Volume 33 Issue 9
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YIN Yi Hong, HU Wei, ZHU Zheng Yu, HAN Min, XIE Zhao Hong, BI Jian Zhong. The SOST (Sclerostin) Gene +219 Site Methylation is the Risk Factor of Acute Cerebral Ischemia in the Han Chinese Population: A Case-control Study[J]. Biomedical and Environmental Sciences, 2020, 33(9): 708-712. doi: 10.3967/bes2020.092
Citation: YIN Yi Hong, HU Wei, ZHU Zheng Yu, HAN Min, XIE Zhao Hong, BI Jian Zhong. The SOST (Sclerostin) Gene +219 Site Methylation is the Risk Factor of Acute Cerebral Ischemia in the Han Chinese Population: A Case-control Study[J]. Biomedical and Environmental Sciences, 2020, 33(9): 708-712. doi: 10.3967/bes2020.092

The SOST (Sclerostin) Gene +219 Site Methylation is the Risk Factor of Acute Cerebral Ischemia in the Han Chinese Population: A Case-control Study

doi: 10.3967/bes2020.092
Funds:  The study was supported by The Fundamental Research Funds of Shandong University [NO: 2016JC022]
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  • Author Bio:

    YIN Yi hong, born in 1972, MM, deputy Chief Physician, majoring in cerebrovascular disease, cognitive impairment, and dystonia

    HU Wei, born in 1978, MD, deputy Chief Physician, majoring in vascular arteriosclerosis and vascular intervention

  • Corresponding author: XIE Zhao Hong, Tel: 86-15153169802, E-mail: xie_zhaohong@126.com; BI Jian Zhong, Tel: 86-1515317811, E-mail: bjz@sdu.edu.cn
  • Received Date: 2020-03-05
  • Accepted Date: 2020-06-18
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  • [1] Lecoffre C, de Peretti C, Gabet A, et al. National Trends in Patients Hospitalized for Stroke and Stroke Mortality in France, 2008 to 2014. Stroke, 2017; 48, 2939−45. doi:  10.1161/STROKEAHA.117.017640
    [2] Honghong Wang HZ. The summaries of ischemic cerebrovascular disease. Chin J Clin, 2011; 39, 7−10. (In Chinese)
    [3] Hiltunen MO, Ylä-Herttuala S. DNA methylation, smooth muscle cells, and atherogenesis. Arterioscler Thromb Vasc Biol, 2003; 23, 1750−3.
    [4] Tsaousi A, Mill C, George SJ. The Wnt pathways in vascular disease: lessons from vascular development. Curr Opin Lipidol, 2011; 22, 350−7. doi:  10.1097/MOL.0b013e32834aa701
    [5] Register TC, Hruska KA, Divers J, et al. Sclerostin is positively associated with bone mineral density in men and women and negatively associated with carotid calcified atherosclerotic plaque in men from the African American-Diabetes Heart Study. J Clin Endocrinol Metab, 2014; 99, 315−21. doi:  10.1210/jc.2013-3168
    [6] Krishna SM, Seto SW, Jose RJ, et al. Wnt Signaling Pathway Inhibitor Sclerostin Inhibits Angiotensin II-Induced Aortic Aneurysm and Atherosclerosis. Arterioscler Thromb Vasc Biol, 2017; 37, 553. doi:  10.1161/ATVBAHA.116.308723
    [7] Sacco RL, Kasner SE, Broderick JP, et al. An Updated Definition of Stroke for the 21st Century A Statement for Healthcare Professionals From the American Heart Association/American Stroke Association. Stroke, 2013; 44, 2064−89. doi:  10.1161/STR.0b013e318296aeca
    [8] Liu ZH, Song-Bin QU. Estrogen and Ischemic Cerebrovascular Disease. Progress in Modern Biomedicine, 2012; 12, 195.
    [9] Ying-Ying X U, Xiao-Yan Z, Zhao-Hong X, et al. Study of Relationship Between DNA Methylation of Estrogen Receptor-α Gene and Ischemic Stroke. Chinese Journal of Arteriosclerosis, 2015; 23, 1021−5. (In Chinese)
    [10] Reppe S, Noer A, Grimholt RM, et al. Methylation of bone SOST, its mRNA, and serum sclerostin levels correlate strongly with fracture risk in postmenopausal women. J Bone Miner Res, 2015; 30, 249−56. doi:  10.1002/jbmr.2342
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The SOST (Sclerostin) Gene +219 Site Methylation is the Risk Factor of Acute Cerebral Ischemia in the Han Chinese Population: A Case-control Study

