Skip to main content

Advertisement

SpringerLink
Log in

High CpG island methylation of p16 gene and loss of p16 protein expression associate with the development and progression of tetralogy of Fallot

  • RESEARCH ARTICLE
  • Published:
Journal of Genetics Aims and scope Submit manuscript

Abstract

We examined CpG island methylation in p16 gene and its effect on p16 protein expression in tetralogy of Fallot (ToF) patients to explore its potential implications in the development and progression of ToF. The study subjects consisted of 75 healthy controls and 63 ToF patients recruited at Linyi People’s Hospital between January 2012 and June 2014. The 4 mL of peripheral venous blood of each subject was obtained and saved in ethylene diamine tetraacetic acid (EDTA) tubes. Methylation-specific polymerase chain reaction (MSP) was employed to detect CpG island methylation in p16 promoter region and Western blotting was used to detect p16 expression of all subjects. Real-time fluorescence quantitative polymerase chain reaction (FQ-PCR) was performed to test p16 mRNA expression. The results showed that p16-methylation rates in ToF group were significantly higher than the control group (ToF group, 58.73%; control group, 13.33%; P < 0.001). Remarkably, Western blotting and FQ-PCR results derived from RVOT revealed that p16 protein expression was significantly lower in ToF group compared to the control group (0.76 ± 0.21 versus 2.31 ± 0.35; P < 0.001), and p16 gene expression was also markedly decreased in ToF group (1.212 ± 0.152 versus 1.346 ± 0.191, P < 0.001). Additionally, our analysis suggested that CpG island methylation in p16 promoters in ToF patients was negatively correlated with p16 protein and gene expression (both P < 0.05). Our study reports that high CpG island methylation of p16 gene and loss of p16 protein expression associate with the development and progression of ToF, which may have significant therapeutic applications for ToF.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1
Figure 2
Figure 3
Figure 4

Similar content being viewed by others

References

  • An S., Chen Y., Gao C., Qin B., Du X., Meng F. et al. 2015 Inactivation of INK4a and ARF induces myocardial proliferation and improves cardiac repair following ischemia-reperfusion. Mol. Med. Rep. 12, 5911–5916.

    CAS  PubMed  Google Scholar 

  • Azad M., Kaviani S., Noruzinia M., Mortazavi Y., Mobarra N., Alizadeh S. et al. 2013 Gene expression status and methylation pattern in promoter of P15INK4b and P16INK4a in cord blood CD34 (+ ) stem cells. Iran J. Basic. Med. Sci. 16, 822–828.

    CAS  PubMed  PubMed Central  Google Scholar 

  • Baker D. J., Wijshake T., Tchkonia T., LeBrasseur N. K., Childs B. G., van de Sluis B. et al. 2011 Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders. Nature 479, 232–236.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ball A. J. and Levine F. 2005 Telomere-independent cellular senescence in human fetal cardiomyocytes. Aging Cell 4, 21–30.

    Article  CAS  PubMed  Google Scholar 

  • Bellinger D. C., Rivkin M. J., DeMaso D., Robertson R. L., Stopp C., Dunbar-Masterson C. et al. 2015 Adolescents with tetralogy of Fallot: neuropsychological assessment and structural brain imaging. Cardiol. Young. 25, 338–347.

    Article  PubMed  Google Scholar 

  • Bichell D. P. 2014 Fourth decade after repair of tetralogy of Fallot: taking aim at moving targets. Circulation 130, 1931–1932.

    Article  PubMed  Google Scholar 

  • Bodoor K., Haddad Y., Alkhateeb A., Al-Abbadi A., Dowairi M., Magableh A. et al. 2014 DNA hypermethylation of cell cycle (p15 and p16) and apoptotic (p14, p53, DAPK and TMS1) genes in peripheral blood of leukemia patients. Asian Pac. J. Cancer Prev. 15, 75–84.

    Article  PubMed  Google Scholar 

  • Cao J., Zhou J., Gao Y., Gu L., Meng H., Liu H. et al. 2009 Methylation of p16 CpG island associated with malignant progression of oral epithelial dysplasia: a prospective cohort study. Clin. Cancer Res. 15, 5178–5183.

