Existence of mutations in the homeodomain-encoding region of NKX2.5 gene in Iranian patients with tetralogy of Fallot
Majid Kheirollahi1, Fereshteh Khosravi2, Saeideh Ashouri3, Alireza Ahmadi3
1 Pediatric Inherited Diseases Research Center, Research Institute for Primordial Prevention of Non-Communicable Disease; Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
2 Department of Biology, Islamic Azad University, Yazd, Iran
3 Pediatric Inherited Diseases Research Center, Research Institute for Primordial Prevention of Non-Communicable Disease, Isfahan, Iran
|Date of Submission||26-Sep-2015|
|Date of Decision||03-Jan-2016|
|Date of Acceptance||17-Feb-2016|
|Date of Web Publication||08-Apr-2016|
Pediatric Inherited Diseases Research Center, Research Institute for Primordial Prevention of Non-Communicable Disease, Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan
Source of Support: None, Conflict of Interest: None
Background: Tetralogy of Fallot (TOF), the most common cyanotic heart defect and one of the most common congenital heart diseases, occurs mostly sporadically and nonsyndromically. The underlying molecular genetic mechanism is not known. Therefore, the existence of mutations in the homeodomain-encoding region of NKX2.5 gene in Iranian patients with tetralogy of Fallot is evaluated. Materials and Methods: In the present study, we analyzed the peripheral blood samples of27 patients in order to find any mutation in the 180 bp homeodomain-encoding region of NKX2.5 gene, which is known to be involved in heart development and diseases. DNA was extracted and all the samples were amplified by polymerase chain reaction (PCR) and sequenced. Results: Twenty-seven patients were included in the study. Twenty-five of them were infants and children (6 days to 11 years of age), one was a teenager (14-years of age), and another was a 33-year-old man [mean ± standard deviation (SD): 5.80 ± 3.90 years]. Thirteen patents were males (mean ± SD: 6.587077 ± 5.02 years) and 14 were females (mean ± SD: 5.0726 ± 2.81 years). One synonymous variant, i.e., c.543G>A was identified in one patient. Conclusion: Mutations in the homeodomain-encoding region of NKX2.5 gene may not have an outstanding role in etiology of tetralogy of Fallot patients in Iran.
Keywords: Cardiac defect, Iranian patient, NKX2.5 gene, tetralogy of Fallot (TOF)
|How to cite this article:|
Kheirollahi M, Khosravi F, Ashouri S, Ahmadi A. Existence of mutations in the homeodomain-encoding region of NKX2.5 gene in Iranian patients with tetralogy of Fallot. J Res Med Sci 2016;21:24
|How to cite this URL:|
Kheirollahi M, Khosravi F, Ashouri S, Ahmadi A. Existence of mutations in the homeodomain-encoding region of NKX2.5 gene in Iranian patients with tetralogy of Fallot. J Res Med Sci [serial online] 2016 [cited 2020 Apr 3];21:24. Available from: http://www.jmsjournal.net/text.asp?2016/21/1/24/179893
| Introduction|| |
Tetralogy of Fallot (TOF) is the most prevalent form of cyanotic congenital cardiac defect, which is repaired by congenital heart defect corrective surgery. This condition occurs because of incorrect development of the right side of the heart. , In 1671, TOF, the most common conotruncal cardiac defect, was described by Niels Stenson for the first time. Then, in 1784 its detailed anatomical description was provided by William Hunter at St. Georges Hospital Medical School in London, England. , Around 3.5% of all the newborns affected by congenital heart diseases have TOF and men and women are equally affected. Similar to most of other congenital heart diseases, the exact cause of TOF is not known. It seems that most cases are sporadic but with no other first-degree affected relatives, the risk of recurrence in siblings is around 3%. 
A ventricular septal defect between the anterior and posterior limbs of the trabecular septal band, right ventricular outflow tract obstruction, and overriding of the aortic valve due to anterocephalad deviation of the outlet septum are characterizations of TOF. It is considered as the malformation of the cardiac outflow tract. Most patients affected by this disorder have sufficient pulmonary blood flow at birth but there is increasing cyanosis during the first few weeks and months after birth. These days, in countries with developed cardiac services for infants, diagnosis of TOF is not postponed and palliative procedures and complete repairs are done; therefore, severe cyanosis and other consequences of severely reduced pulmonary blood ﬂow are uncommon. 