doi: 10.3967/bes2020.092
Funds:  The study was supported by The Fundamental Research Funds of Shandong University [NO: 2016JC022]
YIN Yi Hong, HU Wei, ZHU Zheng Yu, HAN Min, XIE Zhao Hong, BI Jian Zhong. The SOST (Sclerostin) Gene +219 Site Methylation is the Risk Factor of Acute Cerebral Ischemia in the Han Chinese Population: A Case-control Study[J]. Biomedical and Environmental Sciences, 2020, 33(9): 708-712. doi: 10.3967/bes2020.092
Citation: YIN Yi Hong, HU Wei, ZHU Zheng Yu, HAN Min, XIE Zhao Hong, BI Jian Zhong. The SOST (Sclerostin) Gene +219 Site Methylation is the Risk Factor of Acute Cerebral Ischemia in the Han Chinese Population: A Case-control Study[J]. Biomedical and Environmental Sciences, 2020, 33(9): 708-712. doi: 10.3967/bes2020.092
  • Cerebrovascular disease has been the leading cause of mortality in the world[1]. In China alone, 1.5–2 million new patients are diagnosed with cerebrovascular disease ever year, and 80%–85% of them suffer from acute cerebral ischemia[2]. The acute cerebral ischemia can lead to anoxia and ischemia of brain tissue and subsequent necrosis or encephalomalacia, resulting in focal or global cerebral injury. At present, the treatment differs by patients and mortality remains to be high. Hence, further study on the molecular mechanism of the disease is essential to further improve the prognosis of the patients.

    Studies have shown that DNA methylation can lead to the occurring of arteriosclerosis, and results in subsequent morbidity by regulating the arteriosclerosis gene expression[3].

    In addition, it has been found that Wnt/β-catenin pathway, regulated by factors, such as Skip, SFRPs (secreted frizzled related proteins) and SOST (Sclerostin), plays an important role in angiogenesi[4]. Register et al.[5] discovered that the level of SOST is negatively correlated with the level of carotid artery plaque calcification. In addition, hypermethylation of SOST DNA promoter occurred in patients with aneurysm of aorta[6], which leads to the aberrant activation of the Wnt/β-catenin pathway, further promotes the occurring of atherosclerosis.

    Whether the methylation of SOST gene is different between patients with acute cerebral ischemic and those without has not been reported. We speculate that abnormal methylation of SOST gene may occur more frequently in stroke patients, which, if true, could indicate an important mechanism of stroke. Here, we studied whether there is methylation of SOST gene promoter (Supplementary Table S1, available in www.besjournal.com) in patients with acute ischemic stroke accompanied by atherosclerosis, and whether there is difference in the level of the methylation (Supplementary Figure S1 and Supplementary Table S2, available in www.besjournal.com), and SOST gene methylation between stroke patients and controls.

