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Detection of mutations in GATA4 and Nkx2.5 genes in patients with Fallot’s tetralogy

Hussein, Ibtessam R.a; El-Ruby, Mona O.b; Fahmi, Abdelgawad A.c; El-Desouky, Mohamed A.c; Fayez, Alaa El-Deen G.a

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Middle East Journal of Medical Genetics: January 2012 - Volume 1 - Issue 1 - p 49-52
doi: 10.1097/01.MXE.0000407732.76680.89
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Tetralogy of Fallot (TOF) is defined as a combination of four (tetralogy) congenital abnormalities. Typically, the four defects are ventricular septal defect (VSD), pulmonary stenosis, a misplaced aorta, and a thickened right ventricular wall. TOF is the most common type of congenital heart disease (CHD). It is estimated to occur in 3.3 per 10 000 live births and accounts for 6.8% of all CHD cases (Goldmuntz et al., 2001). T-box, NK, and GATA transcription factors have been known to be associated with a variety of syndromic and isolated CHD cases (Wolf and Basson, 2010). CHD, including TOF, could be related to specific molecular pathways controlled by transcription factors such as Nkx2.5 and GATA4 genes. Nkx2.5, which consists of two exons located at 5q34–q35.3, is a pivotal transcription factor in mammalian heart development (Reamon Buettner and Borlak, 2004). The zinc finger transcription factor GATA4, consisting of seven exons located at 8p23.1–p22, has been implicated as a critical regulator of cardiac cell growth, differentiation, and survival during embryogenesis (Tenhunen et al., 2004). Recent reports have implicated mutations in the transcription factor Nkx2.5 as a cause of TOF (Grepin et al., 1997; Benson et al., 1999; Goldmuntz et al., 2001; Bruneau, 2002; Nemer et al., 2006; Peng et al., 2010). A novel mutation in the GATA4 gene was found in patients with TOF. This mutation, located in the exon encoding GATA4 first zinc finger, reduces its transcriptional activation of downstream target genes (Nemer et al., 2006). Durocher et al. (1997) concluded that GATA4 and Nkx2.5 interact physically and synergistically to activate cardiac transcription through specific common domains between them.

Subjects and methods

This study was carried out on 15 unrelated patients of both sexes [six women (40%), nine men (60%); age range: 8 months to 10 years; mean age 2.6±0.6 years] suffering from nonisolated Fallot’s tetralogy [5/15 (33.3%)] and isolated Fallot’s tetralogy [10/15 (66.7%)]. All patients were evaluated clinically in clinical genetics clinics by historical examination, review of medical records, physical examination, pedigree analysis, consanguinity, and evaluation of their history of drug intake. Two-dimensional transthoracic echocardiography with colorflow Doppler was carried out in cardiology clinics. DNA extraction and mutations detection was carried out in Molecular Genetics & Enzymology Department of the National Research Centre.

DNA extraction

Blood samples were collected using Na2EDTA as anticoagulant. DNA was isolated from peripheral blood leukocytes by the salting out technique according to Miller et al., (1988).

Screening for mutations in NKx2.5 and GATA4 genes

Amplification of exons carrying hot spots for mutations was carried out using specific PCR primers as reported by Goldmuntz et al. (2001) and Okubo et al. (2004), followed by single stranded conformation polymorphism (SSCP). Samples that showed an abnormal pattern using the SSCP technique were then subjected to DNA sequencing for mutation detection. The coding regions of the NKx2.5 gene, including exon/intron boundaries, were amplified from genomic DNA by four PCR reactions. All reactions started with 2 min at 95°C, followed by 35 cycles of 45 s at 95°C, 30 s at 60 or 61°C, and 45 s at 72°C, and finished with a 10 min extension period at 72°C (Goldmuntz et al., 2001). PCR enhancer solution was added at 1×concentration. The coding regions of the GATA4 gene were amplified by PCR. Exons corresponding to human GATA4 gene (Accession No.: NT_077531.3) were amplified using specific primers in the intronic regions. PCR was cycled 35 times at 95°C for 1 min, at 55–56°C for 1 min, and at 72°C for 1 min in 50 μl mixture containing 1× Taq buffer, 0.2 mmol/l each dNTP, 1 μM each primer, and 2 units of Taq polymerase (Okubo et al., 2004).

