STIM1和TRPC1基因多态性与闽西南地区泌尿系含钙结石患者相关性的研究及结石成分分析
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摘要
研究背景
泌尿系统结石是一种全世界范围内的常见病和多发病,初发结石的复发率10年内高达50%0,21。而据统计,在我国,泌尿系结石人群中普遍发病率达1%～5%,在南方城市中的发病率更是高达5%～10%,其中约25%的患者需住院治疗,在泌尿外科住院患者中居首位。随着近年来泌尿系结石发病率的增高,我国现已成为世界上三大结石高发地区之一。另外泌尿系结石易复发,在体外冲击波碎石术(extracorporeal shock wave lithotripsy, ESWL)术后1年内复发率可达4%,5年总的复发率可达15.6%,10年内的复发率为10%,15年的复发率更是高达75%。同时泌尿系结石的发病率仍有继续升高的趋势,给结石患者的工作和生活带来极大的不便,并增加对国民健康的危害以及国家医疗资源的消耗,对家庭和社会造成巨大的经济负担。
近年来,随着科学技术的发展与革新,体外冲击波碎石术、经皮肾镜碎石取石术(percutaneous nephrolithotomy, PCNL)、腹腔镜取石术和输尿管镜碎石术等微创治疗技术也取得了长足的进步与发展,改变了长期以来以开放性手术为主的传统治疗方法,为患者的治疗和痊愈带来福音。但泌尿系结石较高的复发率与残石率使针对其的大部分治疗仍旧停留在“治标不治本”的尴尬阶段,随着治疗技术的进步,其发病率和复发率仍然居于高位,特别是上尿路结石的发病率升高趋势依旧明显。同时结石也是泌尿系统疾病如：肿瘤、尿路感染、尿路梗阻等的诱因,因此预防结石和从病因学角度研究、诊断结石已成为当前的迫切需要。
泌尿系结石的病因简而言之就是能够引起尿石症发生的各种因素的总和。迄今为止,尿石症的病因不明,目前普遍认为泌尿系结石是多种因素共同作用的结果,通常将这些因素分为个体因素与环境因素,也可以认为是内因和外因,个体因素包括遗传因素、年龄、性别、代谢异常、尿路局部病变(感染、梗阻、异物)、个人生活习惯、肥胖等；环境因素包括气候、职业、个人饮食习惯、职业、社会发展状况与人营养水平等。
泌尿系结石成分分析是当今确定结石性质的重要方法之一,是临床结石综合治疗的重要“病理”性诊断,也是结石病因诊断中的关键环节。在诊断方面上,它可以对非含钙结石病因判断提供最为直接的证据,而且对于含钙性结石可进一步缩小结石代谢性评估的范围。在临床治疗上,它是溶石治疗和结石预防的重要根据,也可对尿路结石患者进行个体化治疗提供有益思路,也是结石防治个体化治疗的前提条件。除了外在因素影响结石形成,机体的代谢异常会导致尿液成石因子和抑石因子的变化,对尿路结石形成作用巨大,研究含钙结石患者代谢异常,对预防结石复发有重要意义。研究这类患者合并代谢异常的状况,为今后在制定预防结石复发策略方面提供参考。
近年来,随着芯片技术、测序技术等相关遗传学研究技术的进步,泌尿系结石遗传因素的研究越来越受到关注和重视。在遗传因素研究中,单核苷酸多态性(single nucleotide polymorphisms, SNP)的研究在这些年来受到足够的重视,作为人类可遗传的变异中最常见的一种,用于阐释并寻找某种基因变异与表型(疾病)之间关系(相关性)。SNP(单核苷酸多态性)主要是指在基因组水平上由单个核苷酸的变异所引起的DNA序列多态性。SNP在人类基因组中广泛存在,占所有已知多态性的90%以上。
泌尿系结石按晶体成分分类分为含钙结石(calcium urolithiasis)和非含钙结石(none-calcium urolithiasis),其中以含钙结石为主,同时在泌尿系结石中草酸钙结石最为常见,约占68.7%-90.0%。过去研究表明钙离子通道可能在炎症反应和免疫反应中扮演着重要角色,同时泌尿系结石,特别是草酸钙结石与炎症反应息息相关,因此泌尿系结石与钙离子通道的单核苷酸多态性的研究作为结石遗传因素一部分具有较大的意义。过去研究表明钙离子通道可能是含钙结石发生发展的原始调控阀,而钙池操纵的钙通道(store-operated calcium channel, SOCC)作为非兴奋性细胞Ca2+内流最主要的通道成为目前研究最多、最热门的钙离子通道。经典瞬时感受器电位蛋白(canonical transient receptor potential, TRPC)、肌浆网钙泵(sarcoplasmic reticulum Ca2+-AT-Pase, SERCA)和微管结合蛋白(microtubule end binding protein, EBl)等参与了SOCC介导的钙内流(store-operated Ca2+entry, SOCE),它们与STIM (stromal interaction molecule:基质交感分子)、钙释放激活钙通道蛋白(calcium release-activated calcium channel protein, ORAI)钙池操纵钙内流复合体(store-operated calcium influx complex, SOCIC)。STIM1是内质网钙受体基因库损耗后的接受因子,钙通道ORAI1是一种细胞表面蛋白,当钙池中的钙被消耗之后,STIM1接受钙池充盈状态的信息时,其向细胞膜逐渐靠近,并且通过羧基端与ORAI1一起将细胞膜上的钙离子通道开放,从而介导形成SOCC介导的钙内流(store-operated Ca2+entry, SOCE),进而参与细胞信号的传递,影响生理机能。TRPC与ORAI1, STIM1一起作为SOCC主要构成成分,在人体中与其一起作为一个整体,对疾病的发生发展产生作用。Yii-Her Chou等认为ORAI1基因两个SNP位点(rs12313273和rs6486795)与含钙结石的发生与复发具有相关性。通过病例对照研究分析STIM1、TRPC1基因多态性与泌尿系含钙结石的遗传易感性,可以为泌尿系结石的综合防治提供遗传学上参考信息。
第一章STIM1和TRPC1基因多态性与闽西南地区泌尿系含钙结石患者相关性研究
目的
泌尿系结石中草酸钙结石最为常见,约占68.7%-90.0%。福建地区尿路结石成分主要以草酸钙和磷酸钙结石为主,占95%以上。过去研究表明钙离子通道可能是含钙结石发生发展的原始调控阀,而钙池操纵的钙通道作为非兴奋性细胞Ca2+内流最主要的通道成为目前研究最多、最热门的钙离子通道。遗传因素在泌尿系结石的发病中起到一定的作用。目前关于基因多态性与泌尿系结石成因的研究仍主要以欧美国家的资料为主,国内关于钙池操纵的钙通道蛋白基因多态性研究与泌尿系结石相关性研究仍较少。本课题拟检测STIM1、TRPC1基因的多个SNP位点在正常人群、含钙结石患者中的基因型分布,通过病例对照研究进一步分析STIM1、TRPC1基因多态性与泌尿系含钙结石的遗传易感性,为泌尿系结石的综合防治提供有益的遗传信息参考。
方法
1.选取2013年1月至2014年1月我院收治的福建省闽西南地区并确诊为泌尿系含钙结石患者295名,同期选取本院体检中心收集的198名健康体检者作为对照组,两组均排除高血压、冠状动脉硬化性心脏病、痛风、糖尿病、高脂血症、恶性肿瘤等重要器官疾病。2.采用Generay血液基因组DNA提取试剂盒提取两组的外周血DNA,然后置于-80℃冰箱中保存。3根据公共SNP数据库hapmap数据库(http://hapmap.ncbi.nlm.nih.gov/)以及相关文献,选择STIM1和TRPC1基因中频数≥0.05,且与钙池操纵的钙通道调控相关的8个SNP位点,STIM1的五个SNP位点(rs3750994、rs2304891、 rs3750996、rs3813880、rs10835206), TRPC1三个位点(rs7638459、rs2033912、 rs3821647),5’端兼启动区域位点为rs10835206,内含子位点rs7638459、 rs2033912;同义突变位点为rs2304891、rs3821647;启动区域位点为rs3813880。
4.采用Sequenom MassARRAY法检测STIM1、TRPC1基因的多个SNP位点在正常人群、含钙结石患者中的基因型分布。首先根据SNP位点通过Sequenom公司的Assay Design3.1软件进行引物设计,将合成的引物,通过基质辅助激光解吸电离飞行时间质谱仪(Matrix-assisted laser desorption ionization time of flight mass spectrometry, MALDI-TOF-MS)进行质检提纯,接着进行PCR扩增反应、SAP酶消化反应、单碱基延伸反应、树脂纯化等步骤,最终将样品进行MALDI-TOF-MS反应,用Typer4.0软件检测质谱峰,并根据质谱峰图判读各样本目标位点基因型。
5.应用R statistics program2.15.3统计包、SPSS20.0统计软件包和Excel软件分析。应用SPSS20.0进行两组间基线数据分析处理,计量资料的组间差异比较采用t检验,计数资料采用X2检验,P<0.05为差异具有统计学意义。利用Excel进行各位点等基因及基因型在病例组与正常对照组中的Harding-Weinberg定律检验；应用R statistics program2.15.3分析各位点等位基因及基因型在病例组与正常对照组中的基因多态性分布。采用haploview4.2软件构建单倍体型,计算D’和r2,分析同一基因各位点之间的连锁不平衡关系,并行单倍型分析。
结果
1.泌尿系含钙结石组和对照组基线资料对比：病例组年龄(47.88±14.07)岁,体重指数(body mass index, BMI)(24.33+3.07),其中男176例,女119例；对照组年龄(47.72±14.78)岁、BMI指数(23.89±3.51),其中男120例,女78例。病例组及对照组性别比(P=0.834)、年龄(P=0.143)和BMI指数(P=0.899)构成上差异无统计学意义。
2. STIM1和TRPC1多态性位点基因型检测及分布：8个多态性位点包括STIM1的五个SNP位点(rs3750994、rs230489、rs3750996、rs3813880、 rs10835206), TRPC1三个位点(rs7638459、rs2033912、rs3821647)均成功检测。各位点基因型分布均符合Harding-Weinberg平衡(P<0.05,具体详见正文),说明样本具有良好的群体代表性。
3. TRPC1和STIM1基因多态性与泌尿系含钙结石的相关性：各位点等位基因及基因型在病例组与正常对照组中的基因多态性分布以及利用RSTATISTICS PROGRAM2.15.3统计包进行wilcoxon秩和检验,在显著性水平α=0.05,双侧检验下,STIM1基因上的rs10835206这个位点的基因型在正常组和含钙结石组之间是有显著性差异(Z=24101,P=0.0003355),病例组突变基因型(CT+TT)频率(86.10%vs81.82%)以及突变基因T频率(382vs223)高于正常组且有统计学差异；其他位点病例组与对照组差异无统计学意义(P>0.05)。
4. TRPC1和STIM1多态性位点单体型与含钙结石的关系：利用haploview4.2进行连锁不平衡分析(linkage disequilibrium, LD),经统计分析可知TRPC1中rs7638459与rs2033912为完全连锁不平衡关系(D’=1.0,r2=1.0),但是统计学无显著差异；STIM1中各位点为不完全连锁平衡关系,无法构成单倍体。
