脂蛋白酯酶基因Ser447Ter多态性与缺血性脑卒中关系的Meta分析

目的 探讨脂蛋白脂酶基因(LPL) Ser447Ter多态性与缺血性脑卒中的关系.方法 以“lipoprotein lipase”、“LPL”、“polymorphism”、“stroke”为关键词检索数据库PubMed;以“脂蛋白酯酶”、“多态”、“缺血性脑卒中”、“脑梗死”为关键词检索中国学术期刊全文数据库、维普中文科技期刊数据库、万方数据知识服务平台,搜集相关文献.筛选文献,对符合纳入标准的研究用RevMan5.0软件进行Meta分析.结果 ①缺血性脑卒中组LPL Ser447Ter CG+GG基因型频率比对照组显著降低,突变型CG+GG发生缺血性脑卒中的风险是野生型CC的73％ (OR=0.73,95％CI:0.61～0.88,P=0.0007);②Ser447Ter突变等位基因携带者发生动脉粥样硬化性脑梗死的风险显著低于非携带者(OR=0.44,95％CI:0.32～0.62,P＜ 0.000 01)；③与CC基因型相比,CG+GG基因型血浆总胆固醇(TC)及甘油三酯(TG)水平显著降低(TC:WMD=-0.10,95％CI:-0.13～-0.06,P＜0.00001; TG:WMD=-0.24,95％CI:-0.40～-0.09,P=0.002),而高密度脂蛋白(HDL-c)水平显著升高(WMD=0.09,95％CI:0.02～O.15,P=0.009),其差异均具有统计学意义.结论 脂蛋白脂酶基因Ser447Ter多态性与缺血性脑卒中尤其是动脉粥样硬化性脑梗死的发病相关联,G等位基因是保护基因,且Ser447Ter基因多态性可能通过影响血脂和载脂蛋白水平影响缺血性脑卒中的发生.     

中国成年人双生子中脂蛋白酯酶基因多态性位点单体型与血脂关联分析

目的研究成年人双生子脂蛋白酯酶(LPL)基因S447X和HindⅢ多态性及其单体型与血脂的关联。方法基于双生子登记系统,对双生子进行问卷、体检及血脂和LPL基因S447X、HindⅢ多态性的检测。对两多态性位点进行连锁不平衡检验并用SNPHAP软件进行单体型估计。结果共计入选合格成人双生子987对;LPL基因两多态性位点间存在着紧密的连锁不平衡。在女性双生子中,HindⅢ多态性位点的H-等位基因与三酰甘油(TG)水平下降及高TG血症发生风险降低的相关性在调整了X等位基因的作用后就消失了。两多态性位点的H-X单体型组合和TG水平降低12．9％(95％CI:-20．6～-4．5)、TG／HDL水平降低14．9％(95％CI:-21．4～-6．7)以及高TG血症发生危险降低(OR=0．40,95％CI:0．21～0．79)均显著相关。结论LPL基因S447X和HindⅢ多态性位点的单体型与血脂水平的改善有关,但这种相关主要由S447X多态性决定,而HindⅢ多态性位点通过与X等位基因的连锁不平衡间接产生与血脂的关联。     

中国人脂联素基因45T/G与276G/T多态性与2型糖尿病相关性研究的Meta分析

目的 对中国人脂联素基因45T/G和276G/T多态性与2型糖尿病相关性的研究进行Meta分析.方法 通过文献检索收集2007年12月以前完成或发表的中国人脂联素基因45T/G和276G/T多态性与2型糖尿病相关性的病例对照研究,剔除不符合要求的文献,以漏斗图检验人选文献的偏倚,并根据各入选文献结果的同质性检验结果进行数据合并,计算总OR值,Meta分析采用Review Manager 4.2版软件.结果 共23篇文献符合条件纳入研究,其中涉及45T/G多态性文献20篇,涉及276G/T多态性文献9篇,入选文献无明显偏倚,各文献间质性检验显示有关两个位点45T/G(X2=119.8,P<0.001)、276G/T(X2=31.0,P=0.001)等位基因分布情况的文献之间均存在显著异质性.Meta分析显示45G等位基因携带者增加2型糖尿病的易感性(OR=1.38,95%CI:1.04～1.84,P=0.03),276G/T基因多态性与2型糖尿病易感性无明显相关性(OR=0.83,95%CI:0.61～1.13,P=0.23).结论 脂联素基因45T/G多态性与中国人2型糖尿病易感性相关,45G等位基因可能是2型糖尿病易感基因,276G/T基因多态性与2型糖尿病无明显相关性.     

钙蛋白酶10基因多态性与2型糖尿病的关联研究:Meta分析

目的 探讨钙蛋白酶10(CAPN10)基因SNP43、SNP44位点及主要单倍型、单倍型组合与2型糖尿病(T2DM)的关联性.方法 根据系统评价的原理和规范,检索PubMed及中文期刊数据库,纳入CAPN10基因与T2DM的病例对照研究.根据种族,采用分层Meta分析评估CAPN10基因多态性与T2DM的关联性.同时评估发表偏倚.结果 各种族与T2DM有关联的基因多态性分别是:蒙古人种,SNP43位点G等位基因OR=1.368(95%CI:1.155～1.620),G/G基因型OR=1.437(95%CI:1.186～1.741),111/221单倍型组合OR=2.762(95%CI:1.287～5.927);高加索人种,SNP44位点C等位基因OR=1.144(95%C1:1.023～1.278),111/111单倍型组合OR=1.291(95%CI:1.050～1.586);混血人种,SNP44位点C等位基因OR=1.653(95%CI:1.025～2.665).结论 SNP43位点G等位基因、G/G基因型、111/221单倍型组合是蒙古人种的危险因素;SNP44位点C等位基因、111/111单倍型组合是高加索人种的危险因素;SNP44位点C等位基因是混血人种的危险因素.     

脂联素基因T45G与2型糖尿病关系的Meta分析

[目的]分析并明确中国汉族人群脂联索(adiponectin,AMP1)基因第二外显子45位T/G多态性(T45G)与2型糖尿痛(type 2 diabetes mellitus,T2DM)的相关性. [方法]对以往有关T45G与T2DM相关性研究进行Meta分析.通过文献检索收集中国汉族人群AMP1基因SNP45多态性与T2DM的病例-对照研究,剔除不符台要求的文献,对入选文献进行异质性检验并根据检验结果进行数据合并,采用Stata10.0计算合并比值比(odds ratio,OR)及其95%可信区间(confidence interval,CI)以及发表偏倚. [结果]共16篇文献符合条件纳入研究,采用随机效应模型合并OR值,Meta分析结果为在显性、隐性、加性遗传模式下,合并OR(95%CI)分别为1.61(1.21～2.14)、2.16(1.59～2.92)、1.49(1.22～1.83),OR值具有统计学意义. [结论]不论是显性、隐性还是加性遗传模式,G等位基因均为T2DM的危险因素,提示G等位基因携带者易患T2DM.     

MC4R基因rs17782313 和 rs12970134多态性与2型糖尿病风险相关性的Meta分析

目的 系统评价MC4R附近的rs17782313（T/C）和 rs12970134（G/A）多态性与2型糖尿病（T2DM）风险之间的相关性。方法 检索来自 PubMed、Embase、Web of science、中国知网和万方数据库中已发表的文献。囊括评估两种MC4R多态性与T2DM之间关联的所有研究,检索时限为建库至2021年7月6日。采用Stata 15.0软件,进行meta分析。结果 共纳入16项关于MC4R单核苷酸多态性rs17782313 和 rs12970134的研究（33 902例T2DM病例和61 886例健康对照）。Meta分析结果表明,MC4R基因rs17782313 和 rs12970134多态性的风险等位基因（C vs T/A vs G）与T2DM的患病相关（OR = 1.05,95%CI:1.01～1.09）。基因型分析显示,MC4R附近的rs17782313和 rs12970134的多态性（TC + CC vs TT / GA + AA vs GG）与T2DM相关（OR = 1.08,95%CI:1.01～1.15）,并且未检测到发表偏倚。结论 MC4R基因附近的多态性（rs17782313 和 rs12970134）与T2DM的患病风险相关,即C或A等位基因和TC + CC或GA + AA基因型会增加T2DM患病风险。     

TCF7L2和KCNQ1基因多态性与2型糖尿病易感性的Meta分析

目的对TCF7L2,KCNQ1基因多态性与2型糖尿病(T2DM)相关性的研究进行Meta分析。方法通过检索PubMed和CNKI数据库中有关TCF7L2,KCNQ1基因多态性与T2DM易感性关联研究的文献,用漏斗图检验入选文献的偏倚,根据异质性检验结果采用固定效应模型或随机效应模型进行Meta分析,此过程均在软件包Launch Stata11.0上完成。结果该研究共纳入20篇文献(:1)KCNQ1基因rs2237892位点与T2DM发病风险相关性的研究共纳入7篇,得到7764例T2DM患者和8937例对照,Meta分析结果显示,rs2237892位点C等位基因能够增加T2DM的发病风险(OR=1.300,95%CI:1.234～1.366);CC/TC基因型是T2DM的危险基因型(OR=1.453,95%CI:1.279～1.651)。(2)TCF7L2基因rs7903146位点与T2DM发病风险相关性研究12篇,共得到11443例2型糖尿病患者和11003例对照,Meta分析结果显示,rs7903146位点T等位基因能够增加T2DM的发病风险(OR=1.379,95%CI:1.261～1.508),TT/TC基因型是T2DM的危险基因型(OR=1.469,95%CI:1.333～1.620)。(3)TCF7L2基因rs12255372位点与T2DM发病风险相关性的研究12篇,共得到12173例T2DM患者和10869例对照;Meta分析结果显示,rs12255372位点T等位基因能够增加T2DM的发病风险(OR=1.381,95%CI:1.275～1.495),TT/TG基因型是T2DM的危险基因型(OR=1.463,95%CI:1.337～1.602)。结论 TCF7L2基因rs7903146位点TT/TC基因型和rs12255372位点TT/TG基因型,以及KCNQ1基因rs2237892位点CC/TC基因型均能够增加T2DM的发病风险。     

过氧化物酶体增殖物激活受体γ 2 rs1801282位点与2型糖尿病肾病易感性的系统评价

目的系统评价PPAR-γ 2基因rs1801282 CG多态性与2型糖尿病肾病(T2DN)遗传易感性。方法应用PubMed、中国知识资源总库(CNKI)和万方数据库,检索2017年3月前发表的rs1801282多态性与T2DN易感性关联研究。应用Stata 12.0对研究数据统计分析,以合并OR值及相应95%置信区间(95%CI)评价rs1801282与T2DN易感性关系。结果根据纳入、排除标准,纳入16篇病例-对照研究,研究对象9 183例。等位基因比较显示,与rs1801282 C相比,G等位基因可显著降低T2DM患者发生DN的风险,合并效应的OR值及95%CI为0.74(0.62~0.89),P=0.002;以种族进行亚组分析,发现G等位基因与高加索人T2DN发病风险降低有关,OR值及95%CI为0.75(0.60~0.94),P=0.012。基因型分析显示,显性遗传模型(CG+GG vs.CC)下,合并效应的OR值及95%CI为0.72(0.59~0.87),P=0.001;亚组分析发现,rs1801282多态位点与高加索人T2DN遗传易感性有关,OR值及95%CI为0.71(0.56~0.91),P=0.006。结论 PPAR-γ 2基因rs1801282位点与T2DN发病有关,G等位基因可能是机体防止DN发生的保护性遗传因素。     

细胞周期依赖性激酶抑制基因(CDKN2A/B)多态性与2型糖尿病的Meta分析

[目的]综合评价细胞周期依赖性激酶抑制基因(CDKN2A/B)多态性rs10811661与2型糖尿病(T2DM)的易感性关系。[方法]通过Pubmed、OMIM、ISI、Embase数据库全面检索CDKN2A/B基因多态性rs10811661与T2DM相关的论文,通过q值检查各研究间的异质性;用漏斗图和累积Meta分析评估发表偏倚;以OR值为统计量,在基因分型水平上做Meta分析。[结果]本研究共纳入14篇文献,包括27548病例和36502对照样本。Meta分析结果显示:T等位基因相对于C等位基因的合并OR值为1.22(95%CI:1.18～1.26),TT纯合子和TC杂合子与CC纯合子相比的合并OR值分别为1.52(95%CI:1.40～1.66),1.27(95%CI:1.17～1.39)。[结论]CDKN2A/B基因多态性rs10811661与2型糖尿病关联有统计学意义。     

白细胞介素-6基因-174C/G和-634C/G多态性与尘肺易感性的Meta分析

目的探索白细胞介素-6(interleukin-6,IL-6)基因-174C/G和-634C/G多态性与尘肺易感性的关系。方法以尘肺、白细胞介素-6及多态性为关键词,检索中文数据库Sinomed、万方医学、中国知网和维普;以pneumoconiosis、interleukin-6及polymorphism为关键词,检索英文数据库Pubmed、Embase、Cochrane Library和Web of Science。采用Revman Manager 5.2软件进行效应值的合并和文献质量评价。结果共纳入7篇文献(9组病例-对照研究),包括IL-6基因-174C/G位点病例660例,对照848例;IL-6基因-634C/G位点病例344例,对照362例。Meta分析结果表明,IL-6基因-174C/G位点多态性与尘肺易感性之间无关联(CC对CG+GG,OR=1.05(95%CI 0.76~1.45),CG对GG+CC,OR=0.79(95%CI 0.40~1.55),C对G,OR=0.95(95%CI 0.80~1.14))。IL-6基因-634 C/G多态性位点与尘肺发病之间有关联(CC对CG+GG,OR=2.12(95%CI 1.56~2.88),CG对GG+CC,OR=0.40(95%CI 0.27~0.58),C对G,OR=1.67(95%CI 1.33~2.11))。结论IL-6基因-174C/G位点多态性与尘肺易感性之间没有相关性,-634C/G位点多态性与尘肺易感性之间有相关性,-634C/G位点CC基因型是尘肺的易感基因型。     

Gene- gene interaction between PPARG and APOE gene on late-onset Alzheimer’s disease: A case- control study in Chinese han population

Aims

The aim was to investigate the impact of PPARG and APOE gene single nucleotide polymorphisms (SNPs) and additional gene- gene interaction on late-onset Alzheimer’s disease (LOAD) risk based on Chinese Han population.

Methods

A total of 928 participants (466 males, 462 females), with a mean age of 81.3 ± 16.4 years old, were included in the study, including 460 LOAD patients and 468 normal controls participants. Logistic regression was performed to investigate association between SNP and LOAD risk and generalized multifactor dimensionality reduction (GMDR) was used to analysis the gene-gene interaction.

Results

Logistic regression analysis showed that LOAD risk was significantly higher in carriers of G allele of the rs405509 polymorphism than those with AA (AG+ GG versus AA, adjusted OR (95%CI) =1.54(1.20-1.89), and higher in carriers of G allele of the rs1805192 polymorphism than those with CC (CG+ GG versus CC, adjusted OR (95%CI) =1.32(1.16-2.43). We also found that there was a potential gene–gene interaction between rs405509 and rs1805192. Participants with AG or GG of rs405509 and CG or GG of rs1805192 genotype have the highest AD risk, compared to participants with AA of rs405509 and CC of rs1805192 genotype, OR (95%CI) was 2.62(1.64 -3.58), after covariates adjustment.

Conclusions

G allele of the rs405509 of APOE and G allele of the rs1805192 of PPAR G polymorphism were associated with increased LOAD risk, and participants with AG or GG of rs405509 and CG or GG of rs1805192 genotype have the highest AD risk.

     

Plasma triglyceride and HDL-cholesterol concentrations in Vietnamese girls are affected by lipoprotein lipase, but not apolipoprotein CIII polymorphism

Lipoprotein lipase (LPL) and apolipoprotein CIII (apoCIII) play an important role in HDL-cholesterol and triglyceride metabolism. This study examined the effects of LPL S447X and apoCIII SstI polymorphisms on the plasma lipid and lipoprotein concentrations in Vietnamese girls. Three hundred and fifty-one Vietnamese girls, from 7 to 9 y of age, were randomly selected from the urban and rural areas of Ho Chi Minh City, Vietnam. The presence of LPL S447X and apoCIII polymorphism was determined using the Invader assay, and the plasma lipid concentrations were measured by an enzymatic method. Dietary intakes and anthropometrics were assessed for each child. The frequencies of LPL 447X (11.9%) and apoCIII S2 (32.6%) polymorphisms in the Vietnamese were similar to those of other Asian populations. The frequency of LPL 447X was the same as that in Caucasians but the frequency of apoCIII S2 was approximately 3.8 times that in Caucasians. Overall, LPL 447X polymorphism was associated with a higher HDL-cholesterol concentration (7.4%, P = 0.007) and a lower triglyceride concentration (-13.6%, P = 0.04) than LPL 447S. The apoCIII S2 polymorphism was not associated with an increase in the plasma triglyceride concentration in Vietnamese girls with a low fat intake. However, due to the high frequency of the apoCIII SstI polymorphism and the increasingly westernized diet in Vietnam, attention should be paid to the interaction of genotype with the Vietnamese diet.     

载脂蛋白A5 c.553G>T基因多态性与新疆汉族人群冠心病相关性研究

目的 研究新疆汉族人群载脂蛋白A5(ApoA5)c.553G＞T多态性与冠心病及血脂水平的相关性.方法 采用聚合酶链反应-限制性片段长度多态性方法,检测486例冠心病患者和501例对照的ApoA5 c.553G＞T多态性,同时测定血脂水平.结果 ApoA5 c.553G＞T的不同基因型在冠心病组和对照组分布频率的差异具有统计学意义(x2=8.757,P=0.013).通过非条件logistic回归校正年龄、性别、吸烟史、血脂、高血压和糖尿病史等影响因素后,T等位基因携带者(TT+GT基因型)的个体患冠心病的风险是GG型的1.753倍(OR=1.753,95%CI:1.030～2.983,P＜0.05).冠心病组和对照组c.553G＞T基因型亚组间甘油三酯(TG)水平的差异有统计学意义(t=-5.242,P＜0.01;t=-3.499,P=0.001),T等位基因携带者较GG型有更高的TG水平;冠心病组T等位基因携带者较GG型有更高的胆固醇(TC)水平(t=-2.465,P=0.014).结论 ApoA5c.553G＞T多态性对新疆汉族人群血清TG、TC水平有影响,且与冠心病的发生有一定相关性,T等位基因可能是冠心病的一个危险因素.

Abstract：

Objective The aim is to investigate the association between coronary heart disease (CHD) and c.553G＞T polymorphism of apolipoprotein A5 (ApoA5) gene and the influence of serum lipid level in the Hah ethnic population of Xinjiang. Methods The polymorphism of ApoA5 gene in 486 patients with CHD and 501 controls was analyzed by methods of polymerase chain reaction and restriction fragment length polymorphism analysis. Level of serum lipid in each patient was detected at the same time. Results There was significant difference in the distribution of genotypes between CHD group and controls group ( x2 = 8.757, P= 0.013 ). Non-conditioned logistic regression analyses, after adjusted for age, gender, smoking, total serum cholesterol, presence of hypertension and diabetes, revealed that individuals who carried T allele (TT + GT genotype) had an increased risk of CHD, compared to GG genotype (OR= 1.753,95%CI: 1.030-2.983, P＜0.05 ). There was also a remarkable difference noticed in the level of serum triglyceride by genotypes in CHD group and control group (t=5.242, P＜0.01; t=-3.499, P=0.001 ). Individuals in the two groups who carried T allele had higher level of serum triglyceride than those carried GG genotype. Individuals in CHD group who carried T allele had higher level of serum total cholesterol than those carried GG genotype (t=-2.465, P=0.014). Conclusion It seemed that the c.553G＞T polymorphism of ApoA5 gene had influenced on the level of serum triglyceride and the total cholesterol among Han population in Xinjiang. c.553G＞T polymorphism was associated with the development of CHD, while T allele might be an influencing risk factor on CHD.     

FTO基因单核苷酸多态性与2型糖尿病发病风险的相关性研究

目的探讨FTO(fat mass and obesity associated)基因单核苷酸多态性(single nucleotide polymorphism,SNP)与中国人群Ⅱ型糖尿病(Type Ⅱ Diabetes Mellitus,T2DM)易感性的关系。方法利用Sequenom Mass ArrayiPLEX系统对238例T2DM患者及239例健康对照的FTO基因单核苷酸多态性位点rs8050136进行基因分型,并对检测结果根据共显性、显性模型、超显性和隐性模型进行χ2检验和非条件Logistic回归分析。结果 FTO基因rs8050136位点在病例组和对照组的基因型频率分布差异在显性模型和超显性模型中有显著性(χ2=8.603,P=0.003;χ2=5.428,P=0.02)。相对CC基因型而言,CA杂合型和CA-AA基因型均能增加T2DM发病的危险性,(OR=1.751,95%CI:1.129～2.717,P=0.012;OR=1.915,95%CI:1.241～2.954,P=0.003);而两组间等位基因频数分布也有显著性差异(χ2=10.614,P=0.001),相对于C位点而言,A位点是T2DM的危险等位位点,(OR=1.933,95%CI:1.298～2.880,P=0.001)。结论 FTO基因单核苷酸多态性rs8050136与T2DM的发病风险相关,CA杂合型和CA-AA基因型可显著增加T2DM的发病风险。     

Seven lipoprotein lipase gene polymorphisms, lipid fractions, and coronary disease: a HuGE association review and meta-analysis

Lipoprotein lipase (LPL) is a key enzyme in lipoprotein metabolism and a major candidate gene for coronary heart disease (CHD). The authors assessed associations between 7 LPL polymorphisms and lipid fractions and CHD risk in population-based cohort, case-control, and cross-sectional studies published by January 2007. Meta-analyses of 22,734 CHD cases and 50,177 controls in 89 association studies focused on the relations of the T-93G (rs1800590), D9N (rs1801177), G188E, N291S (rs268), PvuII (rs285), HindIII (rs320), and S447X (rs328) polymorphisms to high density lipoprotein cholesterol, triglycerides, myocardial infarction, or coronary stenosis. Carriers of 9N or 291S had modestly adverse lipid profiles. Carriers of the less common allele of HindIII or of 447X had modestly advantageous profiles. The combined odds ratio for CHD among carriers was 1.33 (95% confidence interval (CI): 1.14, 1.56) for 9N, 1.07 (95% CI: 0.96, 1.20) for 291S, 0.89 (95% CI: 0.81, 0.98) for the less common HindIII allele, and 0.84 (95% CI: 0.75, 0.94) for 447X. For T-93G (odds ratio (OR) = 1.22, 95% CI: 0.98, 1.52) and PvuII (OR = 0.96, 95% CI: 0.89, 1.04), there were null associations with lipid levels or CHD risk; information on G188E was limited (OR = 2.80, 95% CI: 0.88, 8.87). The study of LPL genotypes confirms the existence of close interrelations between high density lipoprotein cholesterol and triglyceride pathways. The influence of these genotypes on CHD risk warrants further investigation.     

Variation at the lipoprotein lipase and apolipoprotein AI-CIII gene loci are associated with fasting lipid and lipoprotein traits in a population sample from Iceland: Interaction between genotype,gender, and smoking status

The effects of polymorphisms in the lipoprotein lipase (LPL) gene (HindIII and S447X) and in the apolipoprotein (apo) AI-CIII gene cluster (G75A and C1100T) on levels of fasting plasma triglycerides, apoCIII, high density lipoprotein cholesterol (HDL-C), and apoAI were examined in 315 healthy men and women from Iceland. Non-smoking and smoking men and women were examined separately because of the strong effects of smoking status and gender on lipoproteins. For the LPL gene, there were no significant associations between plasma traits and genotypes of the S447X polymorphism, but the LPL-HindIII polymorphism was associated with significant effects on levels of all traits, with the effect of genotype on triglycerides and apoAI being modulated by smoking status, (genotype × smoking interaction, P < .02). The H- allele was generally associated with slightly lower levels of apoCIII, with a lowering effect on triglycerides only in smokers and with a raising effect on ApoAI in non-smoking and smoking men and in non-smoking women. For the apoCIII C1100T polymorphism, smoking and non-smoking men with one or more T alleles had levels of triglycerides roughly 10% higher than those with only the C allele (P = .004 overall). In the non-smoking men, nonlinear additive effects were observed with combinations of genotypes at the LPL and apoAI-CIII loci, with the HDL-C and apoAI raising effect associated with the A75 allele and H- allele seen only in those men with both alleles, and the apoCIII raising effect associated with the H+ and T alleles seen only in those with both alleles. Thus, variations at both of the LPL and apoAI-apoCIII loci influence levels of triglycerides, apoCIII, HDL-C, and apoAI, but these effects are strongly modulated by smoking and are different between men and women. The mechanisms for these interactions between smoking or gender and genes are unknown, but future studies should take such interactions into account. Genet. Epidemiol. 14:265–282,1997. © 1997 Wiley-Liss, Inc.