    Gene identity: 50964
    (2,000 bp up and downstream in transcription start site):
    1 agagcctgtg ctactggaag gtggcgtgcc ctcctctggc tggtaccatg cagctcccac
    61 tggccctgtg tctcgtctgc ctgctggtac acacagcctt ccgtgtagtg gagggccagg
    121 ggtggcaggc gttcaagaat gatgccacgg aaatcatccc cgagctcgga gagtaccccg
    181 agcctccacc ggagctggag aacaacaaga ccatgaaccg ggcggagaac ggagggcggc
    241 ctccccacca cccctttgag accaaaggta tggggtggag gagagaattc ttagtaaaag
    301 atcctgggga ggttttagaa acttctcttt gggaggcttg gaagactggg gtagacccag
    361 tgaagattgc tggcctctgc cagcactggt cgaggaacag tcttgcctgg aggtggggga
    421 agaatggctc gctggtgcag ccttcaaatt caggtgcaga ggcatgaggc aacagacgct
    481 ggtgagagcc cagggcaggg aggacgctgg ggtggtgagg gtatggcatc agggcatcag
    541 aacaggctca ggggctcaga aaagaaaagg tttcaaagaa tctcctcctg ggaatatagg
    601 agccacgtcc agctgctggt accactggga agggaacaag gtaagggagc ctcccatcca
    661 cagaacagca cctgtggggc accggacact ctatgctggt ggtggctgtc cccaccacac
    721 agacccacat catggaatcc ccaggaggtg aacccccagc tcgaagggga agaaacaggt
    781 tccaggcact cagtaacttg gtagtgagaa gagctgaggt gtgaacctgg tttgatccaa
    841 ctgcaagata gccctggtgt gtgggggggt gtgggggaca gatctccaca aagcagtggg
    901 gaggaaggcc agagaggcac ccctgcagtg tgcattgccc acggcctgcc cagggagctg
    961 gcacttgaag gaatgggagt tttcggcaca gttttagccc ctgacatggg tgcagctgag
    1021 tccaggccct ggaggggaga gcagcatcct ctgtgcagga gtagggacat ctgtcctcag
    1081 cagccacccc agtcccaacc ttgcctcatt ccaggggagg gagaaggaag aggaaccctg
    1141 ggttcctggt caggcctgca cagagaagcc caggtgacag tgtgcatctg gctctataat
    1201 tggcaggaat cctgaggcca tgggggcgtc tgaaatgaca cttcagacta agagcttccc
    1261 tgtcctctgg ccattatcca ggtggcagag aagtccactg cccaggctcc tggaccccag
    1321 ccctccccgc ctcacaacct gttgggacta tggggtgcta aaaagggcaa ctgcatggga
    1381 ggccagccag gaccctccgt cttcaaaatg gaggacaagg gcgcctcccc ccacagctcc
    1441 ccttctaggc aaggtcagct gggctccagc gactgcctga agggctgtaa ggaacccaaa
    1501 cacaaaatgt ccaccttgct ggactcccac gagaggccac agcccctgag gaagccacat
    1561 gctcaaaaca aagtcatgat ctgcagagga agtgcctggc ctaggggcgc tattctcgaa
    1621 aagccgcaaa atgccccctt ccctgggcaa atgcccccct gaccacacac acattccagc
    1681 cctgcagagg tgaggatgca aaccagccca cagaccagaa agcagcccca gacgatggca
    1741 gtggccacat ctcccctgct gtgcttgctc ttcagagtgg gggtgggggg tggccttctc
    1801 tgtcccctct ctggtttggt cttaagacta tttttcattc tttcttgtca cattggaact
    1861 atccccatga aacctttggg ggtggactgg tactcacacg acgaccagct atttaaaaag
    1921 ctcccaccca tctaagtcca ccataggaga catggtcaag gtgtgtgcag gggatcaggc
    1981 caggcctcgg agcccaatct ctgcctgccc agggagtatc accatgaggc gcccattcag
