CLINICAL REPORT

Prenatal Diagnosis of Two Fetuses With Deletionsof 8p23.1, Critical Region for CongenitalDiaphragmatic Hernia and Heart DefectsElisabeth A. Keitges,1* Romela Pasion,2 Rachel D. Burnside,2 Carla Mason,3

Antonio Gonzalez-Ruiz,3 Teresa Dunn,4 Meredith Masiello,4 Joseph A. Gebbia,4

Carlos O. Fernandez,5 and Hiba Risheg11Department of Cytogenetics, Laboratory Corporation of America/Dynacare, Seattle, Washington2Department of Cytogenetics, Laboratory Corporation of America, Center for Molecular Biology and Pathology, Research Triangle Park,

North Carolina3Brookwood Maternal Fetal Medicine, Birmingham, Alabama4Medical Genetic Testing Laboratories, CytoGenX Corp., Stony Brook, New York5Premiere Perinatal, LLC, Toms River, New Jersey

Manuscript Received: 4 February 2013; Manuscript Accepted: 14 March 2013

Microdeletions of 8p23.1 are mediated by low copy repeats and

can cause congenital diaphragmatic hernia (CDH) and cardiac

defects. Within this region, point mutations of the GATA4 gene

have been shown to cause cardiac defects. However, the cause of

CDH in these deletions has been difficult to determine due to the

paucity of mutations that result in CDH, the lack of smaller

deletions to refine the region and the reducedpenetrance ofCDH

in these large deletions. Mice deficient for one copy of theGata4

gene have been describedwith CDHand heart defects suggesting

mutations in Gata4 can cause the phenotype in mice. We report

on the SNP microarray analysis on two fetuses with deletions of

8p23.1. The first had CDH and a ventricular septal defect (VSD)

on ultrasonography and a family history of a maternal VSD.

Microarray analysis detected a 127-kb deletion which included

the GATA4 and NEIL2 genes which was inherited from the

mother. The second fetus had an incomplete atrioventricular

canal defect on ultrasonography. Microarray analysis showed a

315-kb deletion that included seven genes, GATA4, NEIL2,

FDFT1, CTSB,DEFB136,DEFB135, andDEFB134. These results

suggest that haploinsufficiency of the two genes in common

within 8p23.1; GATA4 and NEIL2 can cause CDH and cardiac

defects in humans. � 2013 Wiley Periodicals, Inc.

Key words: SNP microarray; GATA4; NEIL2; diaphragmatic

hernia; congenital heart defects

INTRODUCTION

A recurrent class of chromosome rearrangements at 8p23.1 is

mediated by the low copy repeats (LCR’s) 8p-REPD and 8p-

REPP (olfactory receptor gene clusters). Non-allelic homologous

recombination between these repeats results in a large deletion of

approximately 3.8 Mb that contains numerous genes. These dele-

tions cause a constellation of features that include both cardiac

defects (94% of patients) and congenital diaphragmatic hernia

(CDH; 22% of patients) [Wat et al., 2009]. To date, smaller

deletions of this region have not been reported.

Within the 8p23.1 region, theGATA4 gene has been shown to be

important in normal human heart development. Mutations of

GATA4 appear to be sufficient to cause cardiac defects, as a number

of families have been reported with germ-line point mutations that

result in cardiac defects [Rajagopal et al., 2007; Wat et al., 2009;

Yang et al., 2012]. Studies of mice with Gata4 mutations support

this conclusion [Molkentin et al., 1997; Rajagopal et al., 2007; Jay

et al., 2007].

How to Cite this Article:Keitges EA, Pasion R, Burnside RD, Mason

C, Gonzalez-Ruiz A, Dunn T, Masiello M,

Gebbia JA, Fernandez CO, Risheg H. 2013.

Prenatal diagnosis of two fetuses with

deletions of 8p23.1, critical region for

congenital diaphragmatic hernia and heart

defects.

Am J Med Genet Part A 161A:1755–1758.

