|Year : 2015 | Volume
| Issue : 2 | Page : 40-42
Two novel mutations in NKX 2.5 gene un-translated regions in congenital heart diseases patients from Pakistan
Afsheen Arif1, Sitwat Zehra2, Najma Patel3, Abid Azhar1
1 Karachi Institute of Biotechnology, Dr. A. Q. Khan Institute of Biotechnology & Genetic Engineering (KIBGE), University of Karachi-75270, Pakistan
2 Biological Research Department, Dow Diagnostic Research and Reference Laboratory, DUHS, Karachi, Pakistan
3 National Institute of Cardiovascular Diseases, (NICVD), Karachi, Pakistan
|Date of Web Publication||5-Jul-2017|
Karachi Institute of Biotechnology, Dr. A. Q. Khan Institute of Biotechnology & Genetic Engineering (KIBGE), University of Karachi-75270
Source of Support: None, Conflict of Interest: None
Introduction: Congenital heart diseases (CHD) are one of the neglected and challenging areas in undeveloped countries. In Pakistan approximately 40-50,000 children are affected annually. A majority of these patients belongs to rural areas where properly medical facilities are out of reach. In the etiology of these diseases genetic profile, consanguinity and other factors should be examined carefully. The current study aims to check the genetic manipulations in patients for the NKX 2.5 gene specifically the untranslated gene of this gene. NKX 2.5, a transcription factor and first progenitor in cardiac formation, it encodes 324 amino acid and contains a homeodomain (142-200aa) which is highly conserved among vertebrates. To date, no data is available about the mutations in this gene responsible for CHD in Pakistani population. Material and Methods: A cohort of 225, CHD patients who were registered at National Institute of Cardiovascular Diseases (NICVD) from 2006-2009 were included in the study; these are non-syndromic and sporadic cases. Healthy 200 controls were also included with informed consents and detail family history was obtained. DNA was extracted and NKX 2.5 gene was amplified and sequenced to check mutations. Results: The mean age for patients TOF (2.97±1.21), PDA (2.95±2.55), D-TGA (1.84±2.26) and for controls (3.14±1.82) .In present study, two UTR alterations have been reported for NKX 2.5 one at 5' and other at 3'.In our study we look social and genetic aspects for these diseases. Conclusion: CHD is a major cause of child death in the first year of life. Our study concludes a few aspects and will broaden it to other genes as it is a need to find etiology of these diseases and to combat it with the modern genetic therapeutics.
Keywords: Congenital heart diseases (CHD), NKX 2.5, Tetralogy of Fallot (TOF), Transcription factor
|How to cite this article:|
Arif A, Zehra S, Patel N, Azhar A. Two novel mutations in NKX 2.5 gene un-translated regions in congenital heart diseases patients from Pakistan. Acta Med Int 2015;2:40-2
| Introduction|| |
NKX2.5 is well known earliest molecule marker in the cardiac lineage in vertebrates. It is one of the members of NK2 family of homeobox genes and a homolog of the Drosophila tin man. It has highly conserved regions of DNA binding, protein-protein interactions, nuclear translocation, and regulation of other transcription factors. Homeobox genes have been found to play a crucial role in regulating tissue specific gene expression. Mutations in these genes have been reported to cause ASD (atrial septal defects), VSD (ventricular septal defects) with atrial ventricular block, TOF and Tricuspid valve abnormalities. NKX2.5 locus more than 40 mutations identified to date., NKX2.5 has been associated with single cases of heterotaxy. NKX2.5 is a mysterious component of cardiac gene regulatory networks, involving epigenetic, transcriptional, and post-transcriptional mechanisms. In the heart development. NKX2.5 acts at different molecular stages. Cooperating with other cardiac transcription factor, such as GATA4, TBX5, MEF2C and TBX20., Directly regulating the expression of genes in the heart, such as cardiac alpha-actin, homeobox gene IRX4, atrial natriuretic factor, and connexin genes., Being regulated by other signaling and cardiac transcription factors, including bone morphogenetic protein, GATA4 and NFAT factors.