The etiology of TOF is not exactly known. Apart from this, numerous genes have been identified to have a role in inherited and sporadic congenital heart diseases. Most of them encoding transcription factors play a significant role in heart development.  Some evidence suggests that environmental factors may play a role in its etiology.  It has been suggested that almost 10-15% of the patients affected by TOF carry a 22q11 deletion (del22q11) (DiGeorge syndrome) , and trisomy of chromosome 21 (Down syndrome) has been found in 7% of these patients.  Additionally, TOF occurs in patients with Alagille syndrome (with mutations in JAG1), , VACTERL association, and CHARGE syndrome. , Previous studies have shown that the occurrence of mutations in NKX2.5 is a nonsyndromic cause of TOF. ,,,
NKX2.5 protein, with 324 amino acids, belongs to the NK-2 family of homeodomain-containing transcription factors conserved from Drosophila to humans, and is encoded by the NK2 transcription factor related, locus 5 gene (NKX2.5/CSX1) gene, which has been mapped to chromosome 5q34. This gene includes two exons. TAD, NK2-specific domain, and homeodomain are conserved regions of NKX2.5 that play significant roles in its function. Interaction of NKX2.5 with DNA, which can be regarded as the most important function of NKX2.5 transcription factor, takes place through its 60-amino acid homeodomain. This domain is a helix-tern-helix DNA-binding motif and includes three α-helices (helix 1, helix 2, and helix 3, which provide binding specificity to the domain).,
Despite considerable advancements in therapy, there are still some patients (about 0.5-6%) who unexpectedly die from this condition.  The etiology of TOF is not exactly known. Some evidence suggests that environmental factors may play a role in its etiology.  The aim of the present study is to evaluate the existence of mutations in NKX2.5 homeodomain coding region in TOF patients from Iran; so, we screened 27 sporadic Iranian individuals with different TOF phenotypes using DNA sequencing method.
| Materials and methods|| |
Study design and participants
The present study was designed as a case series study. Patients were recruited prospectively from April 2012 to September 2013 by Al Zahra Hospital staff at Isfahan University of Medical Sciences, regardless of their sex or ethnicity. All the patients were evaluated by a cardiologist and the diagnosis of TOF was confirmed by echocardiography. Patients with syndromic heart diseases such as DiGeorge, Down, Alagille, Char, Marfan, Noonan, Holt-Oram, or other conditions related to chromosomal anomalies were excluded from the study. The study was approved by the Ethics Committee of Isfahan University of Medical Sciences and written informed consents, considering surrogate decision-making matter, were obtained and all patients' personal health information were kept confidential.
Procedure and variable assessments
Whole blood samples were collected from the patients. DNA was extracted from 200 μl of whole blood of patients by using the PrimePrepTM Genomic DNA Extraction Kit (Genetbio, Daejeon, Korea) according to the manufacturer's protocol. Primers were designed using the DNA sequence available in GenBank. Then, the homeodomain encoding region including 180 bps was polymerase chain reaction (PCR)-amplified. PCR was performed on 25 μl containing 100 ng of genomic DNA, 12.5 μl of Taq DNA Polymerase Master Mix Red (Ampliqon, Odense M, Denmark), and 8.5 μl of ddH2O. The PCR program was started with an initial denaturation at 95°C for 4 min followed by 30 repetitive cycles with a strand separation step at 95°C for 30 s, an annealing step at 66°C for 1 min, and an extension step at 72°C for 35 s, and was finished with a 5-min extension period at 72°C. PCR products were loaded on agarose gel to be visualized. All PCR products were sequenced.
After excluding syndromic patients and confirmation of diagnosis by the cardiologist, 27 patients were included in the study. Twenty-five of them were infants and children (6 days to 11 years of age), one was a teenager (14 years of age), and another was a 33-year-old man [mean ± standard deviation (SD): 5.80 ± 3.90 years]. Thirteen patents were males (mean ± SD: 6.587077 ± 5.02 years) and 14 were females (mean ± SD: 5.0726 ± 2.81 years). General characteristics of the patients are provided in [Table 1].
|Table 1: General characteristics of patients affected by tetralogy of Fallot|
Click here to view
DNA extraction, PCR-amplification, and sequencing of all the specimens were successfully performed. The PCR primers are given in [Table 2]. All sequences were checked and one synonymous variant was observed in one patient. This variant was c.543G > A; Q181Q [Figure 1].