Conflict of interest: none.�Correspondence to:

Elisabeth A. Keitges, Labcorp, 550 17th Avenue, Suite 200, Seattle, WA

98122. E-mail: keitgee@labcorp.com

Article first published online in Wiley Online Library

(wileyonlinelibrary.com): 21 May 2013

DOI 10.1002/ajmg.a.35965

� 2013 Wiley Periodicals, Inc. 1755

In the mouse, heterozygous Gata4 mutations have also been

shown to result in diaphragmatic hernias [Jay et al., 2007]. How-

ever, in humans, only large microdeletions of 8p23.1 have been

described with CDH. Yu et al. [2013] recently described an inher-

ited missense mutation in GATA4 in a proband diagnosed with

CDHanda seconddenovomissensemutation in a single individual

with CDH and a heart defect. The mutations were predicted to be

pathogenic.

We present two cases with deletions of 8p23.1 includingGATA4,

referred for prenatal microarray analysis due to ultrasound abnor-

malities. Patient 1 presented with abnormal ultrasound findings

that included a CDH and a cardiac defect. The fetus inherited the

deletion from a parent who was born with cardiac defect. Patient 2

was found to have cardiac defects on level II ultrasonography. The

deletions in these cases overlap with only two genes in common,

GATA4 and NEIL2. These results support the recent reports that

GATA4 alone or in conjunction with NEIL2may result in CDH as

well as cardiac defects in humans.

PATIENTS AND METHODS

Clinical ReportsPatient 1. The patient was born at term, to a primigravid

woman. Ultrasonography of the fetus at 22 weeks gestation revealed

a singleton intrauterinepregnancywithadiaphragmatichernia anda

ventricular septal defect (VSD). Amniocentesis was performed and

results of chromosome analysis were normal, 46,XY. Significant

findings on fetal MRI include a large defect in the posterior hemi-

diaphragm. The left lobe of the liver including the left hepatic vein,

left portal vein, stomach, and loops of bowel were intrathoracic. The

heartwasdisplaced tothe rightand the left lungwashypoplastic.Fetal

echocardiogram showed a large conoventricular type VSD, mild

mitral and tricuspid valve regurgitation and a left superior vena cava

pulmonary sinus connection. The patient had repair of the CDH at

day 1 and repair of the VSD at fourmonths of age. Themother had a

VSDdiagnosed during childhood that did not require repair and has

not caused symptoms. The maternal uncle was born with malrota-

tion of the gut that required neonatal surgery.

Patient 2. The fetus was found to have a congenital heart defect

on a routine 19-week gestation level II ultrasonography. An am-

niocentesis was performed and chromosome analysis was normal,

46,XX. The pregnancy was conceived through in vitro fertilization

withdonor egg and sperm.Anechocardiogramshoweda large atrial

septal defect, an incomplete AV canal defect, abnormal tricuspid

valve, severe right atrioventricular valve regurgitation and hyper-

trophied right ventricle. The pregnancywas terminated at 24weeks;

no follow-up pathology exam was performed on the fetus.

Whole Genome SNP-Microarray AnalysisSNPmicroarray analysis was performed on both patients using the

Affymetrix Cytoscan HD platform. Two hundred fifty nanograms

of total genomic DNA extracted from lymphocytes was digested

with NspI and then ligated to NspI adaptors, respectively, and

amplified using a TitaniumTaqwith aGeneAmpPCR System 9700

(Applied Biosystems, Foster City, CA). PCR products were purified

using AMPure beads (Agencourt Biosciences, Beverly, MA) and

quantified using NanoDrop 8000 (Thermo Fisher, Wilmington,

DE). Purified DNAwas fragmented and biotin labeled and hybrid-

ized to the Affymetrix Cytoscan HD (Affymetrix, Santa Clara, CA).

Data were analyzed using Affymetrix Chromosome Analysis Suite

version CytoB-N1.2.0.225.

RESULTS

Patient 1SNPmicroarray analysis was performed on cultured amniotic fluid

cells and identified amale fetus with a 127-kb interstitial deletion of

8p23.1, arr[hg19] 8p23.1 (11,530,791–11,657,980) (Fig. 1). The

deleted region encompassed two genes, GATA4 and NEIL2. No

other significant copy number changes were noted in the array

analysis. Parental follow-up microarray analysis showed the same

8p23.1 deletion in the mother.

