Skip to main content

NKX2-5 variants screening in patients with atrial septal defect in Indonesia



NKX2-5 variant in atrial septal defect patients has been reported. However, it is not yet been described in the Southeast Asian population. Here, we screened the NKX2-5 variants in patients with atrial septal defect (ASD) in the Indonesian population.


We recruited 97 patients with ASD for genetic screening of the NKX2-5 variant using Sanger sequencing.


We identified three variants of NKX2-5: 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 first variant is commonly found (85.6%) and benign. The last two variants are heterozygous at the same locus. These variants are rare (3.1%) and novel. Interestingly, these variants were discovered in familial atrial septal defects with a spectrum of arrhythmia and severe pulmonary hypertension.


Our study is the first report of the NKX2-5 variant in ASD patients in the Southeast Asian population, including a novel heterozygous variant: NM_004387.4:c.413G>A and NM_004387.4:c.561G>C. These variants might contribute to familial ASD risk with arrhythmia and severe pulmonary hypertension. Functional studies are necessary to prove our findings.

Peer Review reports


Congenital heart disease diagnosis and treatment capabilities have dramatically increased over the previous years, but equivalent success rates have yet to be established in developing countries like Indonesia. Since congenital heart disease accounts for almost a third of all congenital birth defects [1], focusing on it is critical to preventing unnecessary causes of death, such as pulmonary hypertension. However, most patients with congenital heart disease in developing countries lately seek medical attention as symptoms and signs of complications begin to manifest.

According to Indonesia’s first hospital-based registry of individuals with congenital heart disorders (CHD) and CHD-related pulmonary hypertension, atrial septal defects (ASD) account for most congenital heart diseases [2]. For decades, these patients may be asymptomatic. Although these patients may be identified coincidentally during routine checkups, there is no specific screening of asymptomatic CHD in Indonesia. Later symptoms will prompt individuals to contact a doctor and seek treatment at a hospital. Unfortunately, the patients’ primary complaints were associated with the development of pulmonary artery hypertension and resulted in a poor clinical outcome.

Genetic testing plays a significant role in medical evaluation. It detects possible genetic abnormalities in CHD and can enhance prognosis by providing vital information on individualized medical treatment and clinical diagnostic assurance [3]. Since 1950, genetic investigations on family congenital heart disease have been studied. Various studies since then have shown that genetic information has a role in structural heart abnormalities. Numerous genes, including NKX2-5, GATA4, TBX5, NOTCH1, and TBX20, have been related to congenital heart disease [4].

The transcription factor NKX2-5 plays an essential role in embryonic cardiogenesis and postnatal cardiac adaptation [5]. Previous studies have reported a genetic variant of NKX2-5 linked to the development of atrial septal defect [6, 7]. The frequency of gene variant of NKX2-5 in ASD patients with familial history could reach up to 8% and only 1–4% in sporadic cases [8]. Familial genetic variants can be detected earlier, so the management is possible to be carried out before complications appear. In addition, gene variants are different among races. Therefore, more study is needed to identify the gene variant of NKX2-5 in several different ethnicities. Here, we screened the NKX2-5 variants in patients with atrial septal defect in Indonesia.

Material and methods

Patient samples

This study was an analytic observational study involving ASD patients examined at RSUP Dr. Sardjito Yogyakarta and included in the COHARD-PH Registry in Yogyakarta [2]. It was sampled using the convenience sampling method. Informed consent was obtained from patients for diagnostic tests and research studies. The ethics committee approved the study of Universitas Gadjah Mada’s Faculty of Medicine, Public Health, and Nursing under the reference number KE/1116/11/2020. Storage and processing of DNA extraction from blood samples and the polymerase chain reaction were carried out at the Integrated Research Laboratory of the Faculty of Medicine, Public Health and Nursing Universitas Gadjah Mada.

DNA extraction and Sanger sequencing

Genomic DNA was isolated from whole blood using a DNA extraction kit (Geneaid). DNA was extracted and kept at − 20 °C until it was analyzed. Polymerase chain reaction (PCR) was performed using a Promega PCR kit. The primers used in this study are described in Additional file 1.

Subsequently, the Sanger sequencing was performed for the screening of NKX2-5 variants. The variants of NKX2-5 were obtained after being compared with the sequences in the NCBI database using SerialCloner and Sequence Scanner software.


Baseline characteristics

We included 97 ASD patients in this study. Subjects consisted of 25 familial ASD patients and 72 sporadic ASD patients. The participants in this study ranged from 13 to 79 years old, with an average of 36.04 years. The mean age in the familial and sporadic groups was 37.64 (± 13.06) years and 35.49 (± 13.88) years, respectively. There were more female patients than males in both groups, 21 patients (84.0%) in familial and 62 patients (86.1%) sporadic (Table 1).