|Table 2: The sequences of primers for the homeodomain region of NKX2.5 gene|
Click here to view
| Discussion|| |
In the present study, we analyzed the sequence encoding homeodomain of NKX2.5 in 27 patients with TOF and found one synonymous variant, i.e., c.543G>A; Q181Q. This silent variant leads to no change in amino acids. This sequence variant was reported in 2009 for the first time  and was observed in one patient with secundum atrial septal defect (ASD). In 2013, Beffagna et al. reported this variant in three out of 100 patients affected by syndromic and nonsyndromic congenital heart diseases (CHDs).  They also observed that p.Q181Q existed as a relatively common variant in the control population consisting of 250 healthy unrelated individuals (with the frequency of 1.6%). Moreover, in another study c.543G>A variant was observed in two patients affected by Down syndrome with congenital heart defects and also in two out of 113 control individuals (with the frequency of 1.8%). 
In another study by Reamon-Buettner et al., c.543G>A was observed in one patient who was also heterozygous for one other sequence alteration (i.e., c356C>A; p.A119E).  They did not observe this variant in the 100 healthy controls. In addition, by using the Vienna RNA folding algorithm, they predicted that this synonymous variant affects NKX2.5 mRNA folding. They also took the advantage of a yeast-based assay to see if the observed variants had clinical significance and observed that p.A119E variant, with the presence of c.543G>A, and c.63A>G in cis, reduced transactivation activities of the protein. These observations show that even for synonymous variants, which are expected to have no functional effect, there are ways to influence gene functions.
Some genes are responsible for TOF, including mutations in NKX2.5, ,,, GATA4 interacts physically with NKX2.5,  GATA6, ,, JAG1, ,,, JAG5,  TBX20,  BVES,  mitochondrial ATP8 gene,  epigenetic changes of some genes such as NKX2.5,  HAND1,  VANGL2,  and single nucleotide polymorphisms of some genes such as PTPN11  and MTHFR.  In addition, TOF has been observed to be concomitant with some syndromes and associations such as Down, Alagille, DiGeorge, and CHARGE syndromes, and VACTERL association. ,,,,,,
| Conclusion|| |
In conclusion, although we did not find any pathological mutation in this group of TOF patients, the significant role of NKX2.5 gene in normal development of the heart and also its mutations in the occurrence of different phenotypes of CHDs have been proved in other studies. In our knowledge, there are limited studies on the genetic bases of CHDs in the Middle East. According to the population-specific distribution of mutations for this disease, this study presents the results of mutation analysis in patients with CHDs in Iran. Apparently, the homeodomain of NKX2.5 gene may not have an outstanding role in Iranian patients with TOF.
We thank the patients and their families who participated in this study.
Financial support and sponsorship
This study was financially supported by the Research Council of the Isfahan University of Medical Sciences (research project number: 222961).
Conflicts of interest
The authors declare no conflict of interest.
| AUTHOR'S CONTRIBUTION|| |
MK contributed in the conception of the work, conducting the study, revising the draft, approval of the final version of the manuscript, and agreed for all aspects of the work. FK contributed in the conception of the work, drafting and revising the draft, approval of the final version of the manuscript, and agreed for all aspects of the work. SA contributed in the conception of the work, conducting the study, revising the draft, approval of the final version of the manuscript, and agreed for all aspects of the work. AA contributed in the conception of the work, revising the draft, approval of the final version of the manuscript, and agreed for all aspects of the work.
| References|| |
Apitz C, Webb GD, Redington AN. Tetralogy of Fallot. Lancet 2009;374:1462-71.
Therrien J, Webb G. Clinical update on adults with congenital heart disease. Lancet 2003;362:1305-13.
Hunter W. Medical Observations and Inquiries. London: William Johnston; 1757. p. 323-57.
Stenson N. Embrio monstro aﬃnis parisiis dissectum. Acta Med Philos Hafniensa 1671-72;1:202-20.
Shinebourne EA, Anderson RH. Fallot′s tetralogy. In: Anderson RH, Baker EJ, McCartney FJ, editors. Pediatric Cardiology. 2 nd
ed. Vol. 2. Philadelphia: Churchill Livingstone; 2002. p. 1213-50.
Bruneau BG. The developmental genetics of congenital heart disease. Nature 2008;451:943-8.