结论
1. STIM1基因多态性与泌尿系含钙结石发病相关,对于STIM1基因多态性检测可能对预测泌尿系含钙结石的发病危险提供有用的遗传信息。STIM1基因多态性也可能成为一个预判泌尿系结石复发的遗传标记。
2. TRPC1中rs7638459与rs2033912为完全连锁不平衡关系,但是统计学无显著差异；STM1中各位点为不完全连锁平衡关系,无法构成单倍体。
3.与钙池操纵的钙通道调节密切相的TRPC1基因的遗传变异和泌尿系含钙结石的发病危险或者临床表型可能没有相关性。
第二章闽西南地区861例尿路结石成分分析及防治对策
目的
尿路结石是我国特别是南方地区最为常见的泌尿系统疾病之一,影响尿路结石形成的因素及机制很多,年龄、性别、遗传、种族、饮食习惯、环境因素和职业对结石的形成影响很大。福建的闽西南地区是典型的南方气候,有较高的结石发病率,不过在我省地区,关于尿路结石患者所患结石类型、各类型结石所占比例、年龄、性别间差异的相关研究匿乏。不同的结石成分也隐含着结石形成原因的相关信息。本课题通过收集整理泌尿外科尿路结石患者结石成分分析的资料,分析本地区结石发病特点、性别年龄差异、地区差异、结石成分构成特点等。通过对本地区尿路结石患者的结石成分进行分析,了解和掌握本地区尿路结石的流行病学和结石构成情况,为尿路结石的临床预防和综合治疗提供有益的帮助。
方法
收集我院2010年6月～2013年6月期间收治福建省闽西南地区泌尿系结石患者共861例,所有患者均采用蓝莫德公司LIIR20型红外光谱仪,采用定性实验进行结石成分分析检测,并以尿路结石患者性别、年龄、结石部位、居住地等为分组依据,分析各组尿路结石的化学成分特征。采用SPSS20.0统计软件记录并分析检出率,对不同性别间各种成分结石的检出率用卡方检验比较,检验水准为a=0.05。
结果
1.本地区尿路结石发病基本情况
患者年龄3-91岁,平均48.92岁,农村463例,城市398例,农村城市比为1.16：1。结石发病男性多于女性。其中男593例,女268例,男女比为2.21：1。随着年龄增长,尿路结石的构成比逐渐升高,≤19岁患者最少,仅1.51%,30-60岁时达高峰,总和为64.12%。
2.各个部位尿路结石发病情况和成分分析结果
各个部位结石性别分布情况如下：上尿路结石711例,下尿路结石150例,上尿路和下尿路结石比为4.74：1。总的结石男女比为2.21：1,膀胱结石女性低于男性,男女性别比为7.31：1,输尿管结石男女性别比为2.61：1。女性肾结石的构成比较高,其男女性别比为1.42：1,另有检出尿道结石17例,均为男性。
尿路结石主要分布在上尿路,肾结石占46.92%。输尿管结石占35.66%,膀胱结石15.45%,尿道结石1.97%。各部位的尿路结石均以碳酸磷灰石+草酸钙结石(COS, calcium oxalate stones)组成的混合结石成分为主(44.72%),单一草酸钙结石成分居次(31.24%)。感染性结石(含六水磷酸镁铵成分)主要存在于膀胱结石和肾结石中(分别为3.48%和3.25%),尿酸(uric acid, UA)结石也是主要分布于膀胱结石和肾结石中(分别为5.69%和3.14%)。
3.总体结石成分分析结果
从结石成分构成看,单纯性结石占41.00%,两种成分混合性结石占49.94%,3种成分及以上混合性结石占9.06%。单纯性结石主要以草酸钙结石为主(31.24%),混合性结石主要以草酸钙和碳酸磷灰石组成的混合性结石为主(44.72%)。其他混合性结石比例按从多到少依次为六水磷酸镁铵+碳酸磷灰石+草酸钙7.67%,尿酸+草酸钙2.44%,六水磷酸镁铵+尿酸铵+碳酸磷灰石1.39%,尿酸铵+草酸钙1.05%,二水磷酸氢钙+草酸钙0.93%,L-胱氨酸+草酸钙0.46%,L-胱氨酸+碳酸磷灰石0.35%。其他单纯性结石比例按从多到少依次为尿酸结石5.34%,尿酸铵结石1.97%,碳酸磷灰石1.74%,二水磷酸氢钙0.70%。
从结石成分检出率来看,总体检出率最高的为草酸钙(88.50%),其次是碳酸磷灰石(55.87%),无水尿酸或尿酸铵(12.20%)、六水磷酸镁铵(9.06%)、二水磷酸氢铵钙(1.63%)、L-胱氨酸(0.81%)等成分结石所占比例较低。593例男性中检出率最高也是草酸钙(88.03%),其次依次是碳酸磷灰石(54.47%)、无水尿酸或尿酸铵(14.00%)、六水磷酸镁铵(6.75%)、二水磷酸氢铵钙(1.69%)、L-胱氨酸(0.84%)。268例女性中检出率最高也是草酸钙(89.55%),其次依次是碳酸磷灰石(58.96%)、六水磷酸镁铵(14.18%)、无水尿酸或尿酸铵(8.21%)、二水磷酸氢铵钙(1.49%)、L-胱氨酸(0.75%)。
男女尿路结石成分存在差异,无水尿酸或尿酸铵中男性检出率高于女性(14.00%vs8.21%,P=0.016),六水磷酸镁铵则相反,男性低于女性(6.75%vs14.18%,P=0.000)。草酸钙、碳酸磷灰石、L-胱氨酸、二水磷酸氢铵钙男女检出率没有显著性差异。
4.复发性结石成分分析情况
我们统计了其中22例复发性结石的成分分析结果,主要为混合性结石为主,感染性结石复发率最高,有10例(45.45%)为六水磷酸镁铵+碳酸磷灰石+草酸钙的混合性结石,其次有8例(36.37%)为草酸钙+碳酸磷灰石的混合性结石。其余为胱氨酸+碳酸磷灰石混合性结石2例,尿酸结石+草酸钙混合性结石2例。
结论
1.本地区结石发病情况为,尿路结石男性发病多于女性,30～60岁组尿路结石构成比达高峰,≤19岁构成比最低,上尿路结石多于下尿路结石,农村发病多于城市。
2.各部位的尿路结石均以碳酸磷灰石+草酸钙组成的混合结石成分为主,单一草酸钙结石成分居次。感染性结石(含六水磷酸镁铵成分)、尿酸性结石主要存在于肾结石和膀胱结石中。
3.从结石成分构成看,多种成分混合性结石占大多数。混合性结石主要以草酸钙和碳酸磷灰石组成的混合性结石为主。结石成分检出率最高的为草酸钙,其次是碳酸磷灰石、无水尿酸或尿酸铵、六水磷酸镁铵、二水磷酸氢铵钙、L-胱氨酸等。
4.男女尿路结石成分存在差异,无水尿酸或尿酸铵中男性检出率高于女性,六水磷酸镁铵则相反,男性低于女性。
5.以六水磷酸镁铵为主要成分的感染性结石复发率最高,其次为草酸钙+碳酸磷灰石的混合性结石。
6.本地区尿路结石化学成分以草酸钙为主,草酸钙结石所占比率高于国内其他省份,结石成分分析对于了解本地区结石成因,指导治疗和预防结石具有重要意义。
Background
Urolithiasis is a worldwild commonly encountered disease, the recurrence rate of primary caculary in10years is attain50%. According to statistics, the incidence of the urolithiasis is1%～5%in our country, while which reaches up to5%～10%. And25%of these patients need to be in hospital, which ranks first in urology inpatients. With the increased incidence of urinary calculi in recent years, China has now become one of the three stone-prone areas of the world. In addition, urinary stones recur easily. After extracorporeal shock wave lithotripsy (ESWL), the1-year recurrence rate is4%, and5-year recurrence rate is15.6%,10-year recurrence rate is10%,15-year recurrence rate is as high as75%.The incidence of urinary tract stones still rise, which greatly inconvenience the work and life of the patients. It also hazards national health and consumes the national resources, brings about huge economic burden to the family and society.
In recent years, with the development of science and technology and innovation, the minimally invasive techniques such as extracorporeal shock wave lithotripsy, percutaneous nephrolithotomy, laparoscope lithotomy and ureteroscopic lithotripsy, had made considerable progress and development. It changed the traditional method of treatment which based on open surgery for a long time and brought the gospel to the patients. However, because urinary calculi has high recurrence rate and residual stone rate, we cannot solve the problem thoroughly. In particular the increased incidence of urinary tract stones is still evident. In the meantime, urolithiasis is the inducement of some urinary system diseases, such as, tumors, urinary tract infection and urinary obstruction. Nowadays, it is very important to prevent urolithiasis and to research urolithiasis in nosazontology.
Until nowadays, the nosazontology of urolithiasis is still not clear. Now widely recognized that urinary calculi are the result of many factors. These factors are usually divided into individual and environmental factors. Individual factors include genetic factors, age, sex, metabolic disorders, urinary local lesions (Infection, obstruction, and foreign bodies), personal habits, obesity, etc. Environmental factors, including climate, occupation, personal eating habits, occupation, social development and human nutrition, etc.
Urinary stones component analysis is one of the important methods to determine the nature of the stones. It is an important "pathological" diagnosis for the comprehensive clinical treatment of stones. On diagnosis, it provides the most direct evidence to judge the etiological diagnosis of non-calcium stones and for calcium stones it can further narrow the scope of the assessment of metabolic. It is important evidence on dissolution therapy and prevention of stones in the clinical treatment. It also provides useful ideas for individualized treatment to urolithiasis patients. In addition to external factors for stone formation, metabolic abnormalities may also lead to changes in the urine stone promoter factor and suppression factor. Metabolism studies in patients with calcium stones are important to prevent stone recurrence. We should research patients with metabolic abnormalities, and it is important in the development of future strategies for the prevention of recurrence of stones.
For the past few years, with advances in chip technology, genetics sequencing technology and other related technologies, people pay more attention to the studies of genetic factors of urinary calculi. In these years scientists have played sufficient attention to single nucleotide polymorphisms (SNP). Single nucleotide polymorphisms is one of the most common human genetic variation, which Interprets the relationship between genetic variation and phenotype (disease). Single nucleotide polymorphisms mainly refers to a DNA sequence polymorphisms in the genome caused by a single nucleotide mutation. SNP is widespread in the human genome, which accounting for over90%of all known polymorphisms. Crystal composition of urinary calculi is divided into calcium stones and non-calcium stones. Calcium stones account for most of the urinary calculi. Calcium oxalate stones are most common in urinary calculi, accounting for about68.7%to90.0%. Past studies have shown that calcium channels may play an important role in inflammation and immune response. Meanwhile urinary stones, especially calcium oxalate stones are closely related with inflammation. Therefore, single nucleotide polymorphism studies of urinary calculi with calcium ion channels have greater significance as part of the genetic factors stones.
Chapter I Associations of Polymorphisms in STIM1/TRPC1Genes with Calcium Urolithiasis Patients in Southwestern Fujian Province
Objective
Calcium oxalate stones are most common in urinary calculi, accounting for about68.7%to90.0%.And as for Fujian province, calcium oxalate stones and calcium phosphate stone are most common in urinary calculi, accounting for about more than95.0%. Past studies have shown that calcium channels may play an important role in inflammation and immune response. Therefore, a single nucleotide polymorphism studies of urinary calculi with calcium ion channels have greater significance as part of the genetic factors stones. Past studies have shown that calcium channel may be the original regulating valve of development of calcium stones. Current research on the correlation of urinary calculi and gene polymorphism is still mainly from West countries, the domestic correlation research of SOCC genetic polymorphisms and urinary stones is still rare. In this study, genotypes of the8SNP loci in STIM1and TRPC1gene will be detected by Sequenom MassARRAY. Through analysis of genetic susceptibility of STIM1, TRPC1gene polymorphism and urinary calcium stones by case-control study, it can provide reference information on the comprehensive prevention and treatment of urinary calculi in genetics.
Methods
1.Clinical data of295patients with identified urinary calculi and198healthy individuals living in Southwestern Fujian province from January2013to January2014were retrospectively collected. Both groups were excluded hypertension, coronary artery disease, gout, diabetes, hyperlipidemia, cancer and other diseases of vital organs.
2. Genomie DNA was extracted by Generay DNA Purification Kit from peripheral blood samples of both groups, and stored at-80℃.
3. Based on the online public hapmap databases (http://hapmap.ncbi.nlm.nih.gov/) and published documents,8DNA polymorphism loci at STIM1/TRPC1genes with a minor allele frequency≥0.05and potential associations with SOCC were selected for our study.5DNA polymorphism loci at STIM1are rs3750994, rs2304891, rs3750996, rs3813880and rs10835206,3DNA polymorphism loci at TRPC1are rs7638459, rs2033912and rs3821647. rs10835206is in5' promoter, rs7638459and rs2033912are in introns, rs2304891and rs3821647are same sense mutation locis, rs3813880is in promoter.
4. Detect the genotypes of the8SNP loci in STIM1and TRPC1genes by Sequenom MassARRAY. Firstly, primer was designed according to the SNP site by Sequenom's Assay Design3.1software. The primers were synthesized by Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) for the purification quality. Followed by a PCR amplification reaction, SAP enzyme digestion reaction, single base extension reaction, resins purification and so on. Finally, the sample was detected by MALDI-TOF-MS, then detected the mass peak detection by Typer4.0software, interpreted each sample target locus genotype based on mass peak figure.
5. Applications of R statistics program2.15.3statistical package, SPSS20.0statistical analysis software packages and Excel for statistical analysis. Baseline between the two groups was analyzed by SPSS20.0statistical analysis software, measurement data between groups were compared using t-test, count data using Chi-square. P<0.05was considered statistically significant. Used Excel for Harding-Weinberg's law test in allele and genotype between the case group and the control group. Analyzed the allele and genotype polymorphism analysis in patients and normal control group by R statistics program2.15.3. Builted haplotype of the genes by calculating D'and r2in Haploview4.2software, analyzed the linkage disequilibrium between all sites in the same gene, then completed parallel haplotype analysis.
Results
1. Baseline data of case group and control group:Mean age of case group was (47.88±14.07) years, body mass index (BMI) was24.33±3.07, male173cases, female119cases. Mean age of control group was (47.72±14.78) years, body mass index (BMI) was23.89±3.51, male120cases, female78cases. The difference of sex ratio (P=0.834), age (P=0.143) and BMI (P=0.899) between Case and control group was not statistically significant (P>0.05).
2. Genotyping and distribution of STIM1and TRPC1genes:5DNA polymorphism loci at STIM1including rs3750994, rs2304891, rs3750996, rs3813880and rs10835206,3DNA polymorphism loci at TRPC1including rs7638459, rs2033912and rs3821647were successfully detected. The distribution of genotypes of each loci in accordance with Hardy-Weinberg Equilibrium (HWE) in case and control group (P>0.05).
3. Association between STIM1/TRPC1gene polymorphisms and Calcium urolithiasis. Analyzed the allele and genotype polymorphism analysis in patients and normal control group by R statistics program2.15.3, using Wilcox non-parametric test. Under the significance level a=0.05, two-tailed test, the genotype of rs1083206between case and control group was significantly different (Z=24101,P=0.0003355). The mutant genotype (CT+TT) frequency(86.10%vs.81.82%) and the mutant gene T frequency(382vs.223) of case group was statistically higher than the control. Other sites of case and control groups showed no significant difference (P>0.05).
4. Relations between TRPC1/STIM1polymorphic loci haplotypes and calcium urolithiasis. Analyzed the linkage disequilibrium between all sites in the same gene. The statistical analysis showed that the rs7638459and rs2033912TRPC1were fully linkage disequilibrium (D'=1.0,r2=1.0), however, no statistically significant difference. All the loci in STIM1were incomplete linkage equilibrium, haploid cannot be constituted.
Conclusion
1. DNA polymorphisms within STIM1gene were associated with calcium urolithiasis patients. Detection of polymorphisms in STIM1gene might provide some useful genetic information for prognosing the risk of calcium urolithiasis. It can provide reference information on the comprehensive prevention and treatment of urinary calculi in genetics.
2. The statistical analysis showed that the rs7638459and rs2033912TRPC1were fully linkage disequilibrium. All the loci in STIM1were incomplete linkage equilibrium, haploid cannot be constituted.
3. The TRPC1gene, which is closely relative to SOCC, maybe not associated with the risk and clinical genotype of calcium urolithiasis.
Chapter Ⅱ Analysis of861urinary tract calculus components in Southwestern Fujian province and discussion on prevention strategy
Objective
Urolithiasis is one of the most common urinary system diseases in china, especially in the southern region. Many factors and mechanisms influence the formation of urinary stones. Age, gender, genetics, race, diet, environmental factors and occupational greatly impact on the formation of stones. The Southwestern of Fujian province is a typical southern climate, which has a higher incidence of urolithiasis. But the studies of stone composition, rate and the difference between sex and age are very few in our province. The different composition of stone yields the underlying reasons of stone formation. The stone composition, rate and the difference-between sex and age were computed with the dates of patients with urinary stone. To get data of the stone composition, rate, difference between different groups and to evaluate the underlying reasons of stone formation. To offer objective dates for reinforcing the examination, treatment of the related patients and study of prophylaxis. To study the constituents of urinary stones in Southwestern Fujian province, and provide methods in prevention and treatment of urinary stones.
Methods
Clinical data of861patients with identified urinary calculi living in Southwestern Fujian province from June2010to June2013were retrospectively collected. Infrared spectrophotometry was carried out for biochemical composition analysis of urinary stones retrieved by endoscopic, open surgery, or spontaneously or by extracorporeal shock wave lithotripsy. Gender, age and stone location among these patients were evaluated, and the results were analyzed with biochemical stone analysis. SPSS20.0statistical software was used to record and analyze the detection rate. The detection rate of various stone components for different genders was compared with a chi-square test, the test level was a=0.05.
Results
1. The basic incidence of urinary calculi in the local region
Patients age3to91years, mean48.92years old,463cases of rural,398cases of urban, the rural-urban ratio is1.16:1.The incidence of male urolithiasis is higher than female, male593cases, female268cases, the sex ratio is2.21:1. The incidence of urinary tract stones gradually increases with age,≤19years old group is lowest for only1.51%, and the30-60years group is highest, total for64.12%.
2. The incidences of urinary tract stones in various parts and component analysis
The incidences of urinary tract stones in various parts are as follows:711cases in upper urinary tract,150cases in lower urinary tract, the upper urinary tract to lower urinary tract radio is4.74:1. The sex ratio of the general is2.21:1, the incidence of female bladder calculi is lower than male, and the sex ratio is7.31:1. While that of urethral calculi is2.61:1, closing to the general incidence. However, the constituent ratio of female renal calculi is higher than other, the sex ratio is1.42:1. There are17cases of urethral calculi, only found in male.
The upper urinary tract stones were more frequently found than the lower urinary tract stones, renal calculi accounts for46.92%, ureteral calculi accounts for35.66%, bladder calculi accounts for15.45%, urethral calculi accounts for1.97%. The combination of dahlliter and calcium oxalate stones(44.72%) is the most common combination calculi of all the parts of urinary tracts, the secondary component is pure calcium oxalate stones(COS)(31.24%). Infection stones, which are mainly consisted of ammonium magnesium phosphate hexahydrate, are mostly found in the bladder calculi and renal calculi (respectively3.48%and3.25%), uric acid is also mainly found in the bladder calculi and renal calculi (respectively5.69%and3.14%).
3. The general urinary calculi component analysis
Pure stones are found in41.00%of the stones and two-components mixed stones are49.94%, the above and three-components mixed stones are9.06%. The pure stones are mainly consisted of COS(31.24%), the mixed stones are mainly consisted of COS and dahllite (44.72%). Other mixed stones were listed from more proportion to less as follows:ammonium magnesium phosphate hexahydrate+dahllite+COS7.67%, uric acid(UA)+COS2.44%, ammonium magnesium phosphate hexahydrate+ammonium urate+dahllite1.39%, ammonium urate+dahllite+COS1.05%, Calcium hydrogen phosphate dihydrate+COS0.93%,L-cystine+COS0.46%, L-cystine+dahllite0.35%. Other pure stones were listed from more proportion to less as follows:UA5.34%, ammonium urate1.97%, dahllite1.74%, Calcium hydrogen phosphate dehydrate0.70%.
The most dominant composition of urinary stones was calcium oxalate (88.50%), followed by carbonate apatite (55.87%), uric acid or ammonium urate (12.20%), ammonium magnesium phosphate hexahydrate(9.06%), calcium hydrophosphate dihydrate(1.63%), and cystine (0.81%). For male, the most dominant composition of urinary stones was calcium oxalate (88.03%), followed by carbonate apatite (54.47%), uric acid or ammonium urate (14.00%), ammonium magnesium phosphate hexahydrate(6.75%), calcium hydrophosphate dihydrate(1.69%), and cystine (0.84%). For female, the most dominant composition of urinary stones was calcium oxalate (89.55%), followed by carbonate apatite (58.96%), ammonium magnesium phosphate hexahydrate(14.18%),uric acid or ammonium urate (8.21%), calcium hydrophosphate dihydrate(1.49%), and cystine (0.75%). Urinary stone composition is different in terms of gender, the detection rate of uric acid stones is higher in male than female (14.00%vs.8.21%, P=0.016), inversely, the detection rate of Infection stones was lower in male than female (6.75%vsl4.18%, P=0.000). While there are no difference in terms of gender for COS, dahllite, L-cystine, calcium hydrophosphate dihydrate.
4. Component analysis of recurrence calculi
22recurrence calculi were studied with the component analysis. All of them were consisted of mixed stones. Infection stone has the highest recurrence rate, there were10cases (45.45%)of mixed stones with ammonium magnesium phosphate hexahydrate+dahllite+COS, and8cases (36.37%) of mixed stones with COS+dahllite, and2cases of mixed stones with cystine+dahllite,2cases of mixed stones with UA+COS.
Conclusion
1. The basic incidence of urinary calculi in the local region:
The incidence of male urolithiasis is higher than female, the incidence of urinary tract stones gradually increases with age,≤19years old group is lowest, and the30-60years group is highest. The incidence of rural urolithiasis is higher than urban, The incidence of upper urinary tract is higher than lower urinary tract.
2. The combination of dahlliter and calcium oxalate stones is the most common combination calculi of all the parts of urinary tracts, the secondary component is pure calcium oxalate stones (COS). Uric acid and infection stones, which are mainly consist of ammonium magnesium phosphate hexahydrate, are mostly found in the bladder calculi and renal calculi.
3. Majority of the stones are mixed stones with2or3components. The pure stones are mainly consisted of COS, the mixed stones are mainly consisted of COS and dahllite. The most dominant composition of urinary stones was calcium oxalate, followed by carbonate apatite, uric acid or ammonium urate, ammonium magnesium phosphate hexahydrate, calcium hydrophosphate dihydrate, and cystine.
4. Urinary stone composition is different in terms of gender, the detection rate of uric acid stones is higher in male than female, inversely, and the detection rate of Infection stones was lower in male than female.
5. Infection stones, which are mainly consist of ammonium magnesium phosphate hexahydrate had the highest recurrence rate, followed by the mixed stones with COS and dahllite.
6. The dominant stone composition in inhabitants of Southwestern Fujian province is calcium oxalate, which significantly higher than other regions of China. Chemical composition analysis of urinary stones is very important in providing information to the etiology, treatment and prevention of urinary stones in the local region.
泌尿系统结石是一种全世界范围内的常见病和多发病,初发结石的复发率10年内高达50%0,21。而据统计,在我国,泌尿系结石人群中普遍发病率达1%～5%,在南方城市中的发病率更是高达5%～10%,其中约25%的患者需住院治疗,在泌尿外科住院患者中居首位。随着近年来泌尿系结石发病率的增高,我国现已成为世界上三大结石高发地区之一。另外泌尿系结石易复发,在体外冲击波碎石术(extracorporeal shock wave lithotripsy, ESWL)术后1年内复发率可达4%,5年总的复发率可达15.6%,10年内的复发率为10%,15年的复发率更是高达75%。同时泌尿系结石的发病率仍有继续升高的趋势,给结石患者的工作和生活带来极大的不便,并增加对国民健康的危害以及国家医疗资源的消耗,对家庭和社会造成巨大的经济负担。
近年来,随着科学技术的发展与革新,体外冲击波碎石术、经皮肾镜碎石取石术(percutaneous nephrolithotomy, PCNL)、腹腔镜取石术和输尿管镜碎石术等微创治疗技术也取得了长足的进步与发展,改变了长期以来以开放性手术为主的传统治疗方法,为患者的治疗和痊愈带来福音。但泌尿系结石较高的复发率与残石率使针对其的大部分治疗仍旧停留在“治标不治本”的尴尬阶段,随着治疗技术的进步,其发病率和复发率仍然居于高位,特别是上尿路结石的发病率升高趋势依旧明显。同时结石也是泌尿系统疾病如：肿瘤、尿路感染、尿路梗阻等的诱因,因此预防结石和从病因学角度研究、诊断结石已成为当前的迫切需要。
泌尿系结石的病因简而言之就是能够引起尿石症发生的各种因素的总和。迄今为止,尿石症的病因不明,目前普遍认为泌尿系结石是多种因素共同作用的结果,通常将这些因素分为个体因素与环境因素,也可以认为是内因和外因,个体因素包括遗传因素、年龄、性别、代谢异常、尿路局部病变(感染、梗阻、异物)、个人生活习惯、肥胖等；环境因素包括气候、职业、个人饮食习惯、职业、社会发展状况与人营养水平等。
泌尿系结石成分分析是当今确定结石性质的重要方法之一,是临床结石综合治疗的重要“病理”性诊断,也是结石病因诊断中的关键环节。在诊断方面上,它可以对非含钙结石病因判断提供最为直接的证据,而且对于含钙性结石可进一步缩小结石代谢性评估的范围。在临床治疗上,它是溶石治疗和结石预防的重要根据,也可对尿路结石患者进行个体化治疗提供有益思路,也是结石防治个体化治疗的前提条件。除了外在因素影响结石形成,机体的代谢异常会导致尿液成石因子和抑石因子的变化,对尿路结石形成作用巨大,研究含钙结石患者代谢异常,对预防结石复发有重要意义。研究这类患者合并代谢异常的状况,为今后在制定预防结石复发策略方面提供参考。
近年来,随着芯片技术、测序技术等相关遗传学研究技术的进步,泌尿系结石遗传因素的研究越来越受到关注和重视。在遗传因素研究中,单核苷酸多态性(single nucleotide polymorphisms, SNP)的研究在这些年来受到足够的重视,作为人类可遗传的变异中最常见的一种,用于阐释并寻找某种基因变异与表型(疾病)之间关系(相关性)。SNP(单核苷酸多态性)主要是指在基因组水平上由单个核苷酸的变异所引起的DNA序列多态性。SNP在人类基因组中广泛存在,占所有已知多态性的90%以上。
泌尿系结石按晶体成分分类分为含钙结石(calcium urolithiasis)和非含钙结石(none-calcium urolithiasis),其中以含钙结石为主,同时在泌尿系结石中草酸钙结石最为常见,约占68.7%-90.0%。过去研究表明钙离子通道可能在炎症反应和免疫反应中扮演着重要角色,同时泌尿系结石,特别是草酸钙结石与炎症反应息息相关,因此泌尿系结石与钙离子通道的单核苷酸多态性的研究作为结石遗传因素一部分具有较大的意义。过去研究表明钙离子通道可能是含钙结石发生发展的原始调控阀,而钙池操纵的钙通道(store-operated calcium channel, SOCC)作为非兴奋性细胞Ca2+内流最主要的通道成为目前研究最多、最热门的钙离子通道。经典瞬时感受器电位蛋白(canonical transient receptor potential, TRPC)、肌浆网钙泵(sarcoplasmic reticulum Ca2+-AT-Pase, SERCA)和微管结合蛋白(microtubule end binding protein, EBl)等参与了SOCC介导的钙内流(store-operated Ca2+entry, SOCE),它们与STIM (stromal interaction molecule:基质交感分子)、钙释放激活钙通道蛋白(calcium release-activated calcium channel protein, ORAI)钙池操纵钙内流复合体(store-operated calcium influx complex, SOCIC)。STIM1是内质网钙受体基因库损耗后的接受因子,钙通道ORAI1是一种细胞表面蛋白,当钙池中的钙被消耗之后,STIM1接受钙池充盈状态的信息时,其向细胞膜逐渐靠近,并且通过羧基端与ORAI1一起将细胞膜上的钙离子通道开放,从而介导形成SOCC介导的钙内流(store-operated Ca2+entry, SOCE),进而参与细胞信号的传递,影响生理机能。TRPC与ORAI1, STIM1一起作为SOCC主要构成成分,在人体中与其一起作为一个整体,对疾病的发生发展产生作用。Yii-Her Chou等认为ORAI1基因两个SNP位点(rs12313273和rs6486795)与含钙结石的发生与复发具有相关性。通过病例对照研究分析STIM1、TRPC1基因多态性与泌尿系含钙结石的遗传易感性,可以为泌尿系结石的综合防治提供遗传学上参考信息。
第一章STIM1和TRPC1基因多态性与闽西南地区泌尿系含钙结石患者相关性研究
目的
泌尿系结石中草酸钙结石最为常见,约占68.7%-90.0%。福建地区尿路结石成分主要以草酸钙和磷酸钙结石为主,占95%以上。过去研究表明钙离子通道可能是含钙结石发生发展的原始调控阀,而钙池操纵的钙通道作为非兴奋性细胞Ca2+内流最主要的通道成为目前研究最多、最热门的钙离子通道。遗传因素在泌尿系结石的发病中起到一定的作用。目前关于基因多态性与泌尿系结石成因的研究仍主要以欧美国家的资料为主,国内关于钙池操纵的钙通道蛋白基因多态性研究与泌尿系结石相关性研究仍较少。本课题拟检测STIM1、TRPC1基因的多个SNP位点在正常人群、含钙结石患者中的基因型分布,通过病例对照研究进一步分析STIM1、TRPC1基因多态性与泌尿系含钙结石的遗传易感性,为泌尿系结石的综合防治提供有益的遗传信息参考。
方法
1.选取2013年1月至2014年1月我院收治的福建省闽西南地区并确诊为泌尿系含钙结石患者295名,同期选取本院体检中心收集的198名健康体检者作为对照组,两组均排除高血压、冠状动脉硬化性心脏病、痛风、糖尿病、高脂血症、恶性肿瘤等重要器官疾病。2.采用Generay血液基因组DNA提取试剂盒提取两组的外周血DNA,然后置于-80℃冰箱中保存。3根据公共SNP数据库hapmap数据库(http://hapmap.ncbi.nlm.nih.gov/)以及相关文献,选择STIM1和TRPC1基因中频数≥0.05,且与钙池操纵的钙通道调控相关的8个SNP位点,STIM1的五个SNP位点(rs3750994、rs2304891、 rs3750996、rs3813880、rs10835206), TRPC1三个位点(rs7638459、rs2033912、 rs3821647),5’端兼启动区域位点为rs10835206,内含子位点rs7638459、 rs2033912;同义突变位点为rs2304891、rs3821647;启动区域位点为rs3813880。
4.采用Sequenom MassARRAY法检测STIM1、TRPC1基因的多个SNP位点在正常人群、含钙结石患者中的基因型分布。首先根据SNP位点通过Sequenom公司的Assay Design3.1软件进行引物设计,将合成的引物,通过基质辅助激光解吸电离飞行时间质谱仪(Matrix-assisted laser desorption ionization time of flight mass spectrometry, MALDI-TOF-MS)进行质检提纯,接着进行PCR扩增反应、SAP酶消化反应、单碱基延伸反应、树脂纯化等步骤,最终将样品进行MALDI-TOF-MS反应,用Typer4.0软件检测质谱峰,并根据质谱峰图判读各样本目标位点基因型。
5.应用R statistics program2.15.3统计包、SPSS20.0统计软件包和Excel软件分析。应用SPSS20.0进行两组间基线数据分析处理,计量资料的组间差异比较采用t检验,计数资料采用X2检验,P<0.05为差异具有统计学意义。利用Excel进行各位点等基因及基因型在病例组与正常对照组中的Harding-Weinberg定律检验；应用R statistics program2.15.3分析各位点等位基因及基因型在病例组与正常对照组中的基因多态性分布。采用haploview4.2软件构建单倍体型,计算D’和r2,分析同一基因各位点之间的连锁不平衡关系,并行单倍型分析。
结果
1.泌尿系含钙结石组和对照组基线资料对比：病例组年龄(47.88±14.07)岁,体重指数(body mass index, BMI)(24.33+3.07),其中男176例,女119例；对照组年龄(47.72±14.78)岁、BMI指数(23.89±3.51),其中男120例,女78例。病例组及对照组性别比(P=0.834)、年龄(P=0.143)和BMI指数(P=0.899)构成上差异无统计学意义。
2. STIM1和TRPC1多态性位点基因型检测及分布：8个多态性位点包括STIM1的五个SNP位点(rs3750994、rs230489、rs3750996、rs3813880、 rs10835206), TRPC1三个位点(rs7638459、rs2033912、rs3821647)均成功检测。各位点基因型分布均符合Harding-Weinberg平衡(P<0.05,具体详见正文),说明样本具有良好的群体代表性。
3. TRPC1和STIM1基因多态性与泌尿系含钙结石的相关性：各位点等位基因及基因型在病例组与正常对照组中的基因多态性分布以及利用RSTATISTICS PROGRAM2.15.3统计包进行wilcoxon秩和检验,在显著性水平α=0.05,双侧检验下,STIM1基因上的rs10835206这个位点的基因型在正常组和含钙结石组之间是有显著性差异(Z=24101,P=0.0003355),病例组突变基因型(CT+TT)频率(86.10%vs81.82%)以及突变基因T频率(382vs223)高于正常组且有统计学差异；其他位点病例组与对照组差异无统计学意义(P>0.05)。
4. TRPC1和STIM1多态性位点单体型与含钙结石的关系：利用haploview4.2进行连锁不平衡分析(linkage disequilibrium, LD),经统计分析可知TRPC1中rs7638459与rs2033912为完全连锁不平衡关系(D’=1.0,r2=1.0),但是统计学无显著差异；STIM1中各位点为不完全连锁平衡关系,无法构成单倍体。
结论
1. STIM1基因多态性与泌尿系含钙结石发病相关,对于STIM1基因多态性检测可能对预测泌尿系含钙结石的发病危险提供有用的遗传信息。STIM1基因多态性也可能成为一个预判泌尿系结石复发的遗传标记。
2. TRPC1中rs7638459与rs2033912为完全连锁不平衡关系,但是统计学无显著差异；STM1中各位点为不完全连锁平衡关系,无法构成单倍体。
3.与钙池操纵的钙通道调节密切相的TRPC1基因的遗传变异和泌尿系含钙结石的发病危险或者临床表型可能没有相关性。
第二章闽西南地区861例尿路结石成分分析及防治对策
目的
尿路结石是我国特别是南方地区最为常见的泌尿系统疾病之一,影响尿路结石形成的因素及机制很多,年龄、性别、遗传、种族、饮食习惯、环境因素和职业对结石的形成影响很大。福建的闽西南地区是典型的南方气候,有较高的结石发病率,不过在我省地区,关于尿路结石患者所患结石类型、各类型结石所占比例、年龄、性别间差异的相关研究匿乏。不同的结石成分也隐含着结石形成原因的相关信息。本课题通过收集整理泌尿外科尿路结石患者结石成分分析的资料,分析本地区结石发病特点、性别年龄差异、地区差异、结石成分构成特点等。通过对本地区尿路结石患者的结石成分进行分析,了解和掌握本地区尿路结石的流行病学和结石构成情况,为尿路结石的临床预防和综合治疗提供有益的帮助。
方法
收集我院2010年6月～2013年6月期间收治福建省闽西南地区泌尿系结石患者共861例,所有患者均采用蓝莫德公司LIIR20型红外光谱仪,采用定性实验进行结石成分分析检测,并以尿路结石患者性别、年龄、结石部位、居住地等为分组依据,分析各组尿路结石的化学成分特征。采用SPSS20.0统计软件记录并分析检出率,对不同性别间各种成分结石的检出率用卡方检验比较,检验水准为a=0.05。
结果
1.本地区尿路结石发病基本情况
患者年龄3-91岁,平均48.92岁,农村463例,城市398例,农村城市比为1.16：1。结石发病男性多于女性。其中男593例,女268例,男女比为2.21：1。随着年龄增长,尿路结石的构成比逐渐升高,≤19岁患者最少,仅1.51%,30-60岁时达高峰,总和为64.12%。
2.各个部位尿路结石发病情况和成分分析结果
各个部位结石性别分布情况如下：上尿路结石711例,下尿路结石150例,上尿路和下尿路结石比为4.74：1。总的结石男女比为2.21：1,膀胱结石女性低于男性,男女性别比为7.31：1,输尿管结石男女性别比为2.61：1。女性肾结石的构成比较高,其男女性别比为1.42：1,另有检出尿道结石17例,均为男性。
尿路结石主要分布在上尿路,肾结石占46.92%。输尿管结石占35.66%,膀胱结石15.45%,尿道结石1.97%。各部位的尿路结石均以碳酸磷灰石+草酸钙结石(COS, calcium oxalate stones)组成的混合结石成分为主(44.72%),单一草酸钙结石成分居次(31.24%)。感染性结石(含六水磷酸镁铵成分)主要存在于膀胱结石和肾结石中(分别为3.48%和3.25%),尿酸(uric acid, UA)结石也是主要分布于膀胱结石和肾结石中(分别为5.69%和3.14%)。
3.总体结石成分分析结果
从结石成分构成看,单纯性结石占41.00%,两种成分混合性结石占49.94%,3种成分及以上混合性结石占9.06%。单纯性结石主要以草酸钙结石为主(31.24%),混合性结石主要以草酸钙和碳酸磷灰石组成的混合性结石为主(44.72%)。其他混合性结石比例按从多到少依次为六水磷酸镁铵+碳酸磷灰石+草酸钙7.67%,尿酸+草酸钙2.44%,六水磷酸镁铵+尿酸铵+碳酸磷灰石1.39%,尿酸铵+草酸钙1.05%,二水磷酸氢钙+草酸钙0.93%,L-胱氨酸+草酸钙0.46%,L-胱氨酸+碳酸磷灰石0.35%。其他单纯性结石比例按从多到少依次为尿酸结石5.34%,尿酸铵结石1.97%,碳酸磷灰石1.74%,二水磷酸氢钙0.70%。
从结石成分检出率来看,总体检出率最高的为草酸钙(88.50%),其次是碳酸磷灰石(55.87%),无水尿酸或尿酸铵(12.20%)、六水磷酸镁铵(9.06%)、二水磷酸氢铵钙(1.63%)、L-胱氨酸(0.81%)等成分结石所占比例较低。593例男性中检出率最高也是草酸钙(88.03%),其次依次是碳酸磷灰石(54.47%)、无水尿酸或尿酸铵(14.00%)、六水磷酸镁铵(6.75%)、二水磷酸氢铵钙(1.69%)、L-胱氨酸(0.84%)。268例女性中检出率最高也是草酸钙(89.55%),其次依次是碳酸磷灰石(58.96%)、六水磷酸镁铵(14.18%)、无水尿酸或尿酸铵(8.21%)、二水磷酸氢铵钙(1.49%)、L-胱氨酸(0.75%)。
男女尿路结石成分存在差异,无水尿酸或尿酸铵中男性检出率高于女性(14.00%vs8.21%,P=0.016),六水磷酸镁铵则相反,男性低于女性(6.75%vs14.18%,P=0.000)。草酸钙、碳酸磷灰石、L-胱氨酸、二水磷酸氢铵钙男女检出率没有显著性差异。
4.复发性结石成分分析情况
我们统计了其中22例复发性结石的成分分析结果,主要为混合性结石为主,感染性结石复发率最高,有10例(45.45%)为六水磷酸镁铵+碳酸磷灰石+草酸钙的混合性结石,其次有8例(36.37%)为草酸钙+碳酸磷灰石的混合性结石。其余为胱氨酸+碳酸磷灰石混合性结石2例,尿酸结石+草酸钙混合性结石2例。
结论
1.本地区结石发病情况为,尿路结石男性发病多于女性,30～60岁组尿路结石构成比达高峰,≤19岁构成比最低,上尿路结石多于下尿路结石,农村发病多于城市。
2.各部位的尿路结石均以碳酸磷灰石+草酸钙组成的混合结石成分为主,单一草酸钙结石成分居次。感染性结石(含六水磷酸镁铵成分)、尿酸性结石主要存在于肾结石和膀胱结石中。
3.从结石成分构成看,多种成分混合性结石占大多数。混合性结石主要以草酸钙和碳酸磷灰石组成的混合性结石为主。结石成分检出率最高的为草酸钙,其次是碳酸磷灰石、无水尿酸或尿酸铵、六水磷酸镁铵、二水磷酸氢铵钙、L-胱氨酸等。
4.男女尿路结石成分存在差异,无水尿酸或尿酸铵中男性检出率高于女性,六水磷酸镁铵则相反,男性低于女性。
5.以六水磷酸镁铵为主要成分的感染性结石复发率最高,其次为草酸钙+碳酸磷灰石的混合性结石。
6.本地区尿路结石化学成分以草酸钙为主,草酸钙结石所占比率高于国内其他省份,结石成分分析对于了解本地区结石成因,指导治疗和预防结石具有重要意义。
Background
Urolithiasis is a worldwild commonly encountered disease, the recurrence rate of primary caculary in10years is attain50%. According to statistics, the incidence of the urolithiasis is1%～5%in our country, while which reaches up to5%～10%. And25%of these patients need to be in hospital, which ranks first in urology inpatients. With the increased incidence of urinary calculi in recent years, China has now become one of the three stone-prone areas of the world. In addition, urinary stones recur easily. After extracorporeal shock wave lithotripsy (ESWL), the1-year recurrence rate is4%, and5-year recurrence rate is15.6%,10-year recurrence rate is10%,15-year recurrence rate is as high as75%.The incidence of urinary tract stones still rise, which greatly inconvenience the work and life of the patients. It also hazards national health and consumes the national resources, brings about huge economic burden to the family and society.
In recent years, with the development of science and technology and innovation, the minimally invasive techniques such as extracorporeal shock wave lithotripsy, percutaneous nephrolithotomy, laparoscope lithotomy and ureteroscopic lithotripsy, had made considerable progress and development. It changed the traditional method of treatment which based on open surgery for a long time and brought the gospel to the patients. However, because urinary calculi has high recurrence rate and residual stone rate, we cannot solve the problem thoroughly. In particular the increased incidence of urinary tract stones is still evident. In the meantime, urolithiasis is the inducement of some urinary system diseases, such as, tumors, urinary tract infection and urinary obstruction. Nowadays, it is very important to prevent urolithiasis and to research urolithiasis in nosazontology.
Until nowadays, the nosazontology of urolithiasis is still not clear. Now widely recognized that urinary calculi are the result of many factors. These factors are usually divided into individual and environmental factors. Individual factors include genetic factors, age, sex, metabolic disorders, urinary local lesions (Infection, obstruction, and foreign bodies), personal habits, obesity, etc. Environmental factors, including climate, occupation, personal eating habits, occupation, social development and human nutrition, etc.
Urinary stones component analysis is one of the important methods to determine the nature of the stones. It is an important "pathological" diagnosis for the comprehensive clinical treatment of stones. On diagnosis, it provides the most direct evidence to judge the etiological diagnosis of non-calcium stones and for calcium stones it can further narrow the scope of the assessment of metabolic. It is important evidence on dissolution therapy and prevention of stones in the clinical treatment. It also provides useful ideas for individualized treatment to urolithiasis patients. In addition to external factors for stone formation, metabolic abnormalities may also lead to changes in the urine stone promoter factor and suppression factor. Metabolism studies in patients with calcium stones are important to prevent stone recurrence. We should research patients with metabolic abnormalities, and it is important in the development of future strategies for the prevention of recurrence of stones.
For the past few years, with advances in chip technology, genetics sequencing technology and other related technologies, people pay more attention to the studies of genetic factors of urinary calculi. In these years scientists have played sufficient attention to single nucleotide polymorphisms (SNP). Single nucleotide polymorphisms is one of the most common human genetic variation, which Interprets the relationship between genetic variation and phenotype (disease). Single nucleotide polymorphisms mainly refers to a DNA sequence polymorphisms in the genome caused by a single nucleotide mutation. SNP is widespread in the human genome, which accounting for over90%of all known polymorphisms. Crystal composition of urinary calculi is divided into calcium stones and non-calcium stones. Calcium stones account for most of the urinary calculi. Calcium oxalate stones are most common in urinary calculi, accounting for about68.7%to90.0%. Past studies have shown that calcium channels may play an important role in inflammation and immune response. Meanwhile urinary stones, especially calcium oxalate stones are closely related with inflammation. Therefore, single nucleotide polymorphism studies of urinary calculi with calcium ion channels have greater significance as part of the genetic factors stones.
Chapter I Associations of Polymorphisms in STIM1/TRPC1Genes with Calcium Urolithiasis Patients in Southwestern Fujian Province
Objective
Calcium oxalate stones are most common in urinary calculi, accounting for about68.7%to90.0%.And as for Fujian province, calcium oxalate stones and calcium phosphate stone are most common in urinary calculi, accounting for about more than95.0%. Past studies have shown that calcium channels may play an important role in inflammation and immune response. Therefore, a single nucleotide polymorphism studies of urinary calculi with calcium ion channels have greater significance as part of the genetic factors stones. Past studies have shown that calcium channel may be the original regulating valve of development of calcium stones. Current research on the correlation of urinary calculi and gene polymorphism is still mainly from West countries, the domestic correlation research of SOCC genetic polymorphisms and urinary stones is still rare. In this study, genotypes of the8SNP loci in STIM1and TRPC1gene will be detected by Sequenom MassARRAY. Through analysis of genetic susceptibility of STIM1, TRPC1gene polymorphism and urinary calcium stones by case-control study, it can provide reference information on the comprehensive prevention and treatment of urinary calculi in genetics.
Methods
1.Clinical data of295patients with identified urinary calculi and198healthy individuals living in Southwestern Fujian province from January2013to January2014were retrospectively collected. Both groups were excluded hypertension, coronary artery disease, gout, diabetes, hyperlipidemia, cancer and other diseases of vital organs.
2. Genomie DNA was extracted by Generay DNA Purification Kit from peripheral blood samples of both groups, and stored at-80℃.
3. Based on the online public hapmap databases (http://hapmap.ncbi.nlm.nih.gov/) and published documents,8DNA polymorphism loci at STIM1/TRPC1genes with a minor allele frequency≥0.05and potential associations with SOCC were selected for our study.5DNA polymorphism loci at STIM1are rs3750994, rs2304891, rs3750996, rs3813880and rs10835206,3DNA polymorphism loci at TRPC1are rs7638459, rs2033912and rs3821647. rs10835206is in5' promoter, rs7638459and rs2033912are in introns, rs2304891and rs3821647are same sense mutation locis, rs3813880is in promoter.
4. Detect the genotypes of the8SNP loci in STIM1and TRPC1genes by Sequenom MassARRAY. Firstly, primer was designed according to the SNP site by Sequenom's Assay Design3.1software. The primers were synthesized by Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) for the purification quality. Followed by a PCR amplification reaction, SAP enzyme digestion reaction, single base extension reaction, resins purification and so on. Finally, the sample was detected by MALDI-TOF-MS, then detected the mass peak detection by Typer4.0software, interpreted each sample target locus genotype based on mass peak figure.
5. Applications of R statistics program2.15.3statistical package, SPSS20.0statistical analysis software packages and Excel for statistical analysis. Baseline between the two groups was analyzed by SPSS20.0statistical analysis software, measurement data between groups were compared using t-test, count data using Chi-square. P<0.05was considered statistically significant. Used Excel for Harding-Weinberg's law test in allele and genotype between the case group and the control group. Analyzed the allele and genotype polymorphism analysis in patients and normal control group by R statistics program2.15.3. Builted haplotype of the genes by calculating D'and r2in Haploview4.2software, analyzed the linkage disequilibrium between all sites in the same gene, then completed parallel haplotype analysis.
Results
1. Baseline data of case group and control group:Mean age of case group was (47.88±14.07) years, body mass index (BMI) was24.33±3.07, male173cases, female119cases. Mean age of control group was (47.72±14.78) years, body mass index (BMI) was23.89±3.51, male120cases, female78cases. The difference of sex ratio (P=0.834), age (P=0.143) and BMI (P=0.899) between Case and control group was not statistically significant (P>0.05).
2. Genotyping and distribution of STIM1and TRPC1genes:5DNA polymorphism loci at STIM1including rs3750994, rs2304891, rs3750996, rs3813880and rs10835206,3DNA polymorphism loci at TRPC1including rs7638459, rs2033912and rs3821647were successfully detected. The distribution of genotypes of each loci in accordance with Hardy-Weinberg Equilibrium (HWE) in case and control group (P>0.05).
3. Association between STIM1/TRPC1gene polymorphisms and Calcium urolithiasis. Analyzed the allele and genotype polymorphism analysis in patients and normal control group by R statistics program2.15.3, using Wilcox non-parametric test. Under the significance level a=0.05, two-tailed test, the genotype of rs1083206between case and control group was significantly different (Z=24101,P=0.0003355). The mutant genotype (CT+TT) frequency(86.10%vs.81.82%) and the mutant gene T frequency(382vs.223) of case group was statistically higher than the control. Other sites of case and control groups showed no significant difference (P>0.05).
4. Relations between TRPC1/STIM1polymorphic loci haplotypes and calcium urolithiasis. Analyzed the linkage disequilibrium between all sites in the same gene. The statistical analysis showed that the rs7638459and rs2033912TRPC1were fully linkage disequilibrium (D'=1.0,r2=1.0), however, no statistically significant difference. All the loci in STIM1were incomplete linkage equilibrium, haploid cannot be constituted.
Conclusion
1. DNA polymorphisms within STIM1gene were associated with calcium urolithiasis patients. Detection of polymorphisms in STIM1gene might provide some useful genetic information for prognosing the risk of calcium urolithiasis. It can provide reference information on the comprehensive prevention and treatment of urinary calculi in genetics.
2. The statistical analysis showed that the rs7638459and rs2033912TRPC1were fully linkage disequilibrium. All the loci in STIM1were incomplete linkage equilibrium, haploid cannot be constituted.
3. The TRPC1gene, which is closely relative to SOCC, maybe not associated with the risk and clinical genotype of calcium urolithiasis.
Chapter Ⅱ Analysis of861urinary tract calculus components in Southwestern Fujian province and discussion on prevention strategy
Objective
Urolithiasis is one of the most common urinary system diseases in china, especially in the southern region. Many factors and mechanisms influence the formation of urinary stones. Age, gender, genetics, race, diet, environmental factors and occupational greatly impact on the formation of stones. The Southwestern of Fujian province is a typical southern climate, which has a higher incidence of urolithiasis. But the studies of stone composition, rate and the difference between sex and age are very few in our province. The different composition of stone yields the underlying reasons of stone formation. The stone composition, rate and the difference-between sex and age were computed with the dates of patients with urinary stone. To get data of the stone composition, rate, difference between different groups and to evaluate the underlying reasons of stone formation. To offer objective dates for reinforcing the examination, treatment of the related patients and study of prophylaxis. To study the constituents of urinary stones in Southwestern Fujian province, and provide methods in prevention and treatment of urinary stones.
Methods
Clinical data of861patients with identified urinary calculi living in Southwestern Fujian province from June2010to June2013were retrospectively collected. Infrared spectrophotometry was carried out for biochemical composition analysis of urinary stones retrieved by endoscopic, open surgery, or spontaneously or by extracorporeal shock wave lithotripsy. Gender, age and stone location among these patients were evaluated, and the results were analyzed with biochemical stone analysis. SPSS20.0statistical software was used to record and analyze the detection rate. The detection rate of various stone components for different genders was compared with a chi-square test, the test level was a=0.05.
Results
1. The basic incidence of urinary calculi in the local region
Patients age3to91years, mean48.92years old,463cases of rural,398cases of urban, the rural-urban ratio is1.16:1.The incidence of male urolithiasis is higher than female, male593cases, female268cases, the sex ratio is2.21:1. The incidence of urinary tract stones gradually increases with