DNA hypermethylation of the NOX5 gene in fetal ventricular septal defect

  • Authors:
    • Chun Zhu
    • Zhang-Bin Yu
    • Xiao-Hui Chen
    • Chen-Bo Ji
    • Ling-Mei Qian
    • Shu-Ping Han
  • View Affiliations

  • Published online on: June 24, 2011     https://doi.org/10.3892/etm.2011.294
  • Pages: 1011-1015
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Ventricular septal defect (VSD) is the most comon form of congenital heart disease (CHD). DNA hypermethylation analysis may provide an insight into the molecular features and pathogenesis of this heart disease. Although aberrant DNA hypermethylation is implicated in the pathophysiology of this heart disease, only a limited number of genes are known to be epigenetically altered in VSD. We previously identified regulation of the NOX5 gene by hypermethylation in VSD fetuses by promoter methylation microarrays. This study was designed to detect the expression of NOX5 mRNA in VSD and normal fetuses. We also verified the results of promoter methylation microarrays by methylation-specific PCR. DNA extraction and nested methylation-specific PCR were performed on myocardial tissue samples from 21 VSD and 15 normal fetuses. The primers specific for methylated vs. unmethylated DNA were designed and amplified by nested PCR. The products were visualized on agarose gel. Hypermethylation of the NOX5 promoter was more frequent in VSD fetuses (66.67%) than in normal fetuses (20%). There was a significant concordance between NOX5 methylation and a decrease in its mRNA expression. Taken together, our results demonstrate that hypermethylation of the NOX5 gene may be involved in the pathogenesis of VSD.

Introduction

Congenital heart diseases (CHDs) are common congenital malformations. They are caused by abnormalities in the embryonic heart and vasculature. The reported incidence of CHDs varies between 12 and 14 per 1,000 live births (1). In China, approximately 100,000 neonates are born with CHDs every year. Only one-fifth of these neonates obtain treatment in the approximately 300 units that perform cardiac surgery (2). Ventricular septal defect (VSD) is the most common form of CHD. Currently, even though many genes related to cardiac development have been identified and correction by surgery has yielded favorable results as the main treatment option, the etiology and pathological mechanisms of the disease are unknown.

The NADPH oxidase (NOX) family consists of the homologs of gp91phox (glycosylated subunit of phagocyte NADPH oxidase flavocytochrome); this family includes the NOX1, NOX2, NOX3, NOX4 and NOX5 genes. ROS are derived by the NOX family as secondary messenger molecules that participate in cell proliferation, transformation, differentiation and apoptosis (35). The embryonic development of the ventricular septum involves a balance between various cell proliferation, differentiation and apoptosis processes. NOX5 is a highly expressed embryonic gene, which may be involved in this process.

Epigenetic mechanisms, such as miRNA expression and histone modification, are crucially responsible for dysregulated gene expression in CHD (6). By contrast, the role of DNA methylation remains unknown. DNA methylation is catalyzed by DNA methyltransferase and involves the addition of a methyl group to the carbon-5 position of the cytosine ring converting it to methyl cytosine (7), which is another well-characterized epigenetic mark. DNA methylation plays an important role during embryogenesis, normal mammalian development, cellular differentiation and chromosome integrity. NOX5, a promoter hypermethylation gene, was found in our laboratory by promoter methylation microarrays (8). The aim of this study was to verify the results of promoter methylation microarrays and determine whether there is concordance between NOX5 methylation and a decline in its mRNA expression in VSD and normal fetuses.

Materials and methods

Tissues and DNA

Fetal myocardial tissue samples were obtained from the Nanjing Maternal and Child Health Hospital. The representative four-chamber view of VSD is shown in Fig. 1. Thirty-six fetuses at 26 weeks of gestation were obtained during surgery for termination of pregnancy owing to trauma of the pregnant women. All samples were collected with the approval of the ethics committee of the appropriate institute, and written consent was provided by each pregnant woman and her family. The specimens were snap frozen in liquid nitrogen immediately and then stored at −80°C until analysis. Genomic DNA was purified from tissue specimens using the conventional proteinase K digestion and phenol/chloroform extraction method. After purification, genomic DNA was treated with sodium bisulfite. The treatment converts unmethylated cytosines to uracils, while leaving the methylated cytosines unaffected (9).