Pierpont ME, Basson CT, Benson DW Jr, Gelb BD, Giglia TM, Goldmuntz E, et al
.; American Heart Association Congenital Cardiac Defects Committee, Council on Cardiovascular Disease in the Young. Genetic basis for congenital heart defects: Current knowledge: A scientific statement from the American Heart Association Congenital Cardiac Defects Committee, Council on Cardiovascular Disease in the Young: Endorsed by the American Academy of Pediatrics. Circulation 2007;115:3015-38.
Karr SS, Brenner JI, Loffredo C, Neill CA, Rubin JD. Tetralogy of fallot: The spectrum of severity in a regional study, 1981-1985. Am J Dis Child 1992;146:121-4.
Marino B, Digilio MC, Grazioli S, Formigari R, Mingarelli R, Giannotti A, et al
. Associated cardiac anomalies in isolated and syndromic patients with tetralogy of Fallot. Am J Cardiol 1996;77:505-8.
Freeman SB, Taft LF, Dooley KJ, Allran K, Sherman SL, Hassold TJ, et al
. Population-based study of congenital heart defects in Down syndrome. Am J Med Genet 1998;80:213-7.
Eldadah ZA, Hamosh A, Biery NJ, Montgomery RA, Duke M, Elkins R, et al
. Familial Tetralogy of Fallot caused by mutation in the jagged1 gene. Hum Mol Genet 2001;10:163-9.
McElhinney DB, Krantz ID, Bason L, Piccoli DA, Emerick KM, Spinner NB, et al
. Analysis of cardiovascular phenotype and genotype-phenotype correlation in individuals with a JAG1 mutation and/or Alagille syndrome. Circulation 2002;106:2567-74.
Greenwood RD, Rosenthal A. Cardiovascular malformations associated with tracheoesophageal fistula and esophageal atresia. Pediatrics 1976;57:87-91.
Kutiyanawala M, Wyse RK, Brereton RJ, Spitz L, Kiely EM, Drake D, et al
. CHARGE and esophageal atresia. J Pediatr Surg 1992;27:558-60.
Goldmuntz E, Geiger E, Benson DW. NKX2.5 mutations in patients with tetralogy of fallot. Circulation 2001;104:2565-8.
Rauch R, Hofbeck M, Zweier C, Koch A, Zink S, Trautmann U, et al
. Comprehensive genotype-phenotype analysis in 230 patients with tetralogy of Fallot. J Med Genet 2010;47:321-31.
McElhinney DB, Geiger E, Blinder J, Benson DW, Goldmuntz E. NKX2.5 mutations in patients with congenital heart disease. J Am Coll Cardiol 2003;42:1650-5.
Benson DW, Silberbach GM, Kavanaugh-McHugh A, Cottrill C, Zhang Y, Riggs S, et al
. Mutations in the cardiac transcription factor NKX2.5 affect diverse cardiac developmental pathways. J Clin Invest 1999;104:1567-73.
Reamon-Buettner SM, Borlak J. NKX2-5: An update on this hypermutable homeodomain protein and its role in human congenital heart disease (CHD). Hum Mutat 2010;31:1185-94.
Bartlett H, Veenstra GJ, Weeks DL. Examining the cardiac NK-2 genes in early heart development. Pediatr Cardiol 2010;31:335-41.
Di Felice V, Zummo G. Tetralogy of fallot as a model to study cardiac progenitor cell migration and differentiation during heart development. Trends Cardiovasc Med 2009;19:130-5.
Draus JM Jr, Hauck MA, Goetsch M, Austin EH 3 rd
, Tomita-Mitchell A, Mitchell ME. Investigation of somatic NKX2-5 mutations in congenital heart disease. J Med Genet 2009;46:115-22.
Beffagna G, Cecchetto A, Dal Bianco L, Lorenzon A, Angelini A, Padalino M, et al
. R25C mutation in the NKX2.5 gene in Italian patients affected with non-syndromic and syndromic congenital heart disease. J Cardiovasc Med (Hagerstown) 2013;14:582-6.
Alcántara-Ortigoza MA, De Rubens-Figueroa J, Reyna-Fabian ME, Estandía-Ortega B, González-del Angel A, Molina-Álvarez B, et al
. Germline mutations in NKX2-5, GATA4, and CRELD1 are rare in a Mexican sample of Down syndrome patients with endocardial cushion and septal heart defects. Pediatr Cardiol 2015;36:802-8.