Roughly 10% of patients with CHDs have conotruncal heart defects, which include TOF, DORV, TA, IAA, and DTGA. This region is also now offered as commercial genetic testing by prevention genetics, USA Isolated non syndromic congenital heart defects via the NKX 2.5 gene. The promoter region of NKX 2.5 is characterized for its cardiac expression contains binding sites for NKX 2.5 (AAGTG), six GATA factor binding sites (GATA) and multiple Smad binding sites (GTCT/AGAC) consistent to the enhancers of murine. The present study aims to screen mutations in the coding and non-coding regions of this gene.
| Materials and Methods|| |
A cohort of 149 patients for Tetralogy of Fallot, 50 patients for Patent Ductus Arteriosus, 26 patients for Dextro Transposition of Great Arteries. All patients were sporadic and non- syndromic of Tetralogy of Fallot (TOF), Patent Ductus Arteriosus (PDA) and Dextro Transposition of Great Arteries (DTGA) . The patients were recruited after the confirmation by the pediatric cardiologist and examining the cardiac murmur, chest X-ray, fetal echocardiography (ECG), complete blood count (CBC), echocardiograms and/or echocardiogram reports (ECHO), cardiac catherization reports, operative notes and MRI of heart. Two hundred (200), age, gender and weight matched healthy unrelated individuals were selected as controls. Informed consents were obtained from the parents/guardians of controls. The following primers were used for the amplification primers for NKX 2.5 gene. NKX 2.5 has two exons and a 1.5kb intronic region. Primers are design to cover entire exon-intron boundaries. The PCR conditions were taken as been described earlier.
| Results|| |
The mean age for patients TOF (2.97±1.21), PDA (2.95±2.55), D-TGA (1.84±2.26) and for controls (3.14±1.82) . The age of patients ranges from one day to 12 years. The mean weight for patients TOF (12.44±6.7), PDA (9.20±4.75), D-TGA (8.55±6.38) and for controls (13.38±6.63) . The weight for PDA and DTGA patients are below average due to the problem in feeding and delayed growth as compared to controls. Gender distribution for patients of TOF (62%) male and (38%) female, PDA (44%) male and (56%) female, D-TGA (58%) male and (42%) female and controls (55%) male and (45%) female. Males are more frequently affected in TOF patients, PDA occurs more frequent in girls than boys the data reflects same pattern. Mostly boys are affected in D-TGA as expected. The genotypic and phenotypic descriptions of the patients are given in [Table 1].
A novel mutation at 145 bp in 5' UTR region, single base pair change was observed. It is a transition mutation from cytosine to thymine. The chromosomal position for this mutation is 172662118. The last intron/exon boundary begins at 511. Multiple sequence alignment was carried out by CLC software to locate the position and a change in nucleotide at position 145 was observed with transcript ENST00000329198 [Figure 1] . This novel mutation was found in one year old female patient suffering from low pressure PDA associated with CoA.
|Figure 1: Multiple sequence alignment of NKX 2.5 gene, control and sequence of Patient 158 for 5' UTR 145 bp|
Click here to view
Another novel mutation at 1355 bp in 3' UTR region, single base pair change was observed. It is a transition mutation from guanine to adenine. The chromosomal position for this mutation is 172659368. The coding sequence position is 1179. The mutation was tested by software mutation taster predicts poly A signal is functional and it could be a polymorphism. This mutation is found in seven year old male patient suffering from TOF. The details are given in [Table 2].
| Discussion|| |
Untranslated regions (UTRs) have important regulatory roles in gene expression process, they can regulate mRNA function and influence gene function. In present study, two UTR alterations have been reported for NKX 2.5 one at 5' and other at 3'. Among the genome, there are many mysterious information not revealed yet so as the role of the promoter and untranslated regions of the genes. Initially they were termed and considered as junk DNA but now the school of thoughts has emerged that insist that there is potential in information in these UTR's and scientist are far behind to explore their role. This study has just drawn some attention and their significant role in the diseases like PDA and TOF. There can other factors too causing these diseases and in future planning to explore them.
| References|| |
Schott JJ, Benson DW, Basson CT, Pease W, Silberbach GM, Moak JP, et al. Congenital heart disease caused by mutations in the transcription factor NKX2-5. Science. 1998; 281:108–111.
Liu XY, Wang J, Yang YQ, Zhang YY, Chen XZ, Zhang W, et al. Novel NKX2-5 mutations in patients with familial arterial septal defects. Pediatr Cardiol. 2011; 32:193–201.