Single stranded conformation polymorphism

Samples were denatured at 94°C for 5 min and placed on ice for 3–5 min. The PCR product was loaded on 7–10% polyacrylamide gel. Gel electrophoresis was run at a constant temperature of 7°C for 3–5 h at 200–400 Volts. The gel was removed, stained with silver stain, and photographed using a gel documentation system.

DNA sequencing

Samples were run on 1.5% agarose gels, and the bands corresponding to the predicted size were cut and purified using a gel extraction kit (QIA quick columns, Qiagen, West Sussex, England) following the manufacturer’s protocol. Purified samples were subjected to cycle sequencing using a Big Dye Terminator v3.1 Kit and injected into a 377 PRISM automated sequencer Genetic Analyzer (Applied Biosystems, Weiterstadt, Germany).


Mutation analysis results have been summarized in Tables 1 and 2 and Figs 1–4. False-positive results using the SSCP method were detected in four cases (26.7%). It was observed that there was no difference in the incidence of the detected single nucleotide polymorphisms (SNPs) and in the mutation between nonisolated (2/5 40%) and isolated (4/10 40%) TOF patients. Differences were observed in the variety of the detected SNPs and mutation between the two types (isolated and nonisolated TOF patients) (Table 3), where SNP 53423, SNP 53164, SNP 53177, silent mutation 371 Ser (A→G), and Arg25Cys were found in isolated cases and only silent mutation 336 Gly (C→G) and SNP 53164 were found in nonisolated ones.

Table 1
Table 1:
Summary of mutation analysis results
Table 2
Table 2:
Summary of the clinical and molecular genetics results of the patients
Figure 1
Figure 1:
Electrophoresis on 7% polyacrylamide gel under 4°C showing single-stranded conformation polymorphism (SSCP) analysis of polymerase chain reaction-amplified products of exon 6. Lane (C) is normal control. Lanes 1, 2, 4, 5, 6, 7, and 8 represent SSCP analysis of exon 6 that displayed a change in electrophoretic band migration pattern in respect to the normal control.
Figure 2
Figure 2:
Partial sequence of GATA4 (exon 6) showing overlapping of T and C bases (reverse strand) indicating alternation between normal and polymorphism sequence.
Figure 3
Figure 3:
Partial sequence of GATA4 (exon 6) showing overlapping of A and G bases indicating alternation between normal and polymorphism sequence.
Figure 4
Figure 4:
Polyacrylamide gel electrophoresis for restriction enzyme assay of HhaI showing abnormal digestion of exon 1A to length 134 base pairs.
Table 3
Table 3:
Types of mutations and single-nucleotide polymorphisms in isolated and nonisolated tetralogy of Fallot


In this study, a nonsynonymous Arg25Cys mutation was detected in fragment 1A of the Nkx2.5 gene using a restriction enzyme assay (HhaI) in a case with isolated TOF, and hence the mutation rate was 1/15 (6.7%). This point mutation was previously reported in sporadic TOF cases, where Benson et al. (1999) found the Arg25Cys mutation in one of 20 (5%) TOF patients. Another study identified the Arg25Cys mutation in three of 114 (2.6%) unrelated TOF patients, and this represented 50% (3/6) of Nkx2.5 mutations found in 114 TOF patients (Goldmuntz et al., 2001). McElhinney et al. (2003) found that, among patients with conotruncal anomalies, Nkx2.5 mutations were detected in 13 of 201 patients; among the 13 patients, nine had TOF, and the Arg25Cys mutation was found in four of those nine TOF cases (44.4%). Reamon Buettner and Borlak (2004) reported that detection of Arg25Cys mutation in VSD patients is not surprising, as VSD is one of the four complications of TOF. In contrast, in our study the Arg25Cys mutation was not detected in 10 patients with VSD by using the HhaI restriction enzyme. This may because the number of VSD patients was small. Previous reports indicated the incidence of Arg25Cys mutation among TOF patients to be from 2 to 5% (Benson et al., 1999; Goldmuntz et al., 2001). This finding is very similar to the results of the present study, in which it was estimated to be one out of 15 (6.7%).

Two synonymous mutations (silent mutations) were found located in exon 6 of the GATA4 gene. Silent mutation 336 Gly (G→C) was detected in one patient suffering from TOF associated with Russel Silver syndrome, and a second silent mutation 371 Ser (A→G) was detected in one isolated TOF patient. To our knowledge, these silent mutations have not been previously reported.

Three known SNPs (NCBI dSNPs) were detected in four patients (4/15 26.7%) inside the GATA4 gene [53423 A→G base (exon 6) was found in two patients, 53177 C→T base (intron 5) was found in two patients, and 53164 T→C base (intron 5) was found in three patients]. These SNPs, previously reported, may be used as markers for molecular diagnosis of TOF; however, this requires further investigation of these SNPs on more TOF families and control individuals.

No difference was observed in the incidence of the detected SNPs and mutation between nonisolated (2/5 40%) and isolated (4/10 40%) TOF patients in relation to group size as illustrated, but differences were observed in the variety of the detected SNPs and mutation between the two groups, where SNP 53423, SNP 53164, SNP 53177, silent mutation 371 Ser (A→G), and Arg25Cys were found in isolated cases but only silent mutation 336 Gly (C→G) and SNP 53164 were found in nonisolated ones.

False-positive results were observed using the SSCP method [4/15 (26.7%)]. This low sensitivity could be explained by the fact that sensitivity of SSCP in detecting a given variation in a known local context varies from one fragment to another. In addition, the formation of a high-order structure depends on the length of the fragment and on physical factors such as temperature and ionic strength. The sequence changes that have little or no effect on conformation in one set of conditions may have a markedly different effect under other conditions (Wagner and John, 2002). Moreover, overloading of the gel sometimes results in abnormal migration of the bands and in reduced resolution. It was also reported that SSCP analysis is more sensitive for short fragments (175–250 bps) because the effect of a single base change on the overall conformation of large fragments (more than 250 bps) is less than that of small fragments. It was also shown that glycerol usually improves the sensitivity of the analysis when it is run at room temperature (Hayashi and Yandell, 1993).


The results of this study indicate that (a) a high incidence ratio of Arg25Cys mutation, at Nkx2.5 gene, is found in manifested Egyptian TOF patients; (b) there are polymorphic sites in exon 6 and intron 5 of the GATA4 gene, which may be used as diagnostic markers for Fallot’s tetralogy; (c) SSCP is not an accurate tool for detection of mutations in GATA4 and Nkx2.5 genes.


Conflicts of interest

There are no conflicts of interest.


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;104:1567–1573
Bruneau BG. Transcriptional regulation of vertebrate cardiac morphogenesis. Circ Res. 2002;90:509–519
Durocher D, Charron F, Warren R, Schwartz RJ, Nemer M. The cardiac transcription factors Nkx2-5 and GATA-4 are mutual cofactors. EMBO J. 1997;16:5687–5696
Goldmuntz E, Geiger E, Benson DW. NKX2.5 mutations in patients with Tetralogy of Fallot. Circulation. 2001;104:2565–2568
Grepin C, Nemer G, Nemer M. Enhanced cardiogenesis in embryonic stem cells overexpressing the GATA-4 transcription factor. Development. 1997;124:2387–2395
Hayashi K, Yandell DW. How sensitive is PCR-SSCP? Hum Mutat. 1993;2:338–346
McElhinney DB, Geiger E, Blinder J, Benson DW, Goldmuntz E. NKX2.5 mutations in patients with congenital heart disease. J Am Col Cardiol. 2003;42:1650–1655
Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988;16:1215
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;27:293–294
Okubo A, Miyoshi O, Baba K, Takagi M, Tsukamoto K, Kinoshita A, et al. A novel GATA4 mutation completely segregated with atrial septal defect in a large Japanese family. J Med Genet. 2004;41:e97
Peng T, Wang L, Zhou SF, Li X. Mutations of the GATA4 and NKX2.5 genes in Chinese pediatric patients with non-familial congenital heart disease. Genetica. 2010;138(11–12):1231–1240
Reamon Buettner SM, Borlak J. Somatic NKX2-5 mutations as a novel mechanism of disease in complex congenital heart disease. J Med Genet. 2004;41:684–690
Tenhunen O, Sarman B, Kerkela R, Szokodi I, Papp L, Toth M, et al. Mitogen-activated protein kinases p38 and ERK 1/2 mediate the wall stress-induced activation of GATA-4 binding in adult heart. J Biol Chem. 2004;279:24852–24860
Wagner L, John F. Screening methods for detection of unknown point mutations. Hum Mutat. 2002;15:300–305
Wolf M, Basson CT. The molecular genetics of congenital heart disease: a review of recent developments. Curr Opin Cardiol. 2010;25:192–197

congenital heart disease; Fallot’s tetralogy; GATA4; Nkx2.5; point mutation

© 2012 Middle East Journal of Medical Genetics