Reamon-Buettner SM, Sattlegger E, Ciribilli Y, Inga A, Wessel A, Borlak J. Transcriptional defect of an inherited NKX2-5 haplotype comprising a SNP, a nonsynonymous and a synonymous mutation, associated with human congenital heart disease. PloS One 2013;8:e83295.
De Luca A, Sarkozy A, Ferese R, Consoli F, Lepri F, Dentici ML, et al
. New mutations in ZFPM2/FOG2 gene in tetralogy of Fallot and double outlet right ventricle. Clin Genet 2011;80:184-90.
Salazar M, Consoli F, Villegas V, Caicedo V, Maddaloni V, Daniele P, et al
. Search of somatic GATA4 and NKX2.5 gene mutations in sporadic septal heart defects. Eur J Med Genet 2011;54:306-9.
Nemer G, Fadlalah F, Usta J, Nemer M, Dbaibo G, Obeid M, et al
. A novel mutation in the GATA4 gene in patients with Tetralogy of Fallot. Hum Mutat 2006;27:293-4.
Lin X, Huo Z, Liu X, Zhang Y, Li L, Zhao H, et al
. A novel GATA6 mutation in patients with tetralogy of Fallot or atrial septal defect. J Hum Genet 2010;55:662-7.
Huang RT, Xue S, Xu YJ, Yang YQ. Somatic mutations in the GATA6 gene underlie sporadic tetralogy of Fallot. Int J Mol Med 2013;31:51-8.
Zheng G, Zhao H, Wei D, Zhou N, Liu X. Identification of novel mutations in GATA6 gene associated with tetralogy of Fallot. Zhonghua Yi Xue Za Zhi 2012;92:2402-5.
Digilio MC, Luca AD, Lepri F, Guida V, Ferese R, Dentici ML, et al
. JAG1 mutation in a patient with deletion 22q11. 2 syndrome and tetralogy of Fallot. Am J Med Genet A 2013;161A:3133-6.
Kola S, Koneti NR, Golla JP, Akka J, Gundimeda SD, Mundluru HP. Mutational analysis of JAG1 gene in non-syndromic tetralogy of Fallot children. Clin Chim Acta 2011;412:2232-6.
Guarnaccia C, Dhir S, Pintar A, Pongor S. The tetralogy of Fallot-associated G274D mutation impairs folding of the second epidermal growth factor repeat in Jagged-1. FEBS J 2009;276:6247-57.
Guida V, Ferese R, Rocchetti M, Bonetti M, Sarkozy A, Cecchetti S, et al
. A variant in the carboxyl-terminus of connexin 40 alters GAP junctions and increases risk for tetralogy of Fallot. Eur J Hum Genet 2013;21:69-75.
Stennard FA, Costa MW, Elliott DA, Rankin S, Haast SJ, Lai D, et al
. Cardiac T-box factor Tbx20 directly interacts with Nkx2-5, GATA4, and GATA5 in regulation of gene expression in the developing heart. Dev Biol 2003;262:206-24.
Wu M, Li Y, He X, Shao X, Yang F, Zhao M, et al
. Mutational and functional analysis of the BVES gene coding region in Chinese patients with non-syndromic tetralogy of Fallot. Int J Mol Med 2013;31:899-903.
Tansel T, Paçal F, Ustek D. A novel ATP8 gene mutation in an infant with tetralogy of Fallot. Cardiol Young 2014;24:531-3.
Sheng W, Qian Y, Wang H, Ma X, Zhang P, Diao L, et al
. DNA methylation status of NKX2-5, GATA4 and HAND1 in patients with tetralogy of fallot. BMC Med Genomics 2013;6:46.
Yuan Y, Gao Y, Wang H, Ma X, Ma D, Huang G. Promoter methylation and expression of the VANGL2 gene in the myocardium of pediatric patients with Tetralogy of Fallot. Birth Defects Res A Clin Mol Teratol 2014;100:973-84.
Goodship JA, Hall D, Topf A, Mamasoula C, Griffin H, Rahman TJ, et al
. A common variant in the PTPN11 gene contributes to the risk of tetralogy of Fallot. Circ Cardiovasc Genet 2012;5:287-92.
Marinho C, Alho I, Guerra A, Rego C, Areias J, Bicho M. The methylenetetrahydrofolate reductase gene variant (C677T) as a susceptibility gene for tetralogy of Fallot. Rev Port Cardiol 2009;28:809-12.
[Table 1], [Table 2]