Our study found several NKX2-5 variants in ASD patients in Indonesia: NM_004387.4:c.63A>G at exon 1, NM_004387.4:c.413G>A, and NM_004387.4:c.561G>C at exon 2. The variant of c.63A>G is considered benign according to the ClinVar database. However, it is associated with a 20% decrease in transactivation activity.10 In this study, it was frequently found that as much as 85.6% of research subjects. This result is the most significant number after research in the population in China, 80.18%, but it was found lower in Germany (59.4%) and Morocco (56.2%).7,8,11,12
For c.413G>A variant, replacement of guanine to adenine is a non-synonym in the form of missense which changes the arginine (Arg) to glutamine (Gln). Whereas, for c.561G>C variant, guanine substitution to cytosine changes the amino acid glutamine (Gln) to histidine (His). These variants change the amino acid sequence and consequently modify the protein structure, which can interfere with the function of the NKX2-5 protein as a transcription factor.
The variants of c.413G>A and c.561G>C lie in amino acids at 138 and 187, respectively, part of the protein homeodomain. Variants change the amino acid arrangement in the homeodomain, which is a critical domain because it binds directly to specific DNA. Several previous studies reported that missense mutations in the homeodomain could cause secundum type ASD and familial conduction disorders.13–17
In this study, we found these last two variants to occur together. It was found in 3 subjects with a family relationship (Figure 2 and supplementary data). Subjects 48 and 72 have sibling relationships, while subjects 72 and 97 are father and daughter. In subjects 48 and 72, it turned out that they had not only double heterozygote variants of c.413G>A and c.561G>C but also had c.63A>G heterozygous variant. It may be related to the more severe phenotype in subject 48, which developed rapid pulmonary hypertension aggravation and led to her death. In addition, these double variants cause a phenotype in the form of ASD accompanied by arrhythmia disorders. Subject 72 had sinus node dysfunction at a young age, while subject 97 had atrial tachycardia during childhood and ablated. The previous study support that mutations in NKX2-5 cause atrial septal formation disorders and are also associated with arrhythmia disorders.18 Therefore, it is necessary to do early genetic screening in the ASD patient family to see the role of the genetic variant of NKX2-5 on the familial ASD phenotype, especially in Indonesia.
Our study did not perform a functional study to determine the pathogenicity of the novel variant on ASD development. Therefore, further functional studies are necessary.