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(2):1185–1194.
Watanabe Y, Benson DW, Yano S, Akagi T, Yoshino M, Murray JC. Two novel frameshift mutations in NKX2.5 result in novel features including visceral inversus and sinus venosus type ASD. J Med Genet. 2002; 39:1–4.
Akazawa H, Komuto I. Cardiac transcription factor Csx/Nkx2-5: Its role in cardiac development and disease. Pharmthera. 2005; 107:252–268.
Bruneau BG, Black BL, The heart's Da Vinci code: A renaissance at Keystone. Development. 2007, 134:1631–1633.
Durocher D, Charron F, Warren R, Schwarte RJ, Nemer M. The cardiac transcription factors Nkx2-5 and GATA-4 are mutual cofactors. EMBO J. 1997; 16(18):5687–5696.
Durocher D, Chen CY, Ardati A, Schwarte RJ, Nemer M. The atrial natriuretic factor promoter is a downstream target for Nkx-2.5 in the myocardium. Mol Cell Biol. 1996; 16(9):4648–4655.
Lien CL, McAnally J, Richardson JA, Olson EN. Cardiac-specific activity of an Nkx2-5 enhancer requires an evolutionarily conserved Smad binding site. Dev Biol. 2002; 244: 257–266.
Oyen N, Poulsen G, Boyd HA, Wohlfahrt J, Peter KA. Jensen. Melbye M. Recurrence of Congenital Heart Defects in Families. Circ. 2009; 120:295–301.
Shiojima I, Oka T, Hiroi Y, Nagai R, Yazaki Y, Komuro I. Transcriptional regulation of human cardiac homeobox gene CSX1. Biochem Biophys Res Commun. 2000; 272: 749–757.
Stallmeyer B, Fenge H, Nowak-Göttl U, Schulze-Bahr E. Mutational spectrum in the cardiac transcription factor gene NKX2.5 (CSX) associated with congenital heart disease. Clini Genet. 2010; 78:533-40. Arif A, Zehra S, Qamarunissa S, Azhar A. “ Tetralogy of Fallot (Cyanotic cardiac malformation), trends and variation in a population based study”. Pak J Mol Biol Biotech, 2012; 4 (4): 221–224. Shen L, Li XF, Shen AD, Wang Q, Liu CX, Guo YJ, et al. Transcription factor HAND2 mutations in sporadic Chinese patients with congenital heart disease. Chin Med J. 2010; 123:1623–1627.
Frank JE, Jacobe KM. Evaluation and management of heart murmurs in children. Am Acad Family Physic. 2011;84:7–10.
Lai WW, Tal G, Girish S, Shirali GS, Peter C. Frommelt PC et al., Guidelines and Standards for Performance of a Pediatric Echocardiogram: A Report from the Task Force of the Pediatric Council of the American Society of Echocardiography. J Am Societ Echocardiol. 2006 Dec; 19(2):1413–1430.
Goldmuntz E, Geiger E, Benson DW. NKX2.5 Mutations in patients with Tetralogy of Fallot. Circ. 2001; 104:2565–2568.
Majumdar R, Yagubyan M, Sarkar G, Bolander ME, Sundt TM. Bicuspid aortic valve and ascending aortic aneurysm are not associated with germline or somatic homeobox NKX2-5 gene polymorphism in 19 patients. J Thorac Cardiovasc Surg. 2006; 131:1301–1305.
Marelli AJ, Mackie AS, Ionescu-Ittu R, Rahme E, Pilote L. Congenital heart disease in the general population: Changing prevalence and age. Circ. 2007; 115:163–172.
Blue GM, Kirk EP, Sholler GF, Harvey RP and Winlaw DS. Congenital heart disease: Current knowledge about causes and inheritance. Med J Aust. 2012; 197:155–159.
Shen L, Li XF, Shen AD, Wang Q, Liu CX, Guo YJ, et al. Transcription factor HAND2 mutations in sporadic Chinese patients with congenital heart disease. Chin Med J. 2010; 123:1623–1627.
Arif A, Zehra S, Qamarunissa S, Azhar A. “Tetralogy of Fallot (Cyanotic cardiac malformation), trends and variation in a population based study”. Pak J Mol Biol Biotech, 2012; 4 (4): 221–224.
[Table 1], [Table 2]