Methylation-specific PCR

The NOX5 gene sequence was obtained from GenBank (Gene ID 79400). Methyl Primer Express® Software was used to design the primer of methylation-specific PCR. The primer designed for the unmethylated sequences does not amplify the methylated sequence and vice versa. Bisulphite-modified DNA was amplified by nested PCR using the primer sets described in Table I. Primers were purchased from Invitrogen (Carlsbad, CA, USA). Myocardial tissue DNA samples, untreated or methylated in vitro by excess CpG (Sss.I) methyltransferase (NEB, USA), were used as positive controls for unmethylated and methylated DNA, respectively. Distilled water was used as a negative control. The first cycle of nested PCR was carried out in a final volume of 25 μl, containing 2.5 μl 10X buffer, 2.5 μl 10 mM dNTP mixture, 2.0 μl 50 mM MgCl2, 1.0 μl of each of the primers, 0.25 μl (5 U/μl) Taq DNA polymerase and 2.0 μl DNA sample containing 10 ng DNA. The amplification conditions were as follows: an initial incubation at 94°C for 2 min, followed by 36 cycles at 94°C for 30 sec, 57°C for 30 sec and 72°C for 45 sec; and a final extension at 72°C for 7 min. A 20-fold dilution of the product from the first cycle was the template of the second cycle, while the other components were similar. The amplification conditions for the second cycle were as follows: an initial incubation at 94°C for 2 min, followed by 36 cycles at 94°C for 30 sec, 55°C for 30 sec and 72°C for 45 sec; and a final extension at 72°C for 7 min. PCR products were separated in a 1.5% agarose gel, stained with ethidium bromide and visualized under UV illumination. Each experiment was repeated at least three times.

Table I.

Primer sequences for the methylated (M) and unmethylated (U) NOX5 gene.

Table I.

Primer sequences for the methylated (M) and unmethylated (U) NOX5 gene.

PrimersSequencesTemperature (°C)
First cycleM forward 5′-TATAGGGATCGCGTTTAAATTAC-3′57
M reverse 5′-ACTAAAAACTTCATAAACGTCGTC-3′
U forward 5′-TTTTATAGGGATTGTGTTTAAATTAT-3′
U reverse 5′-AAACTAAAAACTTCATAAACATCATCC-3′
Second cycleM forward 5′-TATAGGGATCGCGTTTAAATTAC-3′55
M reverse 5′-TATCAACGAAATACCGTCCTAC-3′
U forward 5′-TTTTATAGGGATTGTGTTTAAATTAT-3′
U reverse 5′-TATCAACAAAATACCATCCTACCTC-3′
RT-PCR

Total RNA from the myocardial tissue samples was extracted using the TRIzol method (Invitrogen). cDNA was synthesized from 1 μg of total RNA using an AMV Reverse Transcriptase kit (Promega A3500; Promega, Madison, WI, USA). An aliquot (10%) of the resulting cDNA was amplified for PCR with the primers listed in Table II. The number of cycles and reaction temperatures used in the PCR assay were optimized to provide a linear relationship between the amount of input template and the amount of PCR product.

Table II.

Primer sequences for RT-PCR.

Table II.

Primer sequences for RT-PCR.

GenePrimersTemperature (°C)
NOX5Forward 5′-AAGACTCCATCACGGGGCTGCA-3′65
Reverse 5′-CCCTTCAGCACCTTGGCCAGAG-3′
GAPDHForward 5′-CCATGTTCGTCATGGGTGTGAACCA-3′60
Reverse 5′-GCCAGTAGAGGCAGGGATGATGTTC-3′
Statistical analysis

Each experiment was performed at least three times. For the analysis, data were classified using Fisher’s exact test and Student’s t-test. A P-value <0.05 (2-sided) was regarded as statistically significant. All data were analyzed with SPSS 13.0 for Windows (SPSS Inc., Chicago, IL, USA).

Results

Hypermethylation of the NOX5 promoter in VSD fetuses

The results of the methylation status analysis of the CpG islands in the NOX5 promoter region in the myocardial tissue of normal and VSD fetuses are shown in Fig. 2. The NOX5 promoter was hypermethylated in 66.67% of the VSD fetuses and in only 20% of the normal fetuses (Table III). Statistical analysis revealed that promoter hypermethylation of the NOX5 gene was strongly correlated with the study groups (P=0.008).

