肥胖與KCNJ11基因多態性在中國漢族人群糖尿病視網膜病變發生中的作用

吳慧慧　劉乃嘉　李燕良 　楊　震　陶曉明　杜艷萍　王宣春　張朝云　胡仁明　鹿　斌 　聞　杰,2△

(1復旦大學附屬華山醫院內分泌科　上海　200040; 2上海市靜安區中心醫院內分泌科　上海　200040;3上海交通大學附屬新華醫院內分泌科　上海　200020; 4復旦大學附屬華東醫院內分泌科　上海　200040)

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肥胖與KCNJ11基因多態性在中國漢族人群糖尿病視網膜病變發生中的作用

吳慧慧1▲劉乃嘉1▲李燕良1楊震3陶曉明4杜艷萍4王宣春1張朝云1胡仁明1鹿斌1聞杰1,2△

(1復旦大學附屬華山醫院內分泌科上海200040;2上海市靜安區中心醫院內分泌科上海200040;3上海交通大學附屬新華醫院內分泌科上海200020;4復旦大學附屬華東醫院內分泌科上海200040)

目的確認在中國漢族人群中KCNJ11基因多態性是否為糖尿病視網膜病變(diabetic retinopathy,DR)的獨立危險因素,探討其與肥胖的交互作用對DR的影響。 方法以580名2型糖尿病(type 2 diabetes mellitus,T2DM)患者為研究對象(475名非DR患者和105名DR患者),利用iPLEX技術進行KCNJ11 rs5219的基因型測定,DR的確診由經驗豐富的眼科醫師根據非散瞳眼底數碼成像的圖像分析來判斷。通過構建單因素、多因素回歸模型來研究肥胖或KCNJ11基因多態性與DR的關系及兩者間的交互作用。結果KCNJ11基因多態性是DR的獨立危險因素。而KCNJ11 rs5219等位基因為G的肥胖患者(BMI≥28.0 kg/m2),發生DR的風險更高(等位基因分析:P<0.05,基因型分析:P<0.01)。結論在中國漢族糖尿病患者中,KCNJ11基因多態性與DR顯著相關,并與肥胖有交互作用。

KCNJ11基因;肥胖;糖尿病視網膜病變;中國漢族人群

糖尿病視網膜病變(diabetic retinopathy,DR)是糖尿病最常見的微血管并發癥之一,是一種具有特異性改變的眼底病變。其發生發展與多種因素有關,如高血壓、血糖控制不良等[1]。該疾病的家族聚集性及不同種族間發病率的差異[2-5],提示遺傳因素在其發生過程中起著關鍵作用。

鉀離子內向整流通道蛋白J亞單位 11號成員(potassium inwardly-rectifying-channel,subfamily-J,member 11,KCNJ11)是ATP敏感性鉀通道(KATP)的成員之一[6],它在胰腺組織中高表達,并與2型糖尿病(type 2 diabctes mellitus,T2DM)的易感性密切相關[7-9]。關于KCNJ11基因多態性在DR發生過程中的作用則研究甚少。已有數據證實,肥胖是DR的獨立危險因素[10-11]。本研究旨在探討KCNJ11基因多態性與DR發病風險之間的關系,及其與肥胖間是否存在交互作用。

資 料 和 方 法

研究對象選取就診于復旦大學附屬華山醫院內分泌科門診的580例T2DM患者(105例DR,475例非DR)。本研究經復旦大學附屬華山醫院倫理委員會批準,患者及家屬簽署知情同意書。入組患者均為居住在上海市區的漢族人群。

問卷調查內容包括一般情況、性別、年齡、飲酒史、病史及藥物治療史等。

檢測方法所有受試者于清晨空腹、脫鞋后測定身高和體重,計算體質指數(body mass index,BMI)。血壓為受試者休息5 min后,取坐位,右臂血壓重復測3次的平均值。病程是初次診斷為糖尿病到患者入組之間的時間段。

所有受試者均空腹采血,采用日立7600全自動生化分析儀檢測總膽固醇、血尿酸、尿素氮、肌酐、三酰甘油。空腹采血后行75g口服葡萄糖耐量試驗(oral glucose tolerance test,OGTT),并于2 h后再次采血測血糖。空腹血糖及餐后血糖均采用葡萄糖氧化酶過氧化物酶法測定,糖化血紅蛋白(hemoglobin A1C,HbA1C)采用高壓液相離子交換層析法測定(美國伯樂公司全自動分析儀及試劑盒)。

免散瞳數碼眼底成像受試者先于暗室內休息5 min以達到視覺適應,然后由同一眼科醫師使用日本免散瞳眼底數碼照相機,拍攝以黃斑為中心的45°眼底后極部彩色照片。照片以1 024×768的像素保存于電腦,之后由同一醫師閱片診斷[12-13]。

診斷標準BMI≥28.0 kg/m2即視為肥胖[14]。所有受試者均符合1999年WHO糖尿病診斷標準:空腹血糖≥7.0 mmol/L, OGTT 2 h后血糖≥11.1 mmol/L,或自述有降糖藥物治療史。排除標準:1型糖尿病、妊娠期糖尿病及繼發性糖尿病(胰腺炎、血色沉著病等)患者,或合并心、肝功能障礙者,急性感染者。存在至少一種以下損傷者即可確診為DR:微動脈瘤、點狀或火焰狀出血、硬性滲出及DR的激光治療。

SNP測定利用傳統的苯酚-氯仿抽提法從580例受試者的外周血淋巴細胞中提取基因組DNA,并用基質輔助激光解吸/電離質譜檢測其KCNJ11 rs5219的基因型。該人群的基因型分布符合Hardy-Weinberg平衡(P>0.05)。

結　　　果

本研究發現在580名受試者中,DR患者血尿素氮、肌酐、HbA1C、空腹及餐后血糖均高于非DR患者,差異有統計學意義(P均<0.05)。同時,DR患者的糖尿病病程明顯長于非DR患者,前者患病年齡也大于后者(P均<0.05)。但兩組患者的性別、年齡、身高、體重、收縮壓、舒張壓、C肽、總膽固醇、三酰甘油、血尿酸水平差異均無統計學意義(P均>0.05,表1)。

在校正以上因素后,Logistic回歸分析提示KCNJ11rs5219的等位基因(A/G)及基因型(AA/AG/GG)在DR組和非DR組的分布情況差異有統計學意義(等位基因分析P=0.004,基因型分析P=0.001),即rs5219的基因分型與DR發生之間存在顯著的相關性(表2、3)。但BMI與DR發生之間并無相關性(95%CI:0.942～1.030,P>0.05),這與以往的研究不符,可能與樣本量太小有關。MLR回歸模型分析提示,rs5219與DR的相關性僅存在于肥胖人群(BMI≥28.0kg/m2)中，等位基因分析、ORInt=2.463,95%CI為1.001～6.071,P<0.05;基因型分析：ORInt=2.555,95%CI為1.298～5.029,P<0.01,提示KCNJ11rs5219與肥胖在DR的發生過程中存在交互作用(表4)。

表1　受試者的基線特征

SBP:Systolic blood pressure;DBP:Diastolic blood pressure;FPG:Fasting plasma glucose;CP:C peptide;PPG:Postprandial plasma glucose;TC:Total cholesterol;TG:Triglyceride;HbA1C:Hemoglobin A1C;BUN:Blood urea nitrogen;CR:Serum creatinine;UA:Uric acid.1 mmHg=0.133 kPa.

討　　　論

世界范圍內約有30%的糖尿病患者會發生眼部血管閉塞、視網膜缺血或新生血管形成,嚴重影響視力甚至致盲[15]。DR作為糖尿病最常見的眼部并發癥,主要表現為血管通透性增加、糖尿病性黃斑水腫和/或增值性病變[16],其發病與糖脂代謝及血壓控制情況密切相關。

KCNJ11是ATP敏感性鉀通道的重要組成蛋白,該通道是典型的代謝門控傳感器,其功能障礙與葡萄糖刺激的胰島素分泌有關[17]。有研究證實,KCNJ11基因缺失小鼠葡萄糖刺激的胰島素分泌減少;而人類KCNJ11基因發生突變則可導致多種胰島素相關性疾病,如新生兒糖尿病、先天性高胰島素血癥及移植后糖尿病[18-21]。機制可能是KCNJ11基因變異體可降低離子通道對ATP的敏感性,進而減少胰島β細胞的活性,導致ATP消耗過多及胰島素釋放障礙[7,22-23]。大量研究證實,肥胖是DR發生的獨立危險因素[10-11],而KCNJ11突變基因攜帶者的BMI顯著高于野生基因攜帶者[24],但關于該基因變異體對DR發生的影響以及其是否與肥胖存在交互作用尚未見報道。

表2　rs5912的等位基因在DR和非DR組的分布情況

There were significantly differences in allele rate between the two groups (P=0.004).

表3　rs5912的基因型在DR和非DR組的分布情況

There were significantly differences in allele rate between the two groups (P=0.001).

表4　 KCNJ11 rs5219與BMI的交互作用

Adjusted for variables of age,sex,SBP,FPG,PPG,TC,TG,HbA1C,duration,age of onset,family history of DM and alcohol.

本研究對580名T2DM患者進行KCNJ11基因型的測定,首次發現KCNJ11 rs5219與DR顯著相關(校正年齡、性別、BMI及血脂等)。同時,HbA1C、糖尿病病程與DR的相關性也得到證實 (P均< 0.05),這與以往的研究結果一致[25-26]。盡管肥胖早已被證實是DR的獨立危險因素,但我們的實驗卻未觀察到二者間的相關性,可能需要更大的樣本量來進一步研究。MLR回歸模型分析發現,在肥胖的T2DM患者(BMI≥28.0 kg/m2)中,野生型G-等位基因攜帶者 (AG和GG) 的DR發生率顯著高于A-等位基因攜帶者 (AA和AG) (等位基因分析:P<0.05,基因型分析:P<0.01),提示KCNJ11基因突變可降低肥胖的T2DM病患者的DR發病風險,即該基因多態性與肥胖在DR的發生中存在交互作用。我們推測KCNJ11 rs5219可能與肥胖之間存在一定的相關性,進而通過肥胖來參與影響DR的發生。

KCNJ11基因多態性是DR的獨立危險因素,并在其發生過程中與肥胖有交互作用。但本研究的受試者均來自上海地區,并不能代表整個中國漢族人群,因而需要更大的樣本量以及更廣泛的基礎研究來闡明其機制。

[1]GHOLAMHOSSEIN Y,BEHROUZ H,ASGHAR Z.Diabetic retinopathy risk factors:plasma erythropoietin as a risk factor for proliferative diabetic retinopathy[J].KoreanJOphthalmol,2014,28(5):373-378.

[2]THOMAS RL,DISTILLER L,LUZIO SD,etal.Ethnic differences in the prevalence of diabetic retinopathy in persons with diabetes when first presenting at a diabetes clinic in South Africa[J].DiabetesCare,2013,36(2):336-341.

[3]SIVAPRASAD S,GUPTA B,CROSBY-NWAOBI R,etal.Prevalence of diabetic retinopathy in various ethnic groups:a worldwide perspective[J].SurvOphthalmol,2012,57(4):347-370.

[4]SIVAPRASAD S,GUPTA B,GULLIFORD MC,etal.Ethnic variations in the prevalence of diabetic retinopathy in people with diabetes attending screening in the United Kingdom (DRIVE UK)[J].PLoSOne,2012,7(3):e32182.

[5]CHORNY A,LIFSHITS T,KRATZ A,etal.Prevalence and risk factors for diabetic retinopathy in type 2 diabetes patients in Jewish and Bedouin populations in southern Israel[J].Harefuah,2011,150(12):906-910,935.

[6]INAGAKI N,GONOI T,CLEMENT JP,etal.Reconstitution of IKATP:an inward rectifier subunit plus the sulfonylurea receptor[J].Science,1995,270(5239):1166-1170.

[7]LI YY.The KCNJ11 E23K gene polymorphism and type 2 diabetes mellitus in the Chinese Han population:a meta-analysis of 6,109 subjects[J].MolBiolRep,2013,40(1):141-146.

[8]QIU L,NA R,XU R,etal.Quantitative assessment of the effect of KCNJ11 gene polymorphism on the risk of type 2 diabetes[J].PLoSOne,2014,9(4):e93961.

[9]ABDELHAMID I,LASRAM K,MEILOUD G,etal.E23K variant in KCNJ11 gene is associated with susceptibility to type 2 diabetes in the Mauritanian population[J].PrimCareDiabetes,2014,8(2):171-175.

[10]DIRANI M,XIE J,FENWICK E,etal.Are obesity and anthropometry risk factors for diabetic retinopathy? The diabetes management project[J].InvestOphthalmolVisSci,2011,52(7):4416-4421.

[11]RAMAN R,RANI PK,GNANAMOORTHY P,etal.Association of obesity with diabetic retinopathy:Sankara Nethralaya Diabetic Retinopathy Epidemiology and Molecular Genetics Study (SN-DREAMS Report no.8)[J].ActaDiabetol,2010,47(3):209-215.

[12]MURGATROYD H,ELLINGFORD A,COX A,etal.Effect of mydriasis and different field strategies on digital image screening of diabetic eye disease[J].BrJOphthalmol,2004,88(7):920-924.

[13]SCANLON PH,FOY C,MALHOTRA R,etal.The influence of age,duration of diabetes,cataract,and pupil size on image quality in digital photographic retinal screening[J].DiabetesCare,2005,28(10):2448-2453.

[14]ZHOU BF,COOPERATIVE META-ANALYSIS GROUP OF THE WORKING GROUP ON OBESITY IN CHINA.Predictive values of body mass index and waist circumference for risk factors of certain related diseases in Chinese adults:study on optimal cut-off points of body mass index and waist circumference in Chinese adults[J].AsiaPacJClinNutr,2002,11 (Suppl 8):S685-S693.

[15]MURGATROYD H,ELLINGFORD A,COX A,etal.Effect of mydriasis and different field strategies on digital image screening of diabetic eye disease[J].BrJOphthalmol,2004,88(7):920-924.

[16]DAS A,STROUD S,MEHTA A,etal.New treatments for diabetic retinopathy[J].DiabetesObesMetab,2015,17(3):219-230.

[17]LIN YW,BUSHMAN JD,YAN FF,etal.Destabilization of ATP-sensitive potassium channel activity by novel KCNJ11 mutations identified in congenital hyperinsulinism[J].JBiolChem,2008,283(14):9146-9156.

[18]NICHOLS CG,KOSTER JC,REMEDI MS.Beta-cell hyperexcitability:from hyperinsulinism to diabetes[J].DiabetesObesMetab,2007,9 (Suppl 2):81-88.

[19]CHANG WL,HUANG CJ,LEI TH,etal.A novel mutation of KCNJ11 gene in a patient with permanent neonatal diabetes mellitus[J].DiabetesResClinPract,2014,104(1):e29-e32.

[20]MARTHINET E,BLOC A,OKA Y,etal.Severe congenital hyperinsulinism caused by a mutation in the Kir6.2 subunit of the adenosine triphosphate-sensitive potassium channel impairing trafficking and function[J].JClinEndocrinolMetab,2005,90(9):5401-5406.

[21]TAVIRA B,COTO E,TORRES A,etal.Association between a common KCNJ11 polymorphism (rs5219) and new-onset posttransplant diabetes in patients treated with Tacrolimus[J].MolGenetMetab,2012,105(3):525-527.

[22]RIEDEL MJ,LIGHT PE.Saturated and cis/trans unsaturated acyl CoA esters differentially regulate wild-type and polymorphic beta-cell ATP-sensitive K+channels[J].Diabetes,2005,54(7):2070-2079.

[23]SCHWANSTECHER C,MEYER U,SCHWANSTE-CHER M.Schwanstecher,K(IR)6.2 polymorphism predisposes to type 2 diabetes by inducing overactivity of pancreatic beta-cell ATP-sensitive K(+) channels[J].Diabetes,2002,51(3):875-879.

[24]NIELSEN EM,HANSEN L,CARSTENSEN B,etal.The E23K variant of Kir6.2 associates with impaired post-OGTT serum insulin response and increased risk of type 2 diabetes[J].Diabetes,2003,52(2):573-577.

[25]HERMANN JM,HAMMES HP,RAMI-MERHAR B,etal.HbA1c variability as an independent risk factor for diabetic retinopathy in type 1 diabetes:a German/Austrian multicenter analysis on 35,891 patients[J].PLoSOne,2014,9(3):e91137.

[26]SHEU SJ,LIU NC,GER LP,etal.High HbA1c level was the most important factor associated with prevalence of diabetic retinopathy in Taiwanese type Ⅱ diabetic patients with a fixed duration[J].GraefesArchClinExpOphthalmol,2013,251(9):2087-2092.

E-mail:wenjie@hotmail.com

The interaction analysis of KCNJ11 gene and obesity on diabetic retinopathy in Chinese Han population

WU Hui-hui1▲, LIU Nai-jia1▲, LI Yan-liang1, YANG Zhen3, TAO Xiao-ming4,DU Yan-ping4,WANG Xuan-chun1, ZHANG Zhao-yun1, HU Ren-ming1, LU Bin1, WEN Jie1,2△

(1DepartmentofEndocrinology,HuashanHospital,FudanUniversity,Shanghai200040,China;2DepartmentofEndocrinology,Jing′anDistrictCenterHospitalofShanghai,Shanghai200040,China;3DepartmentofEndocrinology,XinhuaHospital,ShanghaiJiaoTongUniversity,Shanghai200020,China;4DepartmentofEndocrinology,HuadongHospital,FudanUniversity,Shanghai200040,China)

ObjectiveTo clarify whetherKCNJ11 gene is an independent risk factor for diabetic retinopathy (DR) and its interaction with obesity to DR susceptibility in Chinese population.MethodsWe conducted a study in a cohort of 580 patients with type 2 diabetes mellitus (T2DM),including 475 non-DR and 105 DR subjects,for the genotype of a most common polymorphism-rs5219 ofKCNJ11.Genotyping was performed by iPLEX technology.DR was diagnosed by experienced ophthalmologists based on image analysis of the nonmydriatic digital fundus imaging.The association

KCNJ11 gene;obesity;diabetic retinopathy;Chinese Han population

R587.1

Adoi: 10.3969/j.issn.1672-8467.2016.04.005

2015-09-06;編輯:段佳)

國家自然科學基金(81270903,30900501,81370884),上海市自然科學基金(13140901600)

▲WU Hui-hui and LIU Nai-jia contributed equally to the article

betweenKCNJ11 rs5219 or obesity and DR,as well as the interaction effect ofKCNJ11 and obesity on DR susceptibility were assessed by univariate and multivariate Logistic regression analysis controlling confounders.ResultsThe possible association betweenKCNJ11 rs5219 and DR has been confirmed in this cohort.Interestingly,obese patiens (BMI≥28.0 kg/m2) with G-allele ofKCNJ11 rs5219 had higher risk of DR in Chinese Han population (for allele,P<0.05;for genotype,P<0.01).ConclusionsKCNJ11 gene polymorphism is an independent risk factor for DR,and it may interact with obesity to effect on the development of DR in diabetic patients of Chinese Han population.

*This work was supported by the National Natural Science Foundation of China (81270903,30900501,81370884) and the Natural Science Foundation of Shanghai (13140901600).