Li et al. BMC Medical Genomics (2020) 13:163 https://doi.org/10.1186/s12920-020-00798-7 RESEARCH ARTICLE Open Access The values of AHCY and CBS promoter methylation on the diagnosis of cerebral infarction in Chinese Han population Xiaodong Li1, Shufang Bu1, Ran Ran Pan2, Cong Zhou2, Kun Qu3, Xiuru Ying2, Jie Zhong2, Jianhao Xiao4, Qian Yuan4, Simiao Zhang4, Laura Tipton5, Yunliang Wang4*, Youping Deng5* and Shiwei Duan2* Abstract Background: The goal of our study is to investigate whether the methylation levels of AHCY and CBS promoters are related to the risk of cerebral infarction by detecting the methylation level of AHCY and CBS genes. Methods: We extracted peripheral venous blood from 152 patients with cerebral infarction and 152 gender- and age-matched healthy controls, and determined methylation levels of AHCY and CBS promoters using quantitative methylation-specific polymerase chain reaction. We used the percentage of methylation reference (PMR) to indicate gene methylation level. Results: We compared the promoter methylation levels of two genes (AHCY and CBS) in peripheral blood DNA between the cerebral infarction case group and the control group. Our study showed no significant difference in AHCY promoter methylation between case and control. Subgroup analysis by gender showed that the methylation level of AHCY in males in the case group was lower than that in the control group, but the difference was not statistically significant in females. In a subgroup analysis by age, there was no significant difference in the AHCY methylation level between the case and control in the young group (≤44 years old). However, the level of AHCY gene methylation in the middle-aged group (45–59 years old) was significantly higher and the aged group (≥60 years old) was significantly lower than that in the control groups. However, CBS promoter methylation levels were significantly lower in the case group than in the control group (median PMR: 70.20% vs 104.10%, P = 3.71E-10). In addition, the CBS methylation levels of males and females in the case group were significantly lower than those in the control group (male: 64.33% vs 105%, P = 2.667E-08; female: 78.05% vs 102.8%, P = 0.003). We also found that the CBS levels in the young (23–44), middle-aged (45–59), and older (60–90) groups were significantly lower than those in the control group (young group: 69.97% vs 114.71%; P = 0.015; middle-aged group: 56.04% vs 91.71%; P = 6.744E-06; older group: 81.6% vs 119.35%; P = 2.644E-04). Our ROC curve analysis of CBS hypomethylation showed (Continued on next page) * Correspondence: [email protected]; [email protected]; [email protected] 4Department of Neurology, the Second Affiliated Hospital, Zhengzhou University, Zhengzhou 450014, Henan, China 5Bioinformatics Core, Department of Complementary and Integrative Medicine and John A. Burns School of Medicine, University of Hawai’i, Honolulu, HI 96822, USA 2School of Medicine, Ningbo University, Ningbo, Zhejiang 315211, China Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Li et al. BMC Medical Genomics (2020) 13:163 Page 2 of 10 (Continued from previous page) an area under the curve of 0.713, a sensitivity of 67.4%, and a specificity of 74.0%. Conclusion: Our study suggests that hypomethylation of the CBS promoter may be closely related to the risk of cerebral infarction and may be used as a non-invasive diagnostic biomarker for cerebral infarction. Keywords: Cerebral infarction, DNA methylation, AHCY, CBS, qMSP Background In addition, a meta-analysis of a prospective cohort Stroke is one of the leading causes of death and disabil- study found that after a mean follow-up of 7.3 years, ity [1], with ischemic stroke (cerebral infarction) ac- serum HHcy was reduced by 3 μmol/L, and stroke risk counting for 60 to 80% of all strokes [2]. Cerebral was reduced by 24% [16]. Ashjazadeh et al. found high infarction is a complex disease that is affected by both serum Hcy associated with stroke in the Iranian popula- environmental risk factors and genetic factors [3, 4]. tion [17]. Hankey et al. found elevated serum Hcy in Common risk factors for cerebral infarction include age, stroke patients in Australia as an independent risk factor hypertension, diabetes, hyperlipidemia, smoking, and for ischemic stroke [18]. Hyperhomocysteinemia (HHcy) atrial fibrillation, etc. [5]. DNA methylation is an epigen- is commonly recognized as an independent risk factor etic modification that links the roles between heredity for stroke [19, 20]. and the environment [6]. The mechanism depends on AHCY encodes S-adenosine homocysteine hydrolase the catalysis of DNA methyltransferase (DNMT), which (SAH), whose function is to catalyze the reversible in turn causes the transfer of methyl groups from s- hydrolysis of SAM to produce adenosine (Ado) and adenosylmethionine (SAM) to specific segments in the Hcy (Fig. 1). SAH may control the number of methyl- DNA sequence, thereby changing the function of the ated donors by regulating the level of intracellular genome [7]. Therefore, the level of SAM is closely re- SAM [21]. Plasma Hcy concentrations may increase lated to DNA methylation. Xiao et al. [8] showed that due to genetic defects in related enzymes [22, 23]. PON1 and PON3 methylation were associated with the AHCY methylation may affect the activity of the en- risk of male cerebral infarction. There are several DNA zyme, causing a metabolic disorder of Hcy, leading to methylation studies related to stroke [9]. High homo- HHcy. However, the association between AHCY cysteine (Hcy) leads to hypermethylation of the TM gene methylation and cerebral infarction has not previously and further induction of TM gene silencing, which may been studied. play an important role in the development and progres- In addition, CBS-encoded cystathionine beta-synthase sion of ischemic brain injury [10]. DNA methylation of (CBS) catalyzes the sulfur metabolism of Hcy (Fig. 1). In CDKN2B may play a potential role in arterial calcifica- vivo, Hcy can be irreversibly catalyzed by CBS to pro- tion in patients with cerebral infarction [11]. People with duce cysteine (Fig. 1). At present, many studies have ex- a lower degree of LINE-1 methylation have a higher risk plored the relationship between CBS mutations and of stroke [12]. DNA methylation inhibitor Zebularine stroke, of which T833C point mutation is the most com- confers neuroprotective effects on the ischemic rat brain mon [24–27]. This mutation replaces threonine with iso- and further supports the hypothesis that DNA methyl- leucine, resulting in the inactivation of CBS, blocking transferase promotes delayed ischemic brain injury [13]. Hcy degradation, and ultimately leading to abnormal ac- In the normal fasting state, the human plasma Hcy cumulation of Hcy (HHcy) in the body [28]. concentration is 5–15 μmol. Hcy, as a sulfur-containing The association between AHCY and CBS promoter amino acid in the human body, can be summarized as: methylation and stroke studies has not been reported so (1) further metabolized to cystathionine and cysteine; (2) far. In this study, we examined the levels of AHCY and re-converted to methionine; (3) as a -Based receptors CBS promoter methylation in peripheral blood from pa- participate in choline metabolism; (4) participate in the tients with cerebral infarction and healthy controls. Our folic acid cycle as an essential substrate. Wald et al. have goal is to assess whether promoter methylation of AHCY shown that a 5 μmol/L increase in plasma Hcy leads to a and CBS can be used as a diagnostic biomarker for the 59% increased risk of cerebral infarction; a decrease in risk of cerebral infarction. Hcy of 3 μmol/L reduces the risk of cerebral infarction by 24% [14]. In 2011, the American Heart Association Methods and the Association for Cerebral Infarction jointly issued Sample collection a primary prevention guideline for cerebral infarction, From October 2016 to April 2017, we selected 152 hos- which increases the risk of ischemic cerebral infarction pitalized patients with cerebral infarction from the 960th by two to three times as plasma Hcy levels increase [15]. Hospital of the People’s Liberation Army. Patients were Li et al. BMC Medical Genomics (2020) 13:163 Page 3 of 10 Fig. 1 Hcy metabolic pathway. SAM: S-adenosylmethionine; SAH: S-adenosine homocysteine; Hcy: homocysteine; CBS: cystathionine beta synthase tested by brain CT or MRI, in strict accordance with the ethics committee. In addition, all subjects provided diagnostic criteria of the Fourth National Cerebrovascu- informed consent. lar Disease Conference [29]. The patients included 112 males and 40 females with an age range of 23 to 88 years and an average age of 60.37 ± 12.02 years.