Results
HRM and
Sequencing
Analysis of HRM curves showed that the curve for three patients varied
from the other curve of the sample and healthy subjects (Figure 1). The
RefSeq transcript used was NM_000192.3 and the mutation was named as
following the standard human sequence variant nomenclature using
Mutalyzer program (https://mutalyzer.nl/) and VariantValidator
(https://variantvalidator.org/) (Supplementary1A,1B,2A,2B,3Aand
3B).
The sequencing of these three samples (Figure 2) revealed three
mutations involving two novel mutations and a reported mutation. The
first Novel mutation NM_000192.3:c.44T>G
(NM_000192.3:g.711T>G) (Supplementary1A,1B) was in exon 2
which results in replacing Glutamine amino acid instead of Leucine at
position 15 (p.L15Q) in a 19-year-old girl with ASD with pulmonary
artery stenosis (PS). The second novel mutation was
NM_000192.3c.629C>G (NM_000192.3 g.1296C>G)
(Supplementary2A,2B) in exon 6 which (Figure 1) leads to replacement of
Alanine with Glycine in place 210 of (p.A210G) in a 11-year-old boy with
AVSD atrial and ventricular dysfunction and severe failure three-hinged
valve and right and left ventricular hypertrophy. Reported mutation was
NM_000192.3:c.331G>T (NM_000192.3:g.998G>T)(
(Supplementary3A,3B) in exon 4 causes the replacement of amino acid
Tyrosine amino acid Aspartate at position 111 of protein (p.D111Y) in a
32-year-old woman with a large hole in the atrium and ventricular septal
(large VASD) as well as patent ductus arteriosus (PDA). No mutations
were detected in the control group or other patients.
Structure stability prediction for
each mutation
The results of structure stability prediction for each mutation based on
both value and sign (direction) of energy change using SVM and sequence
information (delta delta G) showed that all three mutations in this
study cause protein instability (Table 3). In addition, prediction of
the sign (direction) of energy change using SVM and neural network with
a smaller sequence window show that these mutations decrease stability
(Table 3). Also, predicting the impact of mutations on protein
conformation, flexibility and stability by DynaMut showed that the D111Y
and A210G mutations cause protein destabilization and an increase in its
flexibility (Table 3).