    Article  CAS  PubMed  Google Scholar 

  • Celebiler Cavusoglu A., Sevinc A. I., Saydam S., Canda T., Baskan Z., Kilic Y. et al. 2010 Promoter methylation and expression changes of CDH1 and P16 genes in invasive breast cancer and adjacent normal breast tissue. Neoplasma 57, 465–472.

    Article  CAS  PubMed  Google Scholar 

  • Dansranjavin T., Krehl S., Mueller T., Mueller L. P., Schmoll H. J. and Dammann R. H. 2009 The role of promoter CpG methylation in the epigenetic control of stem cell related genes during differentiation. Cell Cycle 8, 916–924.

    Article  CAS  PubMed  Google Scholar 

  • Dennis M., Laarkson M., Padang R., Tanous D. J., Robinson P., Pressley L. et al. 2014 Long term followup of aortic root size after repair of tetralogy of Fallot. Int. J. Cardiol. 177, 136–138.

    Article  PubMed  Google Scholar 

  • Doza J. P., Topf A., Bentham J., Bhattacharya S., Cosgrove C., Brook J. D. et al. 2013 Low-frequency intermediate penetrance variants in the ROCK1 gene predispose to tetralogy of Fallot. BMC Genet. 14, 57.

    Article  CAS  Google Scholar 

  • Feng Y. Z. L., Hong L., Shan C., Shi W. and Cai W. 2013 Alteration in methylation pattern of gata-4 promoter region in vitamin A-deficient offspring’s heart. J. Nutr. Biochem. 24, 1373–1380.

    Article  CAS  PubMed  Google Scholar 

  • Fujiwara-Igarashi A., Goto-Koshino Y., Mochizuki H., Sato M., Fujino Y., Ohno K. et al. 2014 Inhibition of p16 tumor suppressor gene expression via promoter hypermethylation in canine lymphoid tumor cells. Res. Vet. Sci. 97, 60–63.

    Article  CAS  PubMed  Google Scholar 

  • Fujiwara S., Noguchi T., Takeno S., Kimura Y., Fumoto S. and Kawahara K. 2008 Hypermethylation of p16 gene promoter correlates with loss of p16 expression that results in poorer prognosis in esophageal squamous cell carcinomas. Dis. Esophagus 21, 125–131.

    Article  CAS  PubMed  Google Scholar 

  • Golubnitschaja O., Moenkemann H., Kim K. and Mozaffari M. S. 2003 DNA damage and expression of checkpoint genes p21 (WAF1/CIP1) and 14-3-3 sigma in taurine-deficient cardiomyocytes. Biochem. Pharmacol. 66, 511–517.

    Article  CAS  PubMed  Google Scholar 

  • Gonzalez A., Rota M., Nurzynska D., Misao Y., Tillmanns J., Ojaimi C. et al. 2008 Activation of cardiac progenitor cells reverses the failing heart senescent phenotype and prolongs lifespan. Circ. Res. 102, 597–606.

    Article  CAS  PubMed  Google Scholar 

  • Goodship J. A., Hall D., Topf A., Mamasoula C., Griffin H., Rahman T. J. et al. 2012 A common variant in the PTPN11 gene contributes to the risk of tetralogy of Fallot. Circ. Cardiovasc. Genet. 5, 287–292.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Goto T., Mizukami H., Shirahata A., Sakata M., Saito M., Ishibashi K. et al. 2009 Aberrant methylation of the p16 gene is frequently detected in advanced colorectal cancer. Anticancer Res. 29, 275–277.

    CAS  PubMed  Google Scholar 

  • Jablonowski Z., Reszka E., Gromadzinska J., Wasowicz W. and Sosnowski M. 2011 Hypermethylation of p16 and DAPK promoter gene regions in patients with non-invasive urinary bladder cancer. Arch. Med. Sci. 7, 512–516.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kirsch R. E., Glatz A. C., Gaynor J. W., Nicolson S. C., Spray T. L., Wernovsky G. et al. 2014 Results of elective repair at 6 months or younger in 277 patients with tetralogy of Fallot: a 14-year experience at a single center. J. Thorac. Cardiovasc. Surg. 147, 713–717.