Patient 2Cultured amniotic fluid cells were processed and SNP microarray

performed. This identified a female fetus with a 315-kb deletion of

8p23.1, arr[hg19] 8p23.1 (11,583,841–11,898,980) (Fig. 1). The

deletion includes seven genes, GATA4, NEIL2, FDFT1, CTSB,

DEFB136, DEFB135, and DEFB134. The deletion of GATA4 was

only partial with loss of exons 3–7. The proximal end of the deletion

occurs at the proximal LCR (8p-REPP). No other significant copy

number changes were seen. Parental follow-up was not possible

since the pregnancy was the result of in vitro fertilization with both

donor egg and sperm.

DISCUSSION

We present two patients diagnosed prenatally with small deletions

of 8p23.1. These two patients are unique in that they have smaller

deletions than the recurrent 8p23.1 deletion that is mediated by

LCRs. Patient 1 was referred for SNPmicroarray analysis due to the

ultrasound findings of CDH and a VSD. A 127-kb deletion limited

to the GATA4 and NEIL2 genes was detected in the fetus and the

mother who also had a VSD at birth. Patient 2 was referred for SNP

analysis due to a cardiac defect seen on ultrasound. A deletion of

seven genes was detected which included GATA4 and NEIL2. A

search of the ISCA database did not find deletions of similar size or

gene content limited to GATA4 and NEIL2. Two patients in the

DECIPHER consortium had deletions that were similar in size to

Patient 2. One of these had a heart defect; the other had no

phenotypic information.

The large microdeletion of 8p23.1 mediated by LCRs surround-

ing the alpha and beta defensin clusters has been reported in

individuals with both CDH and cardiac defects. Three genes within

the region, GATA4, NEIL2, and SOX7, have been implicated in

CDHand/or cardiac defects [Lalani et al., 2013; Longoni et al., 2012;

Wat et al., 2012].Of the three genes, the SOX7 gene is notwithin the

deletion region of either of our patients suggesting that a deletion of

this genemaynot benecessary toproduceCDHinconjunctionwith

8p23.1 deletions.

The six-member family of GATA genes encodes zinc finger

transcription factors [Simon, 1995]. GATA4 is expressed through-

1756 AMERICAN JOURNAL OF MEDICAL GENETICS PART

out embryonic development and in the adult heart whileGATA4, 5,

and 6 are expressed in the visceral endoderm, developing heart, gut

and smooth muscle. Mutations of the GATA4 gene have been

shown to cause cardiac defects and CHD in humans and in mice.

In mice, a heterozygous deletion of exon 2 of the Gata4 gene,

Gata4þ/Dex2 in a C57Bl/6 background was found to have a variable

combination of abnormalities of the diaphragm, heart or lungs in

70% of embryos. The cardiac malformations seen in these mice

included; septal defects, right ventricular hypoplasia, endocardial

cushion defects and cardiomyopathy [Rajagopal et al., 2007; Jay et

al., 2007]. Whereas Gata4 null mutations were lethal with these

mice failing to develop a primitive heart tube and foregut.

Diaphragmatic defects were seen in 29%of theGata4þ/Dex2mice

and were absent in wild type litter mates [Jay et al., 2007]. Expres-

sion studies showed that Gata4 was expressed in the diaphragm

between embryonic day E11.5 andE15.5. Themice developmidline

diaphragmatic hernias which differ from the posterolateral hernia

typically seen in humans with 8p23.1 deletions. Previous studies of

Gata4 mutations in mice in a mixed genetic background failed to

detect CDH as compared to deletion carriers in a C57Bl/6 congenic

strain [Kuoet al., 1997;Molkentin et al., 1997].Therefore, it appears

that while mutations of Gata4 can result in CDH in mice reduced

penetrance and variable expressivity is present [Jay et al., 2007;

Rajagopal et al., 2007].

