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).