Discussion
In the present study, the new missense A210G and L15Q and a previously
reported D111Y mutation were detected in the TBX5 gene of three separate
patients. These mutations were not observed in healthy subjects or the
rest of the patients, all of whom had non-syndromic cardiac septal
abnormalities. The prediction of pathogenic mutations with the
PolyPhen-2 software showed the probable pathogenicity of A210G and D111Y
while predicting the L15Q mutation as a benign mutation. In addition,
the prediction of structural stability for these mutations based on the
SVM method and a neural network approach online at the MUpro site
indicates the role of these mutations in protein structure instability.
Previous studies have shown that mutations in the TBX5 gene do not
follow a specific pattern, leading to various syndromic and
non-syndromic defects in patients. In addition, it shows the existence
of a phenomenon of variability (variable expressivity) in this gene
(Dreßen et al., 2016). Numerous mutations in this gene result in
Holt-Oram syndrome, which includes heart problems and skeletal
abnormalities in the hands and arms (Dreßen et al., 2016). While the
absence of both TBX5 allele (null alleles) usually results in Holt-Oram
syndrome, some patients with missense mutations such as G80R cause
non-syndromic anomalies and also minor limb malformations but severe
heart malformations(Cheng et al., 2005; Postma et al., 2008). Also,
previous studies have shown that mutations in this gene lead to
non-syndromic diseases such as atrial fibrillation (Boogerd et al.,
2010; McDermott et al., 2005; Wang et al., 2016; Yoshida et al., 2015)
or dilated cardiomyopathy(Zhou et al., 2015). Meanwhile, the rate of
mutation prevalence in this gene in patients with ASD non syndromes was
about 4 in 1000, in VSD about 1 in 1000, and in VASD about 3 in 1000
live births (Chung & Rajakumar, 2016).
As studies have shown, some mutations such as the Gly125Arg
gain-of-function mutation, affect the development of the heart and
o15ther organs and cause Holt_Oram syndrome(Basson et al., 1999).
Because this mutated protein interacts with other transcription factors
involved in the formation of the heart such as NKX2.5, TBX3, and GATA4,
they interfere with their ability to activate other genes responsible
for cardiac cell differentiation (Postma et al., 2008). The Pro85Thr
mutation, which causes Holt-Oram syndrome, leads to a significant
decline in the activation of other promoters of the genome (loss of
function) compared to the normal state of the protein, but this mutation
does not disturb the transmission of this protein to the nucleus (Dreßen
et al., 2016). Among 192 non-syndromic cardiac patients in China, only a
H170D mutation in TBX5 was found, leading to non-syndromic atrial
fibrillation as a result of reduced protein activity (Wang et al.,
2016). Also, a study of 111 patients with non-syndromic congenital heart
defects in Japan showed a mutation in the TBX5 gene in only three
patients while their mutations are different from the mutations in the
present study, two mutations were malignant and one benign (Yoshida et
al., 2015). A similar study conducted in heterogeneous populations of
different European countries has shown that among 331 congenital heart
disease (CHD) patients, only the D111Y mutation in the TBX5 gene results
in non-syndromic cardiac abnormalities (Granados‐Riveron et al., 2012).
Studies have shown that 70% of patients with HOS contain abnormalities
in the heart and upper limbs due to mutation in TBX5 and inadequate
haploid (Boogerd et al., 2010; Cross et al., 2000; McDermott et al.,
2005) while the gain of function mutations indicated that this gene
leads to abnormalities only affecting heart formation (Baban et al.,
2014). In addition, mutations in the T-box area and outside it in
patients with only non-syndromic coronary heart disease have been
reported in previous studies (Baban et al., 2014; Bonachea et al., 2014;
Brassington et al., 2003; Fan et al., 2003).
In the present study, mutations were uncovered in the TBX5 gene in
patients with non-syndromic VSAD and non-syndromic ASD without any
skeletal disorders in the Kurdish population of Iran. For the first time
the TBX5 gene in this population has been evaluated in relation to these
patients. The previous studies in these patients were performed with the
screening of NKX2.5 and GATA4 mutations and while no pathogenesis was
detected in NKX2.5 a new mutation was detected in GATA4 (Soheili et al.,
2015; Soheili et al., 2018). However, in this research, three mutations
in TBX5 gene have been identified that have been implicated in
congenital non-syndromic cardiac anomalies and have been discussed under
their phenotypic and genotypic relationships.
Our study here has shown that the D111Y mutation decreases structural
stability and leads to a possibly pathogenic phenotype. However,
Granados-Riveron predicted by structural model when the aspartate acid
residue (D) is replaced with an uncharged amino acid such as tyrosine
(D111Y), a salt bridge is broken down between K126 and D111. Also, this
bridge plays a very important role in the structural change of this
protein when attached to DNA. This mutation in this study caused a large
defect in the heart septal (large VASD) and patent ductus arteriosus
(PDA), while in the Granados-Riveron study, in addition to this clinical
symptoms, both main arteries were attached to the right ventricle (DORV)
(Granados‐Riveron et al., 2012). Therefore, this difference may indicate
the appearance of a variable of clinical symptoms in this mutation. The
new mutation Lue15Gln, which is reported for the first time in this
study, is located in the exon 2 of the TBX5 gene. A missense mutation
Lue15Gln replaced the nonpolar amino acid at the end of the N protein
TBX5 and out of the T-box motif, by glutamine amino acid. Ghosh et al
showed that Amino acids 1–237 of TBX5 are required for DNA binding and
removal of residues 1–54 from the full length TBX5 prevented its
binding to the DNA target (Ghosh et al., 2001). Therefore, although, the
Lue15Gln mutation occurred outside of the T-box and deleterious mutation
prediction by PolyPhen-2 showed that is a benign mutation, structure
stability prediction revealed that this mutation causes protein
instability as well as probably having a negative effect on DNA binding
interaction of TBX5, the function of transcription activation of TBX5,
or potentially causing the loss of synergistic transcription activity
between TBX5 and transcription factors such as NKX2.5. Therefore, this
mutation is associated with ASD and PS in this study; however, this
mutation requires in vitro or in vivo functional studies to clarify the
exact impact on TBX5 protein function. The Ala210Gly mutation was
associated with AVSD, TR, LVH, and RVH in this study. This mutation is
located in the T-box conserved TBX5 gene and in an alpha helix chain of
the DNA binding domain. Structure stability and deleterious mutation
prediction in this study revealed that this mutation leads to protein
instability and is possibly damaging. Since glycine occurs infrequently
in alpha helices, it has more conformational flexibility than the other
amino acid residues and results in alpha helix instability and it could
have an effect on the interaction between TBX5 with DNA. However, as was
the case with D111Y, the Ala210Gly mutation requires in vitro or in vivo
functional studies to clarify its effect.
Conclusion : Previous studies have shown that TBX5 gene
mutations are common in Holt-Oram syndrome or associated with
non-syndromic cardiac abnormalities such as fibroblasts. Based on the
results of this study, mutations in this gene can lead to non-syndrome
septal heart defects and mutations in TBX5 gene is associated with the
variable expressivity.