Materials and
Methods
Patients
In this descriptive study, TBX5 gene mutations in patients with ASD and
VSD were investigated in 100 cases of congenital heart disease in two
hospitals, the Sanandaj Tohid Hospital and Kermanshah Cardiovascular
Center. The target Kurdish population consisted of 50 healthy controls
who had been admitted to hospital (with no history of familial disease
and who showed no cardiac abnormality by echocardiogram ) and 100 cases
(62 females and 38 male) of non-syndromic patients including 57 patients
with VSD (42 membranous, 14 muscular, 1 conal septal), 39 patients with
ASD (36 ostium secundum, 2 sinus venosus, 1 without information) and
four patients possessing AVSD (Atrioventricular) (Table 1).
Extracting Genomic
DNA
Genomic DNA was extracted from whole blood by AccuPrep® Genomic
DNA Extraction Kit from Bioneer Corporation, South Korea.
Real Time PCR and HRM
Analysis
In order to replicate the coding regions of the TBX5 gene (exons 2-9)
specific primers for the reference sequence NM_000192.3 and
NG_007373.1 were designed in NCBI using CLC Workbench 5.5 software.
Gene Reference Sequence ID is LRG_670 (Table 2). The intron/exon
boundary (exons 2 to 8) and exon 9 (containing 2177bp), its 575bp
component, which is part of the coding region, was considered for the
design of the primer. In this study, the Rotor-Gene ™ 6000 (Corbett
Research) and 2x QuantiFast SYBR® Green kits were used. In order to
optimize the PCR reaction as well as HRM, the PCR reaction was performed
for all primers under the same conditions as follows: 95 °C for 5
minutes and initial denaturation and 40 cycles of 95 °C for 10 seconds
and 60 °C for 30 seconds and the HRM step from 72 °C to 95 °C, including
an increase in temperature of 0.1 °C per second. At first, 10 healthy
individuals were identified as sequencing controls and were used as
control (wild type) for HRM.
Sequencing
Patient samples were grouped according to the HRM curve and samples
whose melting patterns differed from healthy subjects or controls
(Figure 1) were sequenced. Also, 25 samples of patients and 25 controls
with normal curves were randomly selected for sequencing. The sequencing
of PCR products by ABI 3730XL DNA Analyzers was performed by Bioneer
(South Korea) and the results were analyzed using the CLC main
workbench.
Predict structural stability of
mutation
The prediction of the structural stability of a protein derived from
amino acid mutation was performed based on energy changes by using the
sequence information online at the MUpro site
(http://www.ics.uci.edu/~baldig/mutation.html) using the
SVM method and the support vector machine and neural network (Cheng,
Randall, & Baldi, 2006; Cheng, Randall, Sweredoski, & Baldi, 2005).
Potential pathogenicity prediction
of TBX5 gene sequence variants using
PolyPhen-2
Potential Pathogenicity of TBX5 sequence variants was tested based on
two standard human datasets, HumDiv and HumVar, which are applied by
PolyPhen-2 Online (http://genetics.bwh.harvard.edu/pph2) (Figure 3). The
HumDiv dataset (Adzhubei et al., 2010) contains 5564 mutations in the
UniProtKB database known to cause Mendelian disease and 7,539 DNA
variants between human proteins and their closely related mammalian
equivalents assumed to be non-damaging. The HumVar dataset (Capriotti,
Calabrese, & Casadio, 2006) consists of 22,196 human disease-causing
mutations from UniProtKB and 21,151 neutral mutations that are common
human nsSNPs (Non-synonymous single nucleotide polymorphisms) (MAF
> 1 %) with no annotated disease involvement. Note that
the HumDiv dataset contains annotated mutations directly associated with
human diseases, while the HumVar dataset is more noisy as its neutral
mutation subset includes many mildly deleterious alleles (Adzhubei et
al., 2010; Plekhanova, Nuzhdin, Utkin, & Samsonova, 2019).