    2041 ataacacaga acaagaaatg tgcccagcag agagccaggt caatgtttgt ggcagctgaa
    2101 cctgtaggtt ttgggtcaga gctcagggcc cctatggtag gaaagtaacg acagtaaaaa
    2161 gcagccctca gctccatccc ccagcccagc ctcccatgga tgctcgaacg cagagcctcc
    2221 actcttgccg gagccaaaag gtgctgggac cccagggaag tggagtccgg agatgcagcc
    2281 cagccttttg ggcaagttct tttctctggc tgggcctcag tattctcatt gataatgagg
    2341 gggttggaca cactgccttt gattcctttc aagtctaatg aattcctgtc ctgatcacct
    2401 ccccttcagt ccctcgcctc cacagcagct gccctgattt attaccttca attaacctct
    2461 actcctttct ccatcccctg tccacccctc ccaagtggct ggaaaaggaa tttgggagaa
    2521 gccagagcca ggcagaaggt gtgctgagta cttaccctgc ccaggccagg gaccctgcgg
    2581 cacaagtgtg gcttaaatca taagaagacc ccagaagaga aatgataata ataatacata
    2641 acagccgacg ctttcagcta tatgtgccaa atggtatttt ctgcattgcg tgtgtaatgg
    2701 attaactcgc aatgcttggg gcggcccatt ttgcagacag gaagaagaga gaggttaagg
    2761 aacttgccca agatgacacc tgcagtgagc gatggagccc tggtgtttga accccagcag
    2821 tcatttggct ccgaggggac agggtgcgca ggagagcttt ccaccagctc tagagcatct
    2881 gggaccttcc tgcaatagat gttcaggggc aaaagcctct ggagacaggc ttggcaaaag
    2941 cagggctggg gtggagagag acgggccggt ccagggcagg ggtggccagg cgggcggcca
    3001 ccctcacgcg cgcctctctc cacagacgtg tccgagtaca gctgccgcga gctgcacttc
    3061 acccgctacg tgaccgatgg gccgtgccgc agcgccaagc cggtcaccga gctggtgtgc
    3121 tccggccagt gcggcccggc gcgcctgctg cccaacgcca tcggccgcgg caagtggtgg
    3181 cgacctagtg ggcccgactt ccgctgcatc cccgaccgct accgcgcgca gcgcgtgcag
    3241 ctgctgtgtc ccggtggtga ggcgccgcgc gcgcgcaagg tgcgcctggt ggcctcgtgc
    3301 aagtgcaagc gcctcacccg cttccacaac cagtcggagc tcaaggactt cgggaccgag
    3361 gccgctcggc cgcagaaggg ccggaagccg cggccccgcg cccggagcgc caaagccaac
    3421 caggccgagc tggagaacgc ctactagagc ccgcccgcgc ccctccccac cggcgggcgc
    3481 cccggccctg aacccgcgcc ccacatttct gtcctctgcg cgtggtttga ttgtttatat
    3541 ttcattgtaa atgcctgcaa cccagggcag ggggctgaga ccttccaggc cctgaggaat
    3601 cccgggcgcc ggcaaggccc ccctcagccc gccagctgag gggtcccacg gggcagggga
    3661 gggaattgag agtcacagac actgagccac gcagccccgc ctctggggcc gcctaccttt
    3721 gctggtccca cttcagagga ggcagaaatg gaagcatttt caccgccctg gggttttaag
    3781 ggagcggtgt gggagtggga aagtccaggg actggttaag aaagttggat aagattcccc
    3841 cttgcacctc gctgcccatc agaaagcctg aggcgtgccc agagcacaag actgggggca
    3901 actgtagatg tggtttctag tcctggctct gccactaact tgctgtgtaa ccttgaacta
    3961 cacaattctc cttcgggacc tcaatttcca ctttgtaaaa tgagggtgga ggtgggaata
    4021 ggatctcgag gagactattg gcatatgatt ccaaggactc cagtgccttt tgaatgggca
      Note. SOST, sclerostin.