Table III.

Hypermethylation status in the two groups.

Table III.

Hypermethylation status in the two groups.

Study groupNo.Hypermethylated casesHypermethylation yes (%)
VSD211466.67
Controls15320.00
Expression of NOX5 gene mRNA in fetal myocardial tissue

The expression levels of NOX5 in fetal myocardial tissue samples were measured by RT-PCR. Fig. 3 shows that the expression levels of NOX5 were much higher in the controls than in the VSD fetuses. The result indicates that there is a significant association between low expression of NOX5 with hypermethylation of the gene in VSD.

Discussion

DNA methylation is the main epigenetic modification in mammals and particularly in humans. The most striking feature of vertebrate DNA methylation patterns is the presence of CpG islands. Earlier studies estimated that ∼60% of human genes are associated with CpG islands (10). Many studies of DNA methylation have been carried out in mammalian systems, in which genomic DNA methylation is found throughout the genome (11). Thus, DNA methylation provides information as to where and when the gene should be expressed, but does not alter the structure or function of a gene (12). DNA methylation patterns are required for normal embryonic development. The DNA methyltransferases DNMT1, DNMT3A and DNMT3B cooperatively regulate cytosine methylation in CpG dinucleotides in mammalian genomes (13); both Dnmt3a and Dnmt3b function as de novo methyltransferases that play important roles in normal development (14). Furthermore, the DNA methylation levels vary throughout the mammalian developmental process (15).

It has been recognized that environmental and genetic factors play important roles in VSD, as in other CHDs. However, recent studies have shown that CHD caused by single gene or single locus defects is more common than expected (16). The interaction between histone de-acetylation and DNA methylation existing between human end-stage cardiomyopathic and heart failure has already been established (17). Most recent studies have focused on the crucial role of the NOX family in cardiac pathophysiology. In this respect, it has been shown that there is increased myocardial NOX family activity in the failing heart (18). Expression of NOX2 has been demonstrated in human cardiomyocytes and was shown to be up-regulated during myocardial infarction (19).

NOX5 was the last discovered gene in the NOX family and it is highly divergent from other members of the family. Evolutionary tree analysis has revealed that NOX5 may represent the gene which is closest to primordial NOX (20), and the gene is unique as it contains EF hand domains in the N-terminal region that bind calcium and permit activation of the enzyme by an increase in intracellular calcium (21). In prior studies, NOX5 expression has been detected within blood vessels of the spleen and lung and also in coronary blood vessels (22,23).

In blood vessels from individuals without coronary artery disease, NOX5 expression is very low, but it is substantially increased in blood vessels of individuals with the disease (24). In this study, we found that NOX5 promoter hypermethylation occurred more often in VSD myocardial tissue by methylation-specific PCR. There was a significant concordance between NOX5 methylation and a decline in its mRNA expression. Thus, the low expression of NOX5 in individuals without coronary artery disease may account for the promoter hypomethylation of the gene. NOX5 promoter hypermethylation in VSD myocardial tissue contributes to transcriptional silencing of the gene. The gene silencing leads to abnormal reduction in ROS production. ROS, as a signaling substance, stimulates the proliferation of mammalian cells. However, the formation of VSD is an extremely complex pathological process, which involves cell proliferation and differentiation during embryogenesis as well as apoptosis (25). NOX5 silencing, which leads to reduction in ROS, may be involved in the pathological process of fetal VSD. On the other hand, in rodents, NOX1 and NOX2 are the primary isoforms expressed in the spleen (26), whereas in humans NOX5 was initially characterized as a gene that is highly expressed in the testis, spleen and lymph nodes; in lymphocytes, this gene may participate in calcium signaling, proliferation, differentiation and apoptosis (21). The immunological profile in CHD children, including levels of IgG and IgA and complement components C3 and C4, was found to be significantly impaired in all children with CHD; T-helper cells were decreased and T-suppressor cells were increased in all groups with CHD as compared to controls (27). The B-cell percentage was increased in cyanotic children, but was not affected in acyanotic children (27). Thus, another possibility is that NOX5 gene silencing in VSD fetuses may result in immune dysfunction and immunoregulatory disorders.

In summary, hypermethylation of the NOX5 promoter was detected in 66.67% of the VSD fetuses using methylation-specific PCR. Our findings indicate that a high frequency of methylation of the NOX5 gene promoter is an important mechanism for NOX5 inactivation in VSD and normal fetuses.

Acknowledgements

This study was supported by grants from the National Natural Science Foundation of China (nos. 30973213, 81070500 and 81070138).

References

1. 

Hoffman JI and Kaplan S: The incidence of congenital heart disease. J Am Coll Cardiol. 39:1890–1900. 2002. View Article : Google Scholar : PubMed/NCBI

2. 

Wenxiang D: Status of paediatric cardiac surgery in China. Heart Lung Circ. 10:16–19. 2001. View Article : Google Scholar

3. 

Suh YA, Arnold RS, Lassegue B, Shi J, Xu X, Sorescu D, Chung AB, Griendling KK and Lambeth JD: Cell transformation by the superoxide-generating oxidase Mox1. Nature. 401:79–82. 1999. View Article : Google Scholar : PubMed/NCBI

4. 

Piao YJ, Seo YH, Hong F, Kim JH, Kim YJ, Kang MH, Kim BS, Jo SA, Jo I, Jue DM, Kang I, Ha J and Kim SS: NOX2 stimulates muscle differentiation via NF-κB/iNOS pathway. Free Radic Biol Med. 38:989–1001. 2005.PubMed/NCBI

5. 

Pedruzzi E, Guichard C, Ollivier V, et al: NAD(P)H oxidase NOX-4 mediates 7-ketocholesterol-induced endoplasmic reticulum stress and apoptosis in human aortic smooth muscle cells. Mol Cell Biol. 24:10703–10717. 2004. View Article : Google Scholar : PubMed/NCBI

6. 

Van Rooij E and Olson EN: MicroRNAs: powerful new regulators of heart disease and provocative therapeutic targets. J Clin Invest. 117:2369–2376. 2007.PubMed/NCBI

7. 

Bird A: DNA methylation pattern and epigenetic memory. Genes Dev. 16:6–21. 2002. View Article : Google Scholar

8. 

Zhu C, Yu ZB, Chen XH, Pan Y, Dong XY, Qian LM and Han SP: Screening for differential methylation status in fetal myocardial tissue samples with ventricular septal defects by promoter methylation microarrays. Mol Med Rep. 4:137–143. 2011.

9. 

Herman JG, Graff JR, Myohanen S, Nelkin BD and Baylin SB: Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA. 93:9821–9826. 1996. View Article : Google Scholar : PubMed/NCBI

10. 

Antequera F and Bird A: Number of CpG islands and genes in human and mouse. Proc Natl Acad Sci USA. 90:11995–11999. 1993. View Article : Google Scholar : PubMed/NCBI

11. 

Suzuki MM and Bird A: DNA methylation landscapes: provocative insights from epigenomics. Nat Rev Genet. 9:465–476. 2008. View Article : Google Scholar : PubMed/NCBI

12. 

Dal C and Guldberyg P: DNA methylation analysis techniques. Biogerontology. 4:233–250. 2003. View Article : Google Scholar

13. 

Tsumura A, Hayakawa T, Kumaki Y, Takebayashi S, Sakaue M, Matsuoka C, Shimotohno K, Ishikawa F, Li E, Ueda HR, Nakayama J and Okano M: Maintenance of self-renewal ability of mouse embryonic stem cells in the absence of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b. Genes Cells. 11:805–814. 2006. View Article : Google Scholar : PubMed/NCBI

14. 

Okano M, Bell DW and Haber DA: DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell. 99:247–257. 1999. View Article : Google Scholar : PubMed/NCBI

15. 

Mayer W, Niveleau A, Walter J, Fundele R and Haaf T: Embryogenesis-demethylation of the zygotic paternal genome. Nature. 403:501–502. 2000. View Article : Google Scholar : PubMed/NCBI

16. 

Burn J, Brennan P, Little J, et al: Recurrence risks in offspring of adults with major heart defects: results from first cohort of British collaborative study. Lancet. 351:311–316. 1998. View Article : Google Scholar : PubMed/NCBI

17. 

Mehregan M, Choy MK, Goddard M, Bennett MR, Down TA and Foo RS: Differential DNA methylation correlates with differential expression of angiogenic factors in human heart failure. Plos One. 5:e85642010. View Article : Google Scholar : PubMed/NCBI

18. 

Heymes C, Bendall JK, Ratajczak P, Cave AC, Samuel JL, Hasenfuss G and Shah AM: Increased myocardial NADPH oxidase activity in human heart failure. J Am Coll Cardiol. 41:2164–2171. 2003. View Article : Google Scholar : PubMed/NCBI

19. 

Krijnen PA, Meischl C, Hack CE, Meijer CJ, Visser CA, Roos D and Niessen HW: Increased Nox2 expression in human cardiomyocytes after acute myocardial infarction. J Clin Pathol. 56:194–199. 2003. View Article : Google Scholar : PubMed/NCBI

20. 

Cheng G, Cao Z and Xu X: Homologs of gp91phox: cloning and tissue expression of NOX3, NOX4, and NOX5. Gene. 269:131–140. 2001. View Article : Google Scholar : PubMed/NCBI

21. 

Bánfi B, Molnár G, Maturana A, Steger K, Hegedûs B, Demaurex N and Krause KH: A Ca(2+)-activated NADPH oxidase in testis, spleen, and lymph nodes. J Biol Chem. 276:37594–37601. 2001.

22. 

BelAiba RS, Djordjevic T, Petry A, Diemer K, Bonello S, Banfi B, Hess J, Pogrebniak A, Bickel C and Görlach A: NOX5 variants are functionally active in endothelial cells. Free Radic Biol Med. 42:446–459. 2007. View Article : Google Scholar : PubMed/NCBI

23. 

Guzik TJ, Chen W, Gongora MC, Guzik B, Lob HE, Mangalat D, Hoch N, Dikalov S, Rudzinski P, Kapelak B, Sadowski J and Harrison DG: Calcium-dependent NOX5 nicotinamide adenine dinucleotide phosphate oxidase contributes to vascular oxidative stress in human coronary artery disease. J Am Coll Cardiol. 52:1803–1809. 2008. View Article : Google Scholar

24. 

Fulton DJ: Nox5 and the regulation of cellular function. Antioxid Redox Signal. 11:2443–2452. 2009. View Article : Google Scholar : PubMed/NCBI

25. 

Kaynak B, von Heydebreck A, Mebus S, Seelow D, Hennig S, Vogel J, Sperling HP, Pregla R, Alexi-Meskishvili V, Hetzer R, Lange PE, Vingron M, Lehrach H and Sperling S: Genome-wide array analysis of normal and malformed human hearts. Circulation. 107:2467–2474. 2003. View Article : Google Scholar : PubMed/NCBI

26. 

Maru Y, Nishino T and Kakinuma K: Expression of Nox genes in rat organs, mouse oocytes, and sea urchin eggs. DNA Seq. 16:83–88. 2005.PubMed/NCBI

27. 

Khalil A, Trehan R and Tiwari A: Immunological profile in congenital heart disease. Indian Pediatr. 31:295–300. 1994.

Related Articles

Journal Cover

September-October 2011
Volume 2 Issue 5

Print ISSN: 1792-0981
Online ISSN:1792-1015

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Zhu C, Yu Z, Chen X, Ji C, Qian L and Han S: DNA hypermethylation of the NOX5 gene in fetal ventricular septal defect. Exp Ther Med 2: 1011-1015, 2011
APA
Zhu, C., Yu, Z., Chen, X., Ji, C., Qian, L., & Han, S. (2011). DNA hypermethylation of the NOX5 gene in fetal ventricular septal defect. Experimental and Therapeutic Medicine, 2, 1011-1015. https://doi.org/10.3892/etm.2011.294
MLA
Zhu, C., Yu, Z., Chen, X., Ji, C., Qian, L., Han, S."DNA hypermethylation of the NOX5 gene in fetal ventricular septal defect". Experimental and Therapeutic Medicine 2.5 (2011): 1011-1015.
Chicago
Zhu, C., Yu, Z., Chen, X., Ji, C., Qian, L., Han, S."DNA hypermethylation of the NOX5 gene in fetal ventricular septal defect". Experimental and Therapeutic Medicine 2, no. 5 (2011): 1011-1015. https://doi.org/10.3892/etm.2011.294