Ó 2013 Wiley Periodicals, Inc.
Birth Defects Research (Part A) 97:463 466 (2013)
CASE REPORT
Microdeletion 11q13.1.q13.2 in a Patient Presenting
with Developmental Delay, Facial Dysmorphism, and
Esophageal Atresia: Possible Role of the GSTP1 Gene
in Esophagus Malformation
Tatiana Ferreira de Almeida1* and Débora Romeo Bertola1,2
1
Hospital de Clı́nicas, Faculdade de Medicina da Universidade de São Paulo, Instituto da Criança,
Unidade de Genética, São Paulo, Brazil
2
Instituto de Biociências, Universidade de São Paulo, Departamento de Genética e Biologia Evolutiva,
São Paulo, Brazil
Received 2 November 2012; Accepted 11 January 2013
BACKGROUND: Esophageal atresia is a major congenital malformation characterized by a complete interruption of the esophageal continuity. It is frequently observed in associations and syndromes. As an isolated
finding, it has a multifactorial etiology whose genetic factors are poorly known. Recently, the GST family,
especially the GSTM1 null genotype (but not the GSTP1 polymorphism I105V), has been associated with
esophageal atresia. These enzymes play a role in phase II detoxification of xenobiotics. Here we present the
clinical and molecular findings observed in a patient suggesting that the loss of the GSTP1 allele might predispose to this malformation. CASE: We describe a patient presenting with esophageal atresia associated
with developmental delay and facial dysmorphism, whose mother used tobacco and alcohol during the first
2 months of her pregnancy. Microdeletion/microduplication analysis was performed using comparative
genomic hybridization and a 180K Agilent array. It detected a de novo 2 Mb chromosome 11q13.1.q13.2 deletion. CONCLUSION: The deleted chromosomal segment includes the GSTP1 gene. We hypothesize that
the deletion of one GSTP1 allele (an isoform highly expressed in embryonic tissues), associated with specific
environmental factors, such as tobacco and alcohol, could cause the esophageal atresia observed in our
patient. Birth Defects Research (Part A) 97:463–466, 2013. Ó 2013 Wiley Periodicals, Inc.
INTRODUCTION
Esophageal atresia (EA) is a major congenital malformation characterized by a complete interruption of the
esophageal continuity with or without a communication
between the esophagus and the trachea. It affects 1:4,220
live births (Nassar et al., 2012). Its mode of inheritance
and pathophysiology are complex (Jacobs et al., 2012).
In approximately 50% of the cases, this anomaly is
associated with other malformations, mainly gastrointestinal atresia or stenosis, anomalies of the urinary tract,
and heart defects. EA could be part of different conditions, such as the VACTERL (vertebral, anal, cardiac, tracheo-esophageal, renal, and limb) association and chromosomal abnormalities (trisomies 21,18, and 13; deletions
22q11.2, 13q13., 16q24.1, 17q21.3.q23). In approximately
23% of the cases (Pedersen et al., 2012) it is observed in
monogenic disorders such as the CHARGE, Feingold,
Rogers, and Opitz syndromes or Fanconi anemia
(Brunner and van Bokhoven, 2005; Felix et al., 2009, Genevieve et al., 2011).
As an isolated malformation, EA is considered to have
a multifactorial etiology, although both the environmental and the genetic factors involved are poorly known (de
*Correspondence to: Tatiana Ferreira de Almeida, Rua Ibiraçu, 76 ap 73, Vila
Madalena, São Paulo-SP, CEP: 05451-040. E-mail: [email protected]
Published online 4 July 2013 in Wiley Online Library (wileyonlinelibrary.
com).
DOI: 10.1002/bdra.23115
Birth Defects Research (Part A): Clinical and Molecular Teratology 97:463 466 (2013)
464
FERREIRA DE ALMEIDA AND BERTOLA
Jong et al., 2010). The environmental factors include
maternal diabetes and phenylketonuria, exposure to
methimazole and diethylstilbestrol, exogenous sex hormones, alcohol, smoking, infectious diseases, and occupational exposure to pesticides (de Jong et al., 2010; Genevieve et al., 2011). The higher concordance rate observed in
monozygotic when compared to dizygotic twins highlights
the role of genetic factors in the development of isolated
EA (Schulz et al., 2012). Studies of foregut development in
animal models suggest that candidate genes, such as the
Wnt2, Wnt2b, and Barx1 genes, might be involved in isolated EA observed in humans (Jacobs et al., 2012).
The glutathione S-transferases (GST) are a family of
enzymes that play an important role in phase II detoxification of xenobiotics, including alcohol and tobacco
(Raijmakers et al., 2001). They catalyze the conjugation of
reduced glutathione on a wide variety of toxic compounds, converting them in less biologically active and
more easily excreted molecules (Ali-Osman et al., 1997).