Table 1 Sinus venosus is part of ASD type as secundum and primum

This study includes three types of defects: secundum defects, primum defects, and sinus venosus defects. The type of secundum defect was the most common in each group, 24 patients (96%) in the familial group and 67 patients (93.1%) in the sporadic group. Primum defect type was only found in the sporadic group, three patients (4.2%). Patients with sinus venosus defect type found one patient (4%) in the familial group and two (2.8%) in the non-familial group. The diameter of the ASD defect in the familial group was 26.3 (± 9.40) mm, while the diameter in the sporadic group was 23.2 (± 7.12) mm. Right atrial dilatation was 46.20 (± 5.72) mm in the familial group and 46.00 (± 8.05) mm in the sporadic group. Patients with ASD with severe pulmonary hypertension may develop Eisenmenger syndrome. Eisenmenger syndrome was diagnosed in one patient (4%) in the familial group and six in the non-familial group (8.3%) (Table 1).

NKX2-5 variants in ASD patients

During this study, 97 patients were subjected to NKX2-5 screening using Sanger sequencing. From this study, three different variants were identified, which are 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 (Fig. 1).

Fig. 1
figure 1

The results of NKX2-5 sequencing show three variants at the codons 63, 413, and 561. The first variant is NM_004387.4:c.63A>G at exon 1, consisting of heterozygous AG and homozygous GG. The second variant is a heterozygous GA (NM_004387.4:c.413G>A), and the last variant is a heterozygous GC (NM_004387.4:c.561G>C). The first variant is a synonymous variant. However, variants at codons 413 and 561 are non-synonymous (arginine replaced with glutamine at 138 and glutamine substituted with histidine at 187). Glu (glutamic acid), Arg (arginine), Gln (glutamine), His (histidine), NH2 (amino-end), TN (transcriptional activation domain), HD (homeodomain), NK (NK2 specific domain), COOH (carboxyl end)

In our population study, the c.63A>G variant consists of 3 kinds of genotypes: homozygous AA (14, 14.4%), heterozygous AG (41, 42.3%), and homozygous GG (42, 43.3%) (Table 2). This variant was frequently observed in patients with ASD. According to ExAC, variant frequency is 40.52% in the control population [9].

Table 2 NKX2-5 variants in our ASD patients

The c.413G>A variant has two types of genotypes, homozygous GG and heterozygous GA. The c.561G>C variant also has two genotypes: homozygous GG and heterozygous GC. Notably, both heterozygous variants have been identified at the same locus. These double heterozygous variants may significantly impact the amino acid missense arrangement, which is very likely to change the structure and function of the NKX2-5 protein. These variants are remarkable because they occurred in three subjects (3.1%) of ASD patients from 1 family (Fig. 2). In the control population, the frequency of c.413G>A is 0.0009% in the GnomAD_exome database, and there is no data for c.561G>C [9].

Fig. 2
figure 2

Patient’s family tree no. 48, 72, and 97. A describes the findings of the NM_004387.4:c.63A>G variant, while B depicts the dual variants of NM_004387.4:c.413G>A and NM_004387.4:c.561G>C. The square symbol denotes male, and the circle denotes female. Patient no. 72, in the index, is marked with an arrow. Black shading describes ASD patients. The + (positive) sign indicates a mutation, the − (negative) sign is no mutation. Subjects without a + or − sign indicate that genetics was not tested. The / (slash) sign indicates that the patient has died


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 lay 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 [13,14,15,16,17].

In this study, we found these last two variants to occur together. It was found in 3 subjects with a family relationship (Fig. 2 and Additional file 1). 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 unfortunately 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 and 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 analysis to determine the pathogenicity of the novel variant on ASD development. Therefore, further functional studies are necessary.


Our study is the first report of the NKX2-5 variants in ASD patients in the Southeast Asian population, including novel heterozygous variants: NM_004387.4:c.413G>A and NM_004387.4:c.561G>C. These variants might contribute to familial ASD risk with arrhythmia and severe pulmonary hypertension. Functional studies are necessary to prove our findings.

Availability of data and material

The genomic DNA sequences generated and/or analyzed during the current study are available in the GenBank repository, under the accession number OM066666-OM066670 and GitHub page (



Atrial septal defect


  1. Zimmerman MS, Smith AGC, Sable CA, et al. Global, regional, and national burden of congenital heart disease, 1990–2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet Child Adolesc Health. 2020;4(3):185–200.

    Article  Google Scholar 

  2. Dinarti LK, Hartopo AB, Kusuma AD, et al. The COngenital HeARt Disease in adult and Pulmonary Hypertension (COHARD-PH) registry: a descriptive study from single-center hospital registry of adult congenital heart disease and pulmonary hypertension in Indonesia. BMC Cardiovasc Disord. 2020;20(1):163.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Chaix MA, Andelfinger G, Khairy P. Genetic testing in congenital heart disease: a clinical approach. World J Cardiol. 2016;8(2):180–91.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Blue GM, Kirk EP, Giannoulatou E, et al. Advances in the Genetics of Congenital Heart Disease: a clinician’s guide. J Am Coll Cardiol. 2017;69(7):859–70.

    Article  CAS  PubMed  Google Scholar 

  5. Akazawa H, Komuro I. Cardiac transcription factor Csx/Nkx2-5: its role in cardiac development and diseases. Pharmacol Ther. 2005;107(2):252–68.

