亞甲基四氫葉酸還原酶C677T基因多態(tài)性與H型高血壓的關(guān)系

張瑩瑩，梁蓉，郭緒昆

(天津市胸科醫(yī)院，天津300222)

亞甲基四氫葉酸還原酶C677T基因多態(tài)性與H型高血壓的關(guān)系

張瑩瑩，梁蓉，郭緒昆

(天津市胸科醫(yī)院，天津300222)

目的探討亞甲基四氫葉酸還原酶(MTHFR)C677T基因多態(tài)性與H型高血壓的關(guān)系。方法273例高血壓患者，其中H型高血壓(血漿Hcy≥10 μmol/L)185例(觀察組)、非H型高血壓88例(對(duì)照組)。取兩組空腹靜脈血，用PCR-RFLP方法觀察MTHFR C677T基因多態(tài)性。采用logistic回歸分析MTHFR C677T基因型與H型高血壓的關(guān)系。結(jié)果觀察組、對(duì)照組MTHFR C677T基因型CC分別為31、35例，CT分別為104、45例，TT分別為50、8例，C等位基因分別為166、115例，T等位基因分別為204、61例，兩組CC、TT基因型及C、T等位基因分布比較P均<0.01。logistic回歸分析結(jié)果顯示，MTHFR C677T基因型突變(OR=2.645，95%CI：1.737～4.025，P<0.01)是H型高血壓的獨(dú)立危險(xiǎn)因素。結(jié)論MTHFR C677T基因型TT是H型高血壓發(fā)病的獨(dú)立危險(xiǎn)因素。

H型高血壓；亞甲基四氫葉酸還原酶；同型半胱氨酸；基因多態(tài)性

高血壓為遺傳及環(huán)境因素共同作用致病，發(fā)病機(jī)制相當(dāng)復(fù)雜。高同型半胱氨酸血癥(HHcy)是高血壓、動(dòng)脈粥樣硬化的重要危險(xiǎn)因素[1]。伴有血漿同型半胱氨酸(Hcy)水平升高的原發(fā)性高血壓，定義為H型高血壓。我國(guó)有2億高血壓患者，H型高血壓患者高達(dá)75%[2]。H型高血壓患者心腦血管事件發(fā)生率約為單純高血壓患者的5倍，為正常人的25～30倍[3]。亞甲基四氫葉酸還原酶(MTHFR)為Hcy代謝過(guò)程的關(guān)鍵酶，編碼該酶的基因位點(diǎn)突變常導(dǎo)致血漿Hcy水平增高。目前，國(guó)內(nèi)外研究對(duì)MTHFR C677T基因多態(tài)性與HHcy及高血壓的相關(guān)性尚存在一定爭(zhēng)議[4～8]。本研究旨在探討MTHFR C677T基因多態(tài)性與H型高血壓發(fā)病的關(guān)系。

1 資料與方法

1.1 臨床資料 選取2016年8～10月在天津市胸科醫(yī)院門(mén)診就診的高血壓患者273例，其中H型高血壓(血漿Hcy≥10 μmol/L)185例(觀察組)、非H型高血壓88例(對(duì)照組)。高血壓診斷標(biāo)準(zhǔn)為收縮壓≥140 mmHg和(或)舒張壓>90 mmHg。排除標(biāo)準(zhǔn)：肝、腎功能不全；自身免疫性疾病；繼發(fā)性高血壓；近3個(gè)月使用過(guò)影響Hcy藥物，如葉酸、甲氨蝶呤、維生素B6、維生素B1等。觀察組男107例、女78例，年齡(63.0±8.0)歲，BMI(25.4±4.5)kg/m2，收縮壓(153.0±10.0)mmHg，舒張壓(93.5±11.0)mmHg，吸煙103例，糖尿病48例，LDL-C(2.94±1.03)mmol/L，尿酸(330.7±89.6)μmol/L，膽固醇(4.46±1.15)mmol/L，甘油三酯(1.71±1.34)mmol/L。對(duì)照組男46例、女42例，年齡(61.2±7.6)歲，BMI(24.7±5.7)kg/m2，收縮壓(120.0±11.0)mmHg，舒張壓(78.8±8.0)mmHg，吸煙45例，糖尿病30例，LDL-C(2.86±0.80)mmol/L，尿酸(294.1±72.8)μmol/L，膽固醇(4.51±0.87)mmol/L，甘油三酯(1.77±1.31)mmol/L。兩組收縮壓、舒張壓、尿酸水平比較P均<0.01。本研究經(jīng)醫(yī)院倫理委員會(huì)批準(zhǔn)，所有研究對(duì)象簽署知情同意書(shū)。

1.2 MTHFR C677T基因多態(tài)性分析 取兩組空腹靜脈血5 mL，乙二胺四乙酸(EDTA)抗凝，采用百泰克離心柱法DP5802提取全血基因組DNA。采用PCR-RFLP方法檢測(cè)MTHFR C677T基因多態(tài)性。MTHFR基因C677T引物序列中正義鏈為5′-CCTTGAACAGGTGGAGGCC-3′，反義鏈為5′-CAAAGAAAAGCTGCGTGAT-3′。PCR反應(yīng)條件：94 ℃預(yù)變性8 min，94 ℃ 60 s，62 ℃ 60 s，72 ℃ 60 s，共35個(gè)循環(huán)，最后于72 ℃再延伸7 min。反應(yīng)結(jié)束，MTHFR基因擴(kuò)增產(chǎn)物經(jīng)限制性內(nèi)切酶Hinf酶切(大連寶生物工程有限公司)，酶切產(chǎn)物經(jīng)2%瓊脂糖凝膠電泳，溴化乙錠染色，紫外透射燈上顯像并拍照記錄結(jié)果。根據(jù)電泳條帶確定基因型。MTHFR基因酶切后可產(chǎn)生3種基因型，無(wú)突變野生CC型產(chǎn)生195 bp片段，突變雜合子CT型產(chǎn)生195、130、65 bp片段，突變純合子TT型產(chǎn)生130、65 bp片段。

1.3 統(tǒng)計(jì)學(xué)方法 采用SPSS18.0統(tǒng)計(jì)軟件。采用Hardy-Weinberg遺傳平衡定律檢測(cè)樣本的群體代表性。計(jì)數(shù)資料比較用χ2檢驗(yàn)；多因素分析采用二元logistic回歸。P<0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 兩組Hardy-Weinberg遺傳平衡定律檢測(cè)結(jié)果 MTHFR C677T基因型在兩組內(nèi)分布頻率符合Hardy-Weinberg遺傳平衡(P均>0.05)。

2.2 兩組MTHFR C677T基因多態(tài)性比較 見(jiàn)表1。

表1 兩組MTHFR C677T基因多態(tài)性比較(例)

2.3 MTHFR C677T基因型與H型高血壓的關(guān)系 以是否患H型高血壓為因變量，以MTHFR C677T基因型由CC→CT→TT有序變化、尿酸水平、甘油三酯水平、性別、年齡等為自變量，帶入logistic回歸模型，分析結(jié)果顯示MTHFR C677T基因型突變(OR=2.645，95%CI：1.737～4.025，P<0.01)為H型高血壓發(fā)病的獨(dú)立預(yù)測(cè)因子。

3 討論

從1969年McCully首次明確提出Hcy是動(dòng)脈粥樣硬化重要的潛在致病因素以來(lái)，Hcy與心腦血管疾病及高血壓的關(guān)系日益受到關(guān)注。研究[9～11]表明，Hcy水平升高與心血管疾病有著密切的關(guān)系，特別是高血壓合并高Hcy水平者危害更大。一般認(rèn)為，空腹血漿Hcy水平為5～15 μmol/L，Hcy水平≥16 μmol/L屬于HHcy，對(duì)于高血壓患者Hcy>10 μmol/L就應(yīng)該嚴(yán)格加以控制。本研究通過(guò)Hcy水平將高血壓患者分組，進(jìn)行對(duì)比分析。

血漿Hcy在體內(nèi)由甲硫氨酸轉(zhuǎn)甲基后生成。甲硫氨酸代謝異常會(huì)引起血漿Hcy增高。MTHFR是甲硫氨酸代謝的關(guān)鍵酶，其C677T基因點(diǎn)突變導(dǎo)致該酶的耐熱性和活性均降低約50%，導(dǎo)致血漿Hcy水平增高。MTHFR C677T基因點(diǎn)突變是否參與了HHcy的形成，并最終引發(fā)高血壓尚無(wú)定論。本研究結(jié)果顯示，兩組人群在MTHFR C677T基因的C、T等位基因分布及基因型分布均存在差異。兩組患者均有3種基因型分布，觀察組CC基因型分布低于對(duì)照組，TT基因型分布高于對(duì)照組；觀察組T等位基因頻率高于對(duì)照組，而C等位基因頻率低于對(duì)照組。提示C677T基因的點(diǎn)突變可能與H型高血壓的發(fā)病有關(guān)。為進(jìn)一步證實(shí)MTHFR C677T基因點(diǎn)突變和H型高血壓發(fā)病的關(guān)系，本研究以是否患高血壓為因變量，基因型由CC→CT→TT有序變化、尿酸水平、甘油三酯水平、年齡、性別等為自變量，帶入二元logistic回歸模型，分析顯示MTHFR C677T基因型突變?yōu)镠型高血壓患病的獨(dú)立預(yù)測(cè)因子。HHcy導(dǎo)致動(dòng)脈粥樣硬化可能與以下機(jī)制有關(guān)：自身氧化作用、改變內(nèi)皮細(xì)胞的基因表達(dá)、抑制一氧化氮合成酶、干擾纖溶酶原的激活位點(diǎn)等直接損傷血管內(nèi)皮細(xì)胞；影響平滑肌細(xì)胞的遷移及增殖；誘導(dǎo)內(nèi)皮細(xì)胞表面的血栓調(diào)節(jié)蛋白減少，導(dǎo)致纖維蛋白原生成纖維蛋白，增強(qiáng)血小板聚集功能和組織因子活性，促進(jìn)血栓形成；導(dǎo)致脂質(zhì)代謝紊亂及促進(jìn)動(dòng)脈粥樣硬化，促進(jìn)脂質(zhì)沉積于動(dòng)脈壁，使泡沫細(xì)胞增加，改變動(dòng)脈壁糖蛋白分子纖維化結(jié)構(gòu)，促進(jìn)斑塊鈣化，促進(jìn)局部巨噬細(xì)胞的聚集，從而促進(jìn)動(dòng)脈粥樣硬化的發(fā)展[9,12]。

本研究同時(shí)發(fā)現(xiàn)血漿尿酸水平在兩組人群中存在差異。HHcy可導(dǎo)致腎血管的動(dòng)脈粥樣硬化或全身血管病變，使腎臟對(duì)血漿尿酸的清除減少，因此導(dǎo)致血尿酸水平升高[13]。血尿酸在正常水平是抗氧化劑，但在高水平時(shí)反而起到促氧化作用，引起內(nèi)皮功能障礙，加重腎損傷，進(jìn)一步升高Hcy水平。因此HHcy和高尿酸血癥可以相互促進(jìn)，共同加劇動(dòng)脈硬化的形成。本研究進(jìn)一步證實(shí)了上述假設(shè)。但Choi等[13]發(fā)現(xiàn)代謝綜合征患者血尿酸水平與Hcy并無(wú)相關(guān)性，考慮與入選人群年齡、糖尿病患者患病比例等因素不同有關(guān)。

本研究尚存在樣本量少等一些不足，同時(shí)血漿Hcy水平除了受MTHFR C677T代謝影響，還受到多種因素的調(diào)節(jié)，如蛋白質(zhì)攝入不足、維生素B12缺乏[14]，及其他代謝酶調(diào)節(jié)[15]等，機(jī)制復(fù)雜。因此，H型高血壓發(fā)病與MTHFR C677T基因多態(tài)性的相關(guān)性還需更大規(guī)模的流行病學(xué)研究。

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CorrelationbetweenMTHFRC677TgenepolymorphismandH-typehypertension

ZHANGYingying,LIANGRong,GUOXukun

(TianjinChestHospital,Tianjin300222,China)

ObjectiveTo explore the relationship between the methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism and H-type hypertension.MethodsA total of 273 consecutive patients with hypertension, including 185 cases of patients with H-type hypertension (observation group, Hcy≥10 μmol/L) and 88 cases of patients with non-H-type hypertension (control group), were enrolled. We collected the fasting venous blood in the two groups, then, the MTHFR C677T gene polymorphism was observed by PCR-RFLP. The relationships between the genotypes of MTHFR C677T and H-type hypertension were analyzed by Logistic regression.ResultsThere were 31 and 35 cases of MTHFR C677T CC genotype in the observation group and control group, and the number of CT genotype was 104 and 45, respectively; the number of TT genotype was 50 and 8, respectively; the number of C allele of the two groups was 166 and 115, respectively; the number of T allele of the two groups was 204 and 61, respectively; Significant difference was found in the distribution of the allelic frequencies of T and C, and CC and TT genotypes between the two groups (P<0.01). Logistic regression analysis showed that MTHFR C677T genotype mutation (OR=2.645, 95%CI: 1.737-4.025,P<0.01) was an independent risk factor for H-type hypertension.Conclusion MTHFR C677T genotype TT is an independent risk factor for H-type hypertension.

H-type hypertension; methylenetetrahydrofolate reductase; homocysteine; gene polymorphism

10.3969/j.issn.1002-266X.2017.38.003

R544.1

A

1002-266X(2017)38-0008-03

天津市衛(wèi)生局科技基金資助項(xiàng)目(2013KZ081)。

張瑩瑩(1977-)，女，博士，副主任醫(yī)師，主要研究方向?yàn)樾难芗膊』A(chǔ)及臨床。E-mail: 1424413836@qq.com

2017-07-13)