In adults, these enzymes play an important role against
oxidative stress and DNA damage (Chen et al., 2012; Liu
et al., 2006). Several studies underline the important role of
the GSTM1, GSTT1, and GSTP1 isoforms in the pathogenesis of some environmentally related diseases such as cancer, respiratory disease, and lung function deficits in children (Filonzi et al., 2010). Nevertheless, the role of GST as
a source of DNA protection in the embryonic tissues is still
unclear (Raijmakers et al., 2001). Some authors suggest
that there is evidence that the disruption of these genes
could play a role in birth defects, such as oral clefts (Shi
et al., 2008), heart defects (Cresci et al., 2011), and hypospadias (van der Zanden et al., 2012), by increasing the susceptibility of the embryonic tissues to environmental factors, especially tobacco (Filonzi et al., 2010; Shi et al., 2007).
Recently, a study of the GST family activity and its
relation with EA showed an association of this disorder
with the GSTM1 genotype, but surprisingly failed to show
a significant association with the GSTP1 polymorphism
(I105V), known to be the most important GST enzyme during fetal development (Raijmakers et al., 2001).
Here we report on a patient with a microdeletion
involving the GSTP1 gene, suggesting that a relationship
might exist between a low activity of this enzyme and
his EA phenotype.
CASE REPORT
The subject is a 1-year-old boy, the first child of nonconsanguineous parents. During the first 2 months of her
pregnancy, the mother used tobacco and alcohol. Fetal
ultrasound showed polyhydramnios and a possible EA.
He was born preterm (34 4/7 weeks), by Cesarean section, with a birth weight of 1345 g, length of 40.5 cm,
and Apgar scores 6/8. Soon after birth, EA without a tracheo-esophageal fistula was confirmed and a gastrostomy/esophagostomy was performed the first day of life.
He was discharged from the hospital after 31 days. The
patient had developmental delay; at 7 months of age he
did not have complete control of his head.
Complementary studies, including abdominal ultrasound, brain magnetic resonance imaging, ophthalmologic evaluation, and a G-banded karyotype, were normal. Echocardiogram disclosed a patent foramen ovale.
The physical examination at 7 months of age showed:
weight 7 kg (5th percentile), length 64 cm (<5th percenBirth Defects Research (Part A) 97:463 466 (2013)
tile), occipital frontal circumference 44.5 cm (50th percentile), a large open anterior fontanelle, frontal bossing, ocular
hypertelorism (inner canthal distance 2.7 cm and outer canthal distance 7.5 cm), downslanting palpebral fissures, horizontal nystagmus, and normal male genitalia and limbs.
As the patient presented with developmental delay, a
major malformation, and some dysmorphic facial features, an array comparative genomic hybridization
(array-CGH) was requested.
METHODS
Array-CGH
Array-CGH was performed using the Agilent Human
Genome CGH Microarray kit 180K (Agilent Technologies,
Santa Clara, CA), hybridized according to the manufacturer’s protocols.
For the location of genes in the deleted genomic segment, the University of California Santa Cruz (UCSC;
http://genome.ucsc.edu/) database and the Database of
Genomic Variants (http://projects.tcag.ca/variation/;
NCBI 36/hg18) were used.
Search for Candidate Genes
Using the UCSC site, a list of genes included in the
deleted region was established. This list originally contained 218 entries for different hg18 gene names. After
filtering, 95 gene entries were kept. Only 11 of these 95
genes were related to a known disease (OMIM). The
search of candidate genes was set up in four steps. First,
we looked for similar deletions in the DECIPHER
(http://decipher.sanger.ac.uk) database; second, a search
in the PubMed database for an association between EA
and chromosome 11 was performed; third, a search in
the PubMed database for an association between each of
the 95 gene symbols and the following entries (EA or
esophagus) was done; and, finally, using the Mouse Genome Informatics (MGI) (http://www.informatics.jax.org) database a search for an association between each of
the 95 gene entries and an abnormal digestive tract phenotype was performed.
RESULTS
The array-CGH showed a de novo 2 Mb chromosome
11
deletion
(array
11q13.1.q13.2
(65,265,47867,229,716)x1dn [hg18]). It was confirmed using fluorescence in situ hybridization (FISH) analysis.
Based on the fact that it was a de novo event, and two
patients with similar deletions were described previously in
the DECIPHER database, the deletion was considered pathogenic. Recently, a third patient showing a microdeletion in
the same chromosome band was described (Floor et al., 2012).
The deletion found in our patient could be larger if one
considers the segment between the first and last probes on
the array (chr11:65,265,469 and chr11:67,330,919). However, as this extended region could not be assessed precisely, we decided to consider only the genes included in
the region where only one signal was observed.
Databases were searched in an effort to find a candidate gene that might be related to the malformation
observed in our patient. The following results were
obtained: no match for EA and chromosome 11; in the
PubMed database only one match for EA and one gene,
HAPLOINSUFFICIENCY OF GSTP1 AND ESOPHAGEAL ATRESIA
namely the GSTP1 gene, out of the 95 deleted genes; and
no match for a specific gene and EA in the MGI database. But with a more extensive search for an abnormal
digestive tract phenotype, an association between the
TBX10 gene and oral cleft came out.
Despite the fact that mutations in TBX1, TBX4, and
TBX5 have been associated with EA, no association
between TBX10 and esophagus or foregut was observed.
In mouse models, gain of function mutations of the
TBX10 gene have been associated only with oral cleft
(Bush et al., 2004).
In the three patients described with an overlapped deletion, none showed EA. Conversely, all of them showed
developmental delay, hypertelorism, and frontal bossing,
as observed in our proband. One of them presented with
an oral cleft. Interestingly, only in this last patient did
the deletion encompass the GSTP1 gene.
Thus, the GSTP1 gene was the only gene deleted in the
region that showed some connection with the malformation observed in our patient.
DISCUSSION
Microarrays are important molecular tools to uncover
copy number variations associated with multiple congenital anomaly syndromes. A microdeletion/microduplication may contain a gene that could be directly related to
a specific malformation, revealing important information
in order to elucidate the genetic factors of complex disorders, such as isolated congenital anomalies.
The patient described here presented with a major gastrointestinal malformation, developmental delay, and facial
dysmorphism, associated with a 11q13.1q13.2 microdeletion.
In this chromosomal region, the GSTP1 gene is a
potential candidate gene for EA. The esophagus is a foregut-derived structure. The separation into the ventral respiratory and the dorsal esophageal parts occurs at fourweek gestation. The mechanism responsible for EA is not
well understood (Jacobs et al., 2012).
Toxic products can cause DNA damage and consequently cause malformations during the embryonic period. Numerous substances taken by a pregnant woman
are known to cause malformations in the fetus, such as
alcohol, smoking, and drugs (Wlodarczyk et al., 2011).
The toxic substances consumed by the mother may affect
organs exposed to the amniotic fluid. During the first
weeks of gestation, the detoxification role of the placenta
is not fully developed and, in order to avoid the entrance
of toxic products in fetal circulation, these substances are
excreted in the amniotic fluid (Raijmakers et al., 2001).
Therefore, the GST family could exert a scavenging effect
on the toxic substances that escape the placental barrier. Raijmakers et al. (2001) showed that, among the different GST
isoforms, the GSTP1 one shows the higher expression in the
embryonic and fetal tissues, especially the tissues exposed
directly to the amniotic fluid such as the esophagus.
Animal models of Gstp1/2 knockout mice fail to show a
higher prevalence of birth defects when compared to
wild type (Henderson et al., 1998). However, the Gstp1/2
knockout mice are generally used as a model of adult
response to xenobiotics in studies of tumorigenesis or
drug metabolism (Henderson and Wolf, 2011). In these
studies, the function of the mouse gene is very similar to
the human ortholog GSTP1. The loss-of-function of
Gstp1/2 enhances the DNA damage leading to a higher
465
risk of developing skin and lung cancer. It is also
involved in the inflammatory response and damage
caused by tobacco smoke (Henderson and Wolf, 2011). A
brief review of the literature did not show any study of
these knockout mice exposed to xenobiotics during the
embryonic life. Therefore, no inference can be made
about the possible birth defects related to a low activity
of Gstp1/2.
Polymorphisms of the GST genes, causing a lower
enzymatic activity, have been implicated as one of the
genetic factors predisposing to the development of
complex disorders. Specifically, the GSTM1 null alleles
leading to reduced catalytic activity and inefficient
detoxification in tissues exposed to the amniotic fluid
could be a factor altering proliferation/apoptotic pattern taking place during the gut–trachea separation
(Filonzi et al., 2010). Interestingly, in the same study,
the GSTP1 polymorphism I105V, known to have a
lower enzymatic activity, did not show a positive association with EA.
One may hypothesize that in the study of Filonzi et al.
(2010) the enzymatic level of GSTP1 did not reach the
threshold required to cause the malformation. In our
patient, the mother used alcohol and tobacco during the
first 2 months of her pregnancy. The presence of only one
copy of the GSTP1 gene could reduce the activity to such a
level that the detoxification of these products in contact
with the esophagus is impaired, causing the atresia. Therefore, we favor the hypothesis that the deletion of one GSTP1
allele, namely the isoform that has a higher expression in
embryonic tissues, associated with specific environmental
factors, such as tobacco and alcohol, could cause EA.
Of the three patients presenting with overlapping deletions, only the patient with a cleft palate had a deletion of
the GSTP1 gene. This could be considered as further evidence for the disruptive role of the GSTP1 gene in organs
exposed to the amniotic fluid. We could speculate that the
exposure to xenobiotics during the embryonic period could
lead to oral cleft or EA.
A weakness of the present study is that we did not
have the opportunity to genotype the isoforms, both in
the patient and his mother. The GSTP1 I105V polymorphism occurs in 38% of individuals in specific populations. If present in our proband or his mother, it would
result in a much lower activity of GSTP1, enhancing the
possibility of DNA damage and foregut malformation.
Moreover, the fact that the null alleles of the GSTM1 and
GSTT1 genes are frequent in different populations (56%
and 18%, respectively), including the Caucasians as our
proband, we cannot rule out that our patient and/or his
mother also might carry one or both of these genotypes.
This in turn would amplify the toxic effects of the xenobiotics (Shi et al., 2007).
This is the first clinical report associating the deletion of
one allele of the GSTP1 gene with EA. Further studies of
microdeletions involving this specific gene will be important to confirm this association.
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