p11.21–18q12.1 (Fig. 1) with an estimated size of 15 Mb.

Analysis of the parents revealed absence of this rearrangement.

Paternity testing was performed with the GenomeStudio genotyp-

ing module (v2009.2, www.illumina.com/). The Reproducibility

and Heritability Report revealed a parent–parent–child heritability

of 99.98%, thus indicating a de novo event (data not shown).

Moreover, haplotype analysis showed the duplication of chromo-

some 18 was of paternal origin. The breakpoint regions could be

defined as ranging from (i) position 10,313,660–10,316,595 and (ii)

position 25,417,450–25,433,122 [according to UCSC human ge-

nome browser assembly build 18 (hg18)].

A review of the literature revealed that cases of partial trisomy 18,

involving euchromatin of both arms are very rare. After exclusion of

patients with additional chromosomal abnormalities, combined

segmental rearrangements of chromosome 18, isochromosome 18p

or 18q, or small supernumerary marker chromosome in the mosaic

state [Marical et al., 2007; Rodr�ıguez et al., 2007], we were able to

identify only three other cases describing duplications of chromo-

some 18 with overlap to the region observed in our patient (Table II)

[Jaffray et al., 1980; Turleau et al., 1980; Wilson et al., 1990]. All

three patients had several phenotypic features of full trisomy 18.

Consistent with the fact that the duplication observed in our case is

the smallest compared to the three previous ones, our patient

showed a much milder phenotype than the three other cases

(Table II).

Mild facial dysmorphism and myopia, as observed in our patient,

are common features of trisomy 18p [Kline et al., 1993; Kosho et al.,

2006]. Given the fact that more than 50% of patients with partial

and nearly 85% of patients with complete trisomy 18 present with

congenital heart defects [Wilson et al., 1990], the occurrence of

PDA in our patient can be considered to be expected. Furthermore,

since more than 50% of patients with trisomy 18p show no

cognitive or behavioral impairment, the normal development in

our patient, without any signs of cognitive, language, or social

impairments seems not to be unusual. However, two of the three

previously described cases with comparable duplications showed

microcephaly and developmental delay. The case of Turleau et al.

[1980] died at 21 days of age because of his congenital diaphrag-

matic hernia; hence, this case could not be assessed for possible

developmental delay.

With regards to chromosome 18 aberrations, the occurrence of

ARM has thus far almost solely been described in association with

complete trisomy 18 (�15% of cases) [Kosho et al., 2006; Lin et al.,

2006]. In the only case with an alternate chromosome 18 aberration

and associated ARM, Chen et al., [1998] describe a patient with

isochromosome 18q; however, it remains unclear whether trisomy

18q or monosomy 18p underlied the formation of ARM in this case.

Thus, to the best of our knowledge, the present duplication of

FIG. 1. Results of genotyping: Illumina Human660Quad-v-1 BeadChip hybridization plots of the entire chromosome 18 showing the duplication

(highlighted) involving 2,892 markers. Log R ratio (top) represents a measure of the signal intensity for each marker and B allele frequency (bottom)

denotes the relative ratio of the fluorescent signals from one allelic probe compared to the other for each SNP. [Color figure can be viewed in the online

issue, which is available at wileyonlinelibrary.com]

SCHRAMM ET AL. 447

chromosomal region 18p11.21–q12.1 represents the first case of

a pure partial trisomy 18 associated with ARM. In our patient, the

duplicated region of chromosome 18 harbors 52 RefSeq genes

and three microRNAs (Fig. 2). Since the exact genomic boundaries

of the duplicated region have only been determined in our case, the

exact region of overlap remains unclear. Since there is no clear

evidence that this is an imprinted region, the significance of the

parental origin of the duplicated chromosomal region cannot be

further interpreted. The present case raises the question of whether

chromosomal region 18p11.21–18q12.1 could be implicated in the

embryonic development of the anorectum, an interesting issue for

further analyses.

ACKNOWLEDGMENTS

We thank all patients and their parents for their cooperation.

Likewise we thank the German self-help organization for people

with anorectal malformations (SoMA e.V.). We thank Pia Uerdin-

FIG. 2. Genomic overview of the maximum duplication as defined by flanking SNP markers identified in the patient and its gene content (hg18). [Color

figure can be viewed in the online issue, which is available at wileyonlinelibrary.com]

TABLE II. Summary of Phenotypic Features of Patients With Trisomy 18p! q12

Jaffray et al.[1980]

Turleau et al.[1980]

Wilson et al.[1990]

Presentcase

Gender F M M FBreakpoints 18pter! q12.2 18pter! q12.2 18pter! q12 18p11.21–18q12.1Maximum size

of duplication (Mb)35.5 �35.5 41.8 15.1

Head and neck(face, eyes,nose, mouth)

Dolichocephaly,malformed ears,

small mouth, high palate,mild retrognathia,

bilateral epicanthus,short neck

Dolichocephaly withprotuberant occiput,

distinctive facies,prominent convex

nasal bridge, short philtrum, severemicroretrognathia,

low set ears

Left-sidedpreauricular pit

Myopia

Cardiovascular — — — Patent ductusarteriosus

Chest — Right-sided diaphragmatic hernia — —Abdominal Umbilical hernia — — Anal atresia

with rectovesicalfistula

Skeletal(spine, pelvis,limbs, hands)

Bilateral talipes equinovarus,fist tightly clenched

over the thumb

Hip hypermobility, bilateraltalipes equinovarus,

dorsiflexion of the1st toe, fist tightly

clenched over the thumb

Bilateralsandal gap

—

Skin, nails, hair — Bilateral single transversepalmar crease

— —

Neurological Microcephaly,psychomotor delay

Death at 21 days of age,no psychomotor

assessment possible

Microcephaly,psychomotor delay

—

Genitourinary — — Glandular hypospadias,left-sided cryptorchidism

—

448 AMERICAN JOURNAL OF MEDICAL GENETICS PART A

gen for her excellent technical assistance and Dr. Christine Schmael

for her expert advice on the manuscript. C.S., M.D., E.B., T.M.B.,

E.S., S.G.-D., S.H., S.H.-C., F.B., M.M.N., M.L., and H.R. are

members of the ‘‘Network for the Systematic Investigation of

the Molecular Causes, Clinical Implications, and Psychosocial

Outcome of Congenital Uro-Rectal Malformations (CURE-Net)’’

which is supported by a research grant (01GM08107) from

the German Federal Ministry of Education and Research

(Bundesministerium f€ur Bildung und Forschung, BMBF). C.S. is

supported by the BONFOR program of the University of Bonn,

grant number O-149.0093.

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