In humans, point mutations have been reported in all seven

exons of the GATA4 gene in patients with heart defects [Yu

et al., 2013]. There is a wide range of anomalies including septal

defects, endocardial cushion defects, ASD, and right ventricular

hypoplasia in both isolated and familial cases [Rajagopal

et al., 2007]. Patients with larger deletions of 8p23.1 that include

GATA4 tend to be associated with more severe cardiac defects than

heterozygous GATA4 point mutation carriers leading to specula-

tion that other genes may contribute to the phenotype [Wat

et al., 2009].

CDH is seen in approximately 22% of patients with large

deletions that include GATA4 and is often found concurrent

with heart defects. Screening of patients with CDH for GATA4

mutations have only detected two cases thus far. In the first case, Yu

et al. [2013] described a family with CDH and amissense mutation

of GATA4 in exon 3 (c.C754T). The mutation was predicted to be

pathogenic and was present in three generations, of which two

members were asymptomatic but identified to have a small CDH

diagnosedbyMRI. Screeningof an additional 96patientswithCDH

found a second missense mutation in exon 4 (c.848G>A). This

mutation had been reported previously in a patient with an isolated

heart defect without a CDH [Reamon-Buettner and Borlak, 2005].

TheNEIL2 gene encodes a DNA glycosylase that is ubiquitously

expressed and initiates the first step in base excision repair due to

FIG. 1. Schematic representation of 8p23.1 between 8p-REPD and 8p-REPP LCR’s. The region between the SOX7 gene and 8p-REPP is drawn to

scale. The deletions seen in Patients 1 and 2 are represented by the horizontal bars. Patient 1 has a 127-kb deletion arr[hg19] 8p23.1

(11,530,791–11,657,980) and Patient 2 has a 315-kb deletion arr[hg19] 8p23.1 (11,583,841–11,898,980). The dotted vertical lines

represent the common region of overlap.

KEITGES ET AL. 1757

damage by reactive oxygen species. The NEIL2 gene has recently

been suggested to form a network with other proteins known to be

associated with CDH and heart defects [Lalani et al., 2013; Longoni

et al., 2012]. A single heterozygous frameshift mutation of NEIL2

that was inherited from a normal mother was found in a patient

with CDH [Longoni et al., 2012]. Expression studies in the mouse

diaphragmatE11.5 andE12.5 foundGata4was expressed, butNeil2

was not [Longoni et al., 2012]. Additional patients with NEIL2

mutations or deletions limited to the NEIL2 gene are needed to

provide direct evidence that the gene contributes to CDH and/or a

cardiac defect.

The reduced penetrance seen with CDH as opposed to cardiac

defects in 8p23.1 is yet to be explained. In this report, Patient 1 had

both a VSD and CDH while his mother, who carried the same

deletion, had only a VSD that did not require surgery. Wat et al.

[2009] described a case of monozygotic twins with an 8p deletion

concordant for a cardiac defect but discordant for CDH which

would suggest that other non-genetic factors or variable expressiv-

ity is important. There is evidence that the phenotypic range of

CDH can vary. A few infants do not show symptoms of CDH at

birth but present after infancy and about 1% of patients are

asymptomatic [Pober et al., 2006]. The family reported by Yu

et al. [2013], had two familymemberswhowere asymptomaticwith

a cryptic CDH detected by MRI. However, numerous mutations

have been reported in all exons of GATA4 in patients with cardiac

defects none of which were reported to have CDH. Therefore,

variable expressivity may be only one of the factors that lead to

reduced penetrance of CDH in 8p23.1 deletions.

The SNP microarray findings in our two patients support the

findings in mice and humans that either mutations or haploinsuf-

ficiency of theGATA4 gene can be associated with heart defects and

CDH. The results provide additional evidence for variable expres-

sivity of CDH as the inherited deletion in case 1 was discordant

between the mother and child. These results do not rule out the

possible involvement of other genes contributing to a more com-

plex heart defect associated with the larger 8p23.1 deletions, nor do

they rule out the possible contribution of haploinsufficiency of

NEIL2 to the phenotype.

ACKNOWLEDGMENTS

This study makes use of data generated by the DECIPHER Con-

sortium. A full list of centers which contributed to the generation of

the data is available fromhttp://decipher.sanger.ac.uk and via email

from decipher@sanger.ac.uk. Funding for the project was provided

by the Wellcome Trust.

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