    Article  PubMed  Google Scholar 

  • Kondo K., Takahashi Y., Hirose Y., Nagao T., Tsuyuguchi M., Hashimoto M. et al. 2006 The reduced expression and aberrant methylation of p16(INK4a) in chromate workers with lung cancer. Lung Cancer 53, 295–302.

    Article  PubMed  Google Scholar 

  • Liu Y. and Sharpless N. E. 2009 Tumor suppressor mechanisms in immune aging. Curr. Opin. Immunol. 21, 431–439.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Luca A. D., Sarkozy A., Ferese R., Consoli F., Lepri F., Dentici M. L. et al. 2011 New mutations in ZFPM2/FOG2 gene in tetralogy of Fallot and double outlet right ventricle. Clin. Genet. 80, 184–190.

    Article  PubMed  Google Scholar 

  • Majid S., Kikuno N., Nelles J., Noonan E., Tanaka Y., Kawamoto K. et al. 2008 Genistein induces the p21WAF1/CIP1 and p16INK4a tumor suppressor genes in prostate cancer cells by epigenetic mechanisms involving active chromatin modification. Cancer Res. 68, 2736–2744.

    Article  CAS  PubMed  Google Scholar 

  • Naeem N., Haneef K., Kabir N., Iqbal H., Jamall S. and Salim A. 2013 DNA methylation inhibitors, 5-azacytidine and zebularine potentiate the transdifferentiation of rat bone marrow mesenchymal stem cells into cardiomyocytes. Cardiovasc. Ther. 31, 201–209.

    Article  CAS  PubMed  Google Scholar 

  • Nosho K., Irahara N., Shima K., Kure S., Kirkner G. J., Schernhammer E. S. et al. 2008 Comprehensive biostatistical analysis of CpG island methylator phenotype in colorectal cancer using a large population-based sample. PLoS One 3, e3698.

    Article  PubMed  PubMed Central  Google Scholar 

  • Nozato T., Ito H., Watanabe M., Ono Y., Adachi S., Tanaka H. et al. 2001 Overexpression of cdk inhibitor p16INK4a by adenovirus vector inhibits cardiac hypertrophy in vitro and in vivo: a novel strategy for the gene therapy of cardiac hypertrophy. J. Mol. Cell Cardiol. 33, 1493–1504.

    Article  CAS  PubMed  Google Scholar 

  • Olaru A. V., Cheng Y., Agarwal R., Yang J., David S., Abraham J. M. et al. 2012 Unique patterns of CpG island methylation in inflammatory bowel disease-associated colorectal cancers. Inflamm. Bowel Dis. 18, 641–648.

    Article  PubMed  Google Scholar 

  • Piepkorn M. 2000 Melanoma genetics: an update with focus on the CDKN2A(p16)/ARF tumor suppressors. J. Am. Acad. Dermatol. 42, 705–722; quiz 723–706.

    Article  CAS  PubMed  Google Scholar 

  • Samowitz W. S., Albertsen H., Herrick J., Levin T. R., Sweeney C., Murtaugh M. A. et al. 2005 Evaluation of a large, population-based sample supports a CpG island methylator phenotype in colon cancer. Gastroenterology 129, 837–845.

    Article  CAS  PubMed  Google Scholar 

  • Schwerzmann M., Samman A. M., Salehian O., Holm J., Provost Y., Webb G. D. et al. 2007 Comparison of echocardiographic and cardiac magnetic resonance imaging for assessing right ventricular function in adults with repaired tetralogy of Fallot. Am. J. Cardiol. 99, 1593–1597.

    Article  PubMed  Google Scholar 

  • Shao L., Li H., Pazhanisamy S. K., Meng A., Wang Y. and Zhou D. 2011 Reactive oxygen species and hematopoietic stem cell senescence. Int. J. Hematol. 94, 24–32.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sheng W., Wang H., Ma X., Qian Y., Zhang P., Wu Y. et al. 2012 LINE-1 methylation status and its association with tetralogy of Fallot in infants. BMC Med. Genomics. 5, 20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sheng W., Qian Y., Wang H., Ma X., Zhang P., Chen L. et al. 2013 Association between mRNA levels of DNMT1, DNMT3A, DNMT3B, MBD2 and LINE-1 methylation status in infants with tetralogy of Fallot. Int. J. Mol. Med. 32, 694–702.

    CAS  PubMed  Google Scholar 

  • Shimoda N., Izawa T., Yoshizawa A., Yokoi H., Kikuchi Y. and Hashimoto N. 2014 Decrease in cytosine methylation at CpG island shores and increase in DNA fragmentation during zebrafish aging. Age (Dordr) 36, 103–115.

    Article  CAS  Google Scholar 

  • Shin K. C., Lee K. H., Lee C. H., Shin I. H., Suh H. S. and Jeon C. H. 2012 MAGE A1-A6 RT-PCR and MAGE A3 and p16 methylation analysis in induced sputum from patients with lung cancer and non-malignant lung diseases. Oncol. Rep. 27, 911– 916.

    CAS  PubMed  Google Scholar 

  • Torella D., Rota M., Nurzynska D., Musso E., Monsen A., Shiraishi I. et al. 2004 Cardiac stem cell and myocyte aging, heart failure, and insulin-like growth factor-1 overexpression. Circ. Res. 94, 514–524.

    Article  CAS  PubMed  Google Scholar 

  • Wachter E., Quante T., Merusi C., Arczewska A., Stewart F., Webb S. et al. 2014 Synthetic CpG islands reveal DNA sequence determinants of chromatin structure. Elife 3, e03397.

    Article  PubMed  PubMed Central  Google Scholar 

  • Wang H. L., Zhou P. Y., Liu P. and Zhang Y. 2014 Role of p16 gene promoter methylation in gastric carcinogenesis: a meta-analysis. Mol. Biol. Rep. 41, 4481–4492.

    Article  CAS  PubMed  Google Scholar 

  • Weinberg C. R. and McElhinney D. B. 2014 Pulmonary valve replacement in tetralogy of Fallot. Circulation 130, 795–798.

    Article  PubMed  Google Scholar 

  • Wolstein J. M., Lee D. H., Michaud J., Buot V., Stefanchik B. and Plotkin M. D. 2010 INK4a knockout mice exhibit increased fibrosis under normal conditions and in response to unilateral ureteral obstruction. Am. J. Physiol. Renal. Physiol. 299, F1486– F1495.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Xu M. W. X., Li Y., Yang X., Hu J., Zheng M. et al. 2014 CITED 2 mutation and methylation in children with congenital heart disease. J. Biomed. Sci. 21, 7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yang C., Gu L. and Deng D. 2014 Distinct susceptibility of induction of methylation of p16ink4a and p19arf CpG islands by X-radiation and chemical carcinogen in mice. Mutat. Res. Genet. Toxicol. Environ. Mutagen. 768, 42–50.

    Article  CAS  PubMed  Google Scholar 

  • Yuan Y. G. Y., Wang H., Ma X., Ma D. and Huang G. 2014 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. 100, 973–984.

    Article  CAS  PubMed  Google Scholar 

  • Zhang Y., Wang R., Song H., Huang G., Yi J., Zheng Y. et al. 2011 Methylation of multiple genes as a candidate biomarker in non-small cell lung cancer. Cancer Lett. 303, 21–28.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We are grateful to our instructors for their valuable advice. We also thank the reviewers for their precious comments on this paper.

Author information

Authors and Affiliations

  1. Department of Pediatrics, Linyi People’s Hospital, No. 27, Jiefang Dong Road, Linyi, 276003, People’s Republic of China

    SI-JU GAO, GUI-FANG ZHANG & RONG-PENG ZHANG

Authors
  1. SI-JU GAO
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. GUI-FANG ZHANG
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. RONG-PENG ZHANG
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to RONG-PENG ZHANG.

Additional information

Corresponding editor: Arun Kumar

[Gao S.-J., Zhang G.-F. and Zhang R.-P. 2016 High CpG island methylation of p16 gene and loss of p16 protein expression associate with the development and progression of tetralogy of Fallot. J. Genet. 95, xx–xx]

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

GAO, SJ., ZHANG, GF. & ZHANG, RP. High CpG island methylation of p16 gene and loss of p16 protein expression associate with the development and progression of tetralogy of Fallot. J Genet 95, 831–837 (2016). https://doi.org/10.1007/s12041-016-0697-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12041-016-0697-z

Keywords

Search

Navigation