Background: Several genes encoding transcription factors are known to be the primary cause of congenital heart disease. NKX2‐5 and GATA4 were the first congenital
heart disease–causing genes identified by linkage analysis. This study designed to
study the association of five single–nucleotide variants of NKX2‐5, GATA4, and
TBX5 genes with sporadic nonsyndromic cases of a congenital cardiac septal defect
in Egyptian children.
Methods: Venous blood samples from 150 congenital heart disease children (including a ventricular septal defect, atrial septal defect, tetralogy of Fallot, and patent
ductus arteriosus) and 90 apparently healthy of matched age and sex were studied by
polymerase chain reaction followed by direct sequencing in order to study two single–nucleotide variants of NKX2‐5 (rs2277923, rs28936670), two single–nucleotide
variants of GATA4 (rs368418329, rs56166237) and one single–nucleotide variant
TBX5 (rs6489957). The distribution of genotype and allele frequency in the congenital heart diseases (CHD) group and control group were analyzed.
Results: We found different genotype frequencies of the two variants of NKX2‐5, as
CT genotype of rs2277923 was present in 58% and 36% in cases and control respectively, and TT genotype present in 6% of the cases. Also regarding missense variant
rs28936670, heterozygous AG presented in 82% of the cases. Also, we observed a
five prime UTR variant rs368418329, GT (42% of the cases) and GG (46% of the
cases) genotypes showed the most frequent presentation in cases. While regarding a
synonymous variant rs56166237, GT and GG were the most presented in cases
(41.4%, 56% respectively) in contrast to control group (20%, 1.7% respectively).
Also, a synonymous variant in TBX5, the distribution of genotype frequency was
significantly different between the CHD group and control group. CT genotype of
TBX5 ‐rs6489957 was found in 12 ASD, 24 VSD, six PDA, three aortic coarctation
and nine fallot that represent 42% of the cases.
Conclusions: Significantly higher frequency of different allelle of five variants was
observed in cases when compared to the control group, with significant risky effect
for the development of septal defect. In addition to two polymorphisms of NKX2‐5
2 of 9 | BEHIRY et al.
1 | BACKGROUND
Congenital heart diseases (CHD) could be a major cause
of morbidity and mortality in children, a previous study revealed that CHD occurred in 80.8% as a solitary lesion, and
in 19.2% of the cases they were combined with other cardiac
lesions (Bassili et al., 2000). The prevalence increased over
time in total CHD birth, from 0.6 to 9.1 per 1,000 live births
from 1930 to 1934. Stabilization occurred all over the last
15 years, reaching 1.35 million newborns with CHD every
year (van der Linde et al., 2011).
Several studies have been made to identify genes that
could be responsible for syndromic and nonsyndromic forms
of CHD, by identifying the human gene mutations associated, it has been estimated that NKX2‐5 (OMIM: 600,584),
mutations account for at least 4% of fallot’s tetralogy cases.
Also authors found that nonsyndromic cardiac septal defects
have been linked to mutations in GATA4 (OMIM: 600,576)
(McDermott, Basson, & Hatcher, 2006). Nineteen mutations
in GATA4 have been studied in patients with atrial septal
defect (ASD), ventricular septal defect (VSD), and Fallot’s
tetralogy (Mattapally, Nizamuddin, Murthy, Thangaraj, and
Banerjee (2015)).
Mutations in genes known to be resposible for cardiac
development such as NKX2‐5, TBX20, GATA4, GATA6, and
MYH6 have been studied in families with isolated, nonsyndromic cardioavascular deformities, these genes are resposible
for cardiac development in animal models. GATA4, NKX2‐5,
and TBX5 (the gene causing Holt‐Oram syndrome) may function in a complex to regulate a group of genes required for
formation of cardiac septa (Ware & Jefferies, 2012).
TBX5 changes cause both skeletal and cardiovascular
phenotypes. It has been revealed that major cardiovascular
yet milder skeletal abnormalities are because of missense
alterations at the amino terminal of the DNA binding domain (Basson et al., 1999). Human NKX2‐5 is imparted in
the cardiovascular primordia from day 7 and is an early sign
of both embryonic heart fields development (Stanley et al.,
2004). Though alterations in NKX2‐5 are linked to an extensive variety of CHDs and thyroid dysgenesis, NKX2‐5 codes
for a homeodomain–containing transcription factor with an
important role in heart development, and mutations affecting
this gene in individuals with congenital heart disease were
reported (Dentice et al., 2006).
NKX2‐5 has a role in nearly almost all phases of heart
development, including the regulation of number of cardiac
progenitor cells, conduction system development, valve formation, and septation. Interestingly, NKX2‐5 acts in combination with other transcription factors that are highly‐conserved
to organize cardiogenesis. Genome–wide expression analysis
has begun to investigate NKX2‐5 dependent genes (Elliott,
Kirk, Schaft, & Harvey, 2010).
Previous authors have identified an unknown mutation in
the TBX5 (OMIM: 601,620) leading to an amino acid change
at position 85 from proline to threonine. The mutation had
dramatically reduced biological activity that lead to clinical
HOS phenotype (Dreßen et al., 2016).
This study planned to study two single–nucleotide variants of NKX2‐5 (rs2277923, rs28936670), two single–nucleotide variants of GATA4 (rs368418329, rs56166237) and one
single–nucleotide variant TBX5 (rs6489957) in sporadic nonsyndromic CHD cases in Egyptian children with congenital
cardiac septal deformity from Qalubeya.
2 | METHODS
This research was accepted by the Research Ethics Committee
of Benha University according to the “World Medical
Association Declaration of Helsinki 1964” (Idänpään‐
Heikkilä, 2001). Written informed consents were obtained
from the Guardians of all participants.
2.1 | Subjects
In this case‐controlled study, we recruited 150 unrelated affected children with non‐syndromic isolated and non‐isolated
cardiac septal defects from Benha University Hospital, they
were 84 females and 66 males with age ranged from days to
4 years with a mean age (7.8 ± 2.2) months during the period
from March 2017 to May 2018. Ninety healthy children with
matched age and sex and without a family history of cardiac
diseases served as a control group. Patients were diagnosed
according to ESC Guidelines for congenital heart disease
(Baumgartner et al., 2010). Syndromic CHD cases diagnosed
by clinical examination and karyotyping or cases with congenital heart malformations without septation defects were
excluded.
(rs2277923, rs28936670) variant in the cardiac septal defect, two variants in GATA4
(rs368418329, rs56166237) and one variant in TBX5 (rs6489957) seem to have a role
in the pathogenesis of congenital heart disease. |