    Table S1.  SOST promoter sequencing

    Figure S1.  The CpG island predication of SOST promoter. 2,000 bp in the Upstream and 2,800 bp (including 1st exon) downstream of TSS were include, respectively. From http://itsa.ucsf.edu/~urolab/methprimer. CpG island: a region abundant of C and G nucleic acid base. TSS: transcription start site. SOST, sclerostin.

    No.Size (bp)Start and end
    Island 15763,514–4,089
      Note. SOST, sclerostin. CpG sites: a region abundant of C and G nucleic acid base.

    Table S2.  Predication of cpg sites around the SOST promoter

    Based on our preliminary study results and the estimation of required sample size (using formula of hypothesis testing for mean: (n = [(tα + tβ)2Ơ2]/§2, tα = 1.645, tβ = 1.298; Ơ: standard deviation; §: mean difference), we collected the peripheral blood samples from 41 patients diagnosed with acute ischemic stroke and 41 healthy volunteers matched on the frequency of age and gender for this case-control study. The patients were recruited consecutively from February 2017 to May 2017 in Anhui provincial hospital (China), and the controls were stratified by age and gender and randomly selected from the physical examination center of Anhui Provincial Hospital. All patients and controls were Han nationality and had no kinship with each other.

    The inclusion criteria were: patients who were diagnosed with acute ischemic stroke (confirmed by magnetic resonance imaging)[7], meanwhile, patients had no consanguinity with each other. The exclusion criteria were as follows: patients were receiving anticoagulation or thrombolysis treatment, patients with atrial fibrillation or a history of atrial fibrillation, patients with cranio-cerebral trauma, idiopathic cerebral infarction, a history of MI (myocardial infarction), hemopathy, tissue-damaging infection or took antithrombotics, patients with osteoporosis or who took medications that have an impact on DNA methylation, such as oxytetracycline, dimethyl sulfoxide, curcumin, quinolone. The inclusion criteria of control group were: volunteers who received routine physical examination in heath management center of the same institute. The exclusion criteria were: patients diagnosed with stroke and acute coronary syndrome; patients receiving anticoagulation or thrombolysis treatment, patients with atrial fibrillation or a history of atrial fibrillation.

    Detection of DNA Methylation We first mixed 200 μL blood, 800 μL SDS lysate and 10 μL (20 mg/μL) PK together. The DNA was extracted using Phenol extraction method and Gel electrophoresis was used to detect the DNA product. DNA methylation detection kit (CW2140, CWBIO) was used to detect the methylation of SOST gene. The DNA solution was preserved under −20 ℃.

    PCR Process PCR Amplifier was used to amplify the DNA fragment. The amplification system included 1 μL DNA template, 0.8 μL primers (5 μmol/L), 2 μL 10×PCR Buffer, 1.6 μL Dntp (2.5 mmol/L), 0.2 μL Taq enzyme. DdH2O supplement was added to reach the total volume of 20 μL. The primers [designed using Methprimer (http://www.urogene.org/methprimer/index1.html)] were SOST (Amplicon size: 332 bp): hSOST-Bf: 5’-TGGTATGAAGTAGAGAGGGGTTTTA-3’, hSOST-Br: 5’-CCACCCCATACCTTTAATCTCA-3’. The reaction protocol was 5 min at 95 ℃ activation, 10 cycles of 30 s at 95 ℃, 30 s at 60 ℃ to 50 ℃ Δ1 ℃ and 30 s at 72 ℃. Then 30 Cycles of 30 s at 95 ℃, 30 s at 50 ℃, and 30 s at 72 ℃. 30 min for 60 ℃ at last.

    Sequencing We confirmed the DNA using gel electrophoresis (2% agarose) and purified the DNA with DNA purify and recycle kit (CW0524, CWBIO). We then finished the process of transformation and colony authentication using T vector connection kit (CW0801, CWBIO), selecting the positive samples for sequencing. The results of DNA methylation were obtained at last.

    Statistical Analysis SPSS19.0 was used for statistical analysis. The formula of C/(C+T) *100 (C: cytosine, T: thymine) was adopted to calculate the rate of methylation. Student T test was used to analyze the difference in SOST gene methylation between patients and controls; chi-square test was used to analyze the categorical data. Logistic regression was applied with P < 0.05 defined as being statistically significant. Stratified analysis by gender and status of menopause were also conducted.

    The characteristics of the patients and controls were shown in Table 1. Blood glucose, total cholesterol levels, the percentages of subjects with IMT ≥ 0.9 mm, and subjects with plaque were significantly different between two groups (P = 0.0499, P = 0.0312, P = 0.0000, respectively).

    ItemCerebral ischemic patients (n = 41)Controls (n = 41)P value
    Gender0.506a
     Male (%)24 (58.54)21 (51.22)
     Female (%)17 (41.46)20 (48.78)
    Ages (years)55.71 ± 8.1156.32 ± 5.820.6968b
    Hypertension (n)No (11)No (36)0.000c
    I stage (3)I stage (5)
    II stage (0)II stage (0)
    III stage (27)III stage (0)
    Blood glucose (mmol/L)6.94 ± 3.765.58 ± 1.540.0499b*
    Triglyceride (mmol/L)1.84 ± 1.181.55 ± 1.050.2586b
    Homocysteine (mmol/L)13.17 ± 5.02
    Total cholesterol (mmol/L)4.41 ± 1.215.03 ± 0.950.023b*
    Carotid artery
     Subjects with IMT ≥ 0.9 mm29130.000a*
     Subjects with plaque26150.000a*
      Note. aTwo sides χ2 test; bTwo sides students T test; cMann-Whitney U test; *Indicating a significant difference. IMT, intima-media thickness.

    Table 1.  General characteristics of the patients and controls

    Overall there was no significant difference in SOST DNA methylation between the patients and controls (P = 0.27, Supplementary Table S3 available in www.besjournal.com), neither was any difference when stratified by gender (P = 0.94 for female, Supplementary Table S4 available in www.besjournal.com, P = 0.21 for male, Supplementary Table S5 available in www.besjournal.com).

    NoGenderAge (year)Methylation rate (%)
    Cases
    1M6495.6
    2M5796.3
    3M5496.3
    4M5598.1
    5M5891.9
    6M6196.9
    7M6291.9
    8M4995.0
    9M4595.0
    10M5293.1
    11M6289.4
    12M4491.9
    13M4790.0
    14M5285.6
    15M4396.3
    16M4191.9
    17F4693.1
    18F5091.9
    19F6188.1
    20F6492.5
    21F5389.4
    22F6083.1
    23F6495.6
    24F6086.3
    25F6695.6
    26F4891.9
    27F6092.5
    28F3894.4
    29F4892.5
    30F5793.8
    31F7193.1
    32M7288.7
    33M6193.1
    34M6190.6
    35F5691.2
    36M6597.5
    37M5795.6
    38F5595.0
    39M5589.4
    40M6393.1
    41M4790.6
    Controls
    C1M6496.3
    C2M5688.1
    C3M5478.7
    C4M5388.7
    C5M5993.8
    C6M6093.8
    C7M6294.4
    C8M5091.9
    C9M4691.2
    C10M5293.1
    C11M6293.1
    C12M5094.4
    C13M4678.1
    C14M5097.5
    C15M5085.6
    C16M5191.2
    C17F5194.4
    C18F5086.9
    C19F6190.0
    C20F6293.8
    C21F5398.8
    C22F6094.4
    C23F6495.6
    C24F5782.5
    C25F5896.3
    C26F6490.6
    C27F5295.0
    C28F6293.8
    C29F4395.6
    C30F5087.5
    C31F6493.1
    C32M6496.3
    C33M5793.8
    C34M5692.5
    C35F5786.3
    C36M5894.4
    C37M6392.5
    C38F5890.6
    C39F6191.9
    C40F6393.1
    C41F5683.1
    P value (T Test 2)0.27
      Note. M, male; F, female.

    Table S3.  Methylation rates between groups

    NoGenderAge (year)Methylation rate (%)
    Cases
    1F4693.1
    2F5091.9
    3F6188.1
    4F6492.5
    5F5389.4
    6F6083.1
    7F6495.6
    8F6086.3
    9F6695.6
    10F4891.9
    11F6092.5
    12F3894.4
    13F4892.5
    14F5793.8
    15F7193.1
    16F5691.2
    17F5595.0
    Controls
    C1F5194.4
    C2F5086.9
    C3F6190.0
    C4F6293.8
    C5F5398.8
    C6F6094.4
    C7F6495.6
    C8F5782.5
    C9F5896.3
    C10F6490.6
    C11F5295.0
    C12F6293.8
    C13F4395.6
    C14F5087.5
    C15F6493.1
    C16F5786.3
    C17F5890.6
    C18F6191.9
    C19F6393.1
    C20F5683.1
    P value (T Test 2) 0.94
      Note. F, female.

    Table S4.  Methylation rates in women

    NoGenderAge (year)Methylation rate (%)
    Cases
    1M6495.6
    2M5796.3
    3M5496.3
    4M5598.1
    5M5891.9
    6M6196.9
    7M6291.9
    8M4995.0
    9M4595.0
    10M5293.1
    11M6289.4
    12M4491.9
    13M4790.0
    14M5285.6
    15M4396.3
    16M4191.9
    17M7288.7
    18M6193.1
    19M6190.6
    20M6597.5
    21M5795.6
    22M5589.4
    23M6393.1
    24M4790.6
    Control
    C1M6496.3
    C2M5688.1
    C3M5478.7
    C4M5388.7
    C5M5993.8
    C6M6093.8
    C7M6294.4
    C8M5091.9
    C9M4691.2
    C10M5293.1
    C11M6293.1
    C12M5094.4
    C13M4678.1
    C14M5097.5
    C15M5085.6
    C16M5191.2
    C17M6496.3
    C18M5793.8
    C19M5692.5
    C20M5894.4
    C21M6392.5
    P value (T Test 2)0.21
      Note. M, male.

    Table S5.  Methylation rates in males

    Since most patients also had hypertension, we conducted a subgroup analysis by the status of hypertension. There was a significant difference between the patients and controls (P = 0.00, Table 1) and hypertension was a risk factor of acute cerebral ischemia. Logistic analysis adjusting for ages and gender also did not show significant association between gender or age with the methylation.

  • Next, we found that 16 CpG sites were located in the SOST DNA sequence (260 bp) from −23 to +237, including −23, −19, −12, +25, +74, +102, +130, +148, +161, +167, +179, +190, +219, +223, +230, +237 sites (Before and after the transcription initiation, Supplementary Figure S2). We found that the mean methylation level at +219 CpG site was significantly higher in stroke patients than controls (0.98 vs. 0.94, P = 0.023, Figure 1AB) . In addition, we compared the level of SOST methylation at +219 CpG site between two groups stratified by gender. Interestingly, we found that the mean methylation level +219 CpG site was significant higher for stroke patients in females (0.98 vs. 0.94, P = 0.04, Figure 3B and 3E), but not in males (P = 0.20, Figure 1D and 1F). This indicated that the association between +219 CpG methylation and acute cerebral ischemic stroke might vary by gender.

    Figure S2.  SOST CpG sites analysis and methylation rates of one case. (A) Sequencing of the amplified DNA fragments. sixteen islands were analyzed. (B) Sixteen island methylation rates of 17th patient (Female, 46 years). The whole methylation rate of 17th patient is 93.1% and rate of the +219 site (13th) is 100%. (C) The island methylation rates of 17th control (Female, 51 years). The whole methylation rate of 17th control is 94.4% and rate of the +219 site (13th) is 90.0%. Each rectangle is a CpG site. The numbers under the CpG positions represent the amounts of methylated and unmethylated colons. SOST, sclerostin. CpG island, a region abundant of C and G nucleic acid base. DNA, deoxyribonucleic acid. TSS, transcription start site.

    Figure 1.  Methylation Level of +219 Site. (A)(D) sixteen sites were analyzed in 41 patients and 41 controls. +219 site (*) level in patients is higher than controls. (B)(E) in female (17 patients vs. 20 controls) a significant difference was identified at +219 site. (C)(F) in male (24 patients vs. 21 controls) no significant differences was identified by +219 site. The results are from Students’ Test. n.s, no significant. *P < 0.05, indicated a significant difference.

    Furthermore, we analyzed the level of +219 CpG methylation in menopause female and found that there was no significant difference between patients and controls (Figure 2 and Supplementary Table S6 available in www.besjournal.com).

    Figure 2.  Mean methylation status of +219 sites in female by menopause. In menopause (11 patients vs. 15 controls) no significant difference was identified. Before the menopause (17 patients vs. 20 controls) a significant difference was identified. The results are from Students’ Test. *P < 0.05, indicated a significant difference; n.s: no significant.

    StatusGroupNumberMeanSTDP
    menopauseControl150.9470.0740.367
    Stroke110.9730.065
    non-menopauseControl200.940.6810.04*
    Stroke170.9820.053
      Note. STD, standard deviation. *Significant differences.

    Table S6.  +219 Site methylation levels in menopause

    We found that the difference in methylation level between patients and control were all 0.05 at the +74 site in females and +74/+130 sites in male, which is higher than 0.03 and 0.04 at +219 site between patients and controls in general and female patients and female controls, respectively. However, the differences were not statistically significant (P values are 0.418, 0.194, and 0.202, respectively). (Supplementary Table S7 available in www.besjournal.com)

    GenderSiteGroupNumberMean ± STDDifferenceP value
    female+74
    controls200.745±0.2160.050.418
    patients170.700±0.162
    male+74
    controls200.719±0.1400.050.194
    patients170.771±0.123
    +130
    controls200.876±0.1600.050.202
    patients170.925±0.846
      Note. STD, standard deviation.

    Table S7.  Methylation levels at +74 and +130 sites in female and male

    In our study, we confirmed that methylation rate at +219 site of SOST gene was higher in acute cerebral ischemic patients in this Chinese population. We found a significant difference in methylation level at +219 site of SOST gene between patients with acute cerebral ischemic stroke and controls in female subjects, which might be the most important finding of our study.

    Interestingly, we discovered that the methylation of +219 site was different by gender in patients with cerebral arterial thrombosis. Most of female subjects in the study (26/37, 70.3%) already had menopause, and there was no significant difference between the patients and controls (Supplementary Table S8 available in www.besjournal.com). Limited to those subjects who had menopause, there was no difference in methylation level of +219 site between patients and controls (Figure 2). Therefore, we speculated that the +219 site methylation might be affected by hormone, and estrogen and androgen level might influence the +219 site methylation in patients with cerebral arterial thrombosis. Reports have shown that estrogen has a protective effect on ischemic brain injury, and its neuroprotective mechanisms present in many ways[8]. However, the impact of hormones on the methylation level of individual CpG site remains unclear. These data suggested that decreased estrogen and increased androgen may play a role in the reduced methylation level at +219 site in menopause women.

    GroupMenopauseNon-menopauseSumP*
    Control155200.495
    Stroke11617
    Sum261137
      Note. *Chi-square test.

    Table S8.  Menopause in stroke and control

    In previous study, HF lin[6] found that the methylation levels of MMP-2 (Matrix metalloproteinase-2) in the peripheral blood of the patients with stroke were lower than controls in its eight CpG sites, and also proved that a significant association was found only in men but not in women. That study also reported that the cerebral ischemic stroke was regulated by several genes, and a complex mechanism was present; and interestingly the significant difference between men and women was consistent with what we found in our study. Shan Jiang et al.[9] also showed that ER-α (estrogen receptor-α) gene promoter methylation rate was significant higher in patients with cerebral ischemic stroke than controls. They suggested that gene methylation was associated with cerebral ischemic stroke, but no report has studied the relationship between SOST gene and the disease. In fact, the relationship between SOST DNA methylation and many diseases have been reported in the literature[10], but no study about its association with cerebral ischemic stroke, especially the individual CpG site methylation and stroke, has been published. To explore the relationship between SOST and artery plaque type disease, we studied the association between SOST and cerebral ischemic stroke, and these findings will help further illuminate the mechanism of the disease.

    Moreover, we also found that the differences in methylation level between patients and controls at +74 site in females and +74/+130 sites in males are all 0.05, which is higher than 0.03 and 0.04 at +219 site between patients and controls overall, between female patients and female controls. But the difference was not significant, suggesting large variations in methylation at these sites.

    This study has several limitations: first, our study did not detect the SOST mRNA expression. Secondly, the sub-group analysis (by gender, menopause status) was based on small number of subjects. Finally, since it is a case-control study, the results are only for hypothesis-generating.

    Further studies are needed to confirm the molecular mechanism, especially the impact of hormone level on +219 methylation, and to test the mRNA level. A larger study is needed to explore the methylation levels of +74 and +130 sites in the sub-group subjects.

    Anyhow, we found higher methylation level of +219 CpG site in patients with cerebral ischemia comparing to controls in female subjects. The +219 CpG site partially elucidated the molecular mechanism of acute cerebral ischemic disease, and provided a molecular target for further studying and possibly developing new treatment for patients with acute cerebral ischemia.

    Ethics approval and consent to participate: The study was approved by the ethics committee of Anhui Provincial Hospital (NO. P-012), and all objects signed the informed consent.

    Conflicts of interest  The authors declare that they have no competing interests.

    Availability of data and materials  The datasets used and/or analyzed during the current study are available from the corresponding author.

    Authors’ contributions  YIN Yi Hong designed the protocol, collected samples, analyzed the data, performed the examination, and wrote the manuscript. HU Wei was a major contributor in collecting samples, analyzing the data, and writing the manuscript. ZHU Zheng Yu was a major contributor in enrolling cases in groups. HAN Min was a major contributor in collecting samples and data. XIE Zhao Hong supervised the designing of protocol and collecting of data. BI Jian Zhong supervised the designing of protocol and writing of the manuscript. All authors read and approved the final manuscript.

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