    Article  CAS  PubMed  Google Scholar 

  6. Behiry EG, Al-Azzouny MA, Sabry D, Behairy OG, Salem NE. Association of NKX2-5, GATA4, and TBX5 polymorphisms with congenital heart disease in Egyptian children. Mol Genet Genom Med. 2019;7(5): e612.

    Article  CAS  Google Scholar 

  7. Wang H, Liu Y, Li Y, et al. Analysis of NKX2-5 in 439 Chinese patients with sporadic atrial septal defect. Med Sci Monit. 2019;25:2756–63.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. El Bouchikhi I, Bouguenouch L, ZohraMoufid F, et al. NKX2–5 molecular screening and assessment of variant rate and risk factors of secundum atrial septal defect in a Moroccan population. Anatol J Cardiol. 2017;17(3):217–23.

    Article  PubMed  Google Scholar 

  9. Sherry ST, Ward M, Sirotkin K. dbSNP-database for single nucleotide polymorphisms and other classes of minor genetic variation. Genome Res. 1999;9(8):677–9.

    Article  CAS  Google Scholar 

  10. Ouyang P, Saarel E, Bai Y, et al. A de novo mutation in NKX2.5 associated with atrial septal defects, ventricular noncompaction, syncope and sudden death. Clin Chim Acta. 2011;412(1–2):170–5.

    Article  CAS  PubMed  Google Scholar 

  11. Posch MG, Perrot A, Schmitt K, et al. Mutations in GATA4, NKX2.5, CRELD1, and BMP4 are infrequently found in patients with congenital cardiac septal defects. Am J Med Genet A. 2008;146A(2):251–3.

    Article  CAS  PubMed  Google Scholar 

  12. Juan W, Xingyuan L, Yiqing Y. Novel NKX2-5 mutations responsible for congenital heart disease. Heart. 2011;97:A205–A205.

    Article  Google Scholar 

  13. Abou Hassan OK, Fahed AC, Batrawi M, et al. NKX2-5 mutations in an inbred consanguineous population: genetic and phenotypic diversity. Sci Rep. 2015;5:8848.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Benson DW, Silberbach GM, Kavanaugh-McHugh A, et al. Mutations in the cardiac transcription factor NKX2.5 affect diverse cardiac developmental pathways. J Clin Invest. 1999;104(11):1567–73.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Hosoda T, Komuro I, Shiojima I, et al. Familial atrial septal defect and atrioventricular conduction disturbance associated with a point mutation in the cardiac homeobox gene CSX/NKX2-5 in a Japanese patient. Jpn Circ J. 1999;63(5):425–6.

    Article  CAS  PubMed  Google Scholar 

  16. Kasahara H, Benson DW. Biochemical analyses of eight NKX2.5 homeodomain missense mutations causing atrioventricular block and cardiac anomalies. Cardiovasc Res. 2004;64(1):40–51.

    Article  CAS  PubMed  Google Scholar 

  17. Schott JJ, Benson DW, Basson CT, et al. Congenital heart disease caused by mutations in the transcription factor NKX2-5. Science. 1998;281(5373):108–11.

    Article  CAS  PubMed  Google Scholar 

  18. Ellesoe SG, Johansen MM, Bjerre JV, Hjortdal VE, Brunak S, Larsen LA. Familial atrial septal defect and sudden cardiac death: identification of a novel NKX2-5 mutation and a review of the literature. Congenit Heart Dis. 2016;11(3):283–90.

    Article  PubMed  Google Scholar 

Download references


We are very grateful to the patients and their families for participation in this research study. We acknowledge Dr. Sardjito Hospital for patient registration support.


This study was funded by the Universitas Gadjah Mada (Rekognisi Tugas Akhir [RTA] 2021 to LKD).

Author information

Authors and Affiliations



The study was conceived by RR, AHS, HM and LKD. RR prepared the manuscript, HM & G provided critical revisions for significant intellectual content. All project-related tasks were facilitated by RR, MGS, DWA, ABH, and LKD. Each author has reviewed and approved the text and agreed to be responsible for all parts of the work, including ensuring that any concerns about the accuracy or integrity of any portion of the work are thoroughly investigated and resolved. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Lucia Kris Dinarti.

Ethics declarations

Ethics approval and consent to participate

The Ethical Committee approved the study of the Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada (Ref #KE/1116/11/2020). The research has been performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from all study participants or legally authorized representatives for a patient who died and a patient under the age of 18.

Consent for publication

The patients and legally authorized representatives for a patient who died and a patient under the age of 18 gave written informed consent for publication.

Competing interests

The authors declare no conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Additional file 1

. Table S1. Primer Ex1-FW and Ex-RV are located in exon 1, while Ex2A-FW, Ex2A-RV, Ex2B-FW, and Ex2B-RV are located in exon 2. Table S2. AA – amino acid change; AT – atrial tachycardia; AVND – atrioventricular node dysfunction; Seq var – sequence variant; SND – sinus node dysfunction; SVT – supraventricular tachycardia.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Rozqie, R., Satwiko, M.G., Anggrahini, D.W. et al. NKX2-5 variants screening in patients with atrial septal defect in Indonesia. BMC Med Genomics 15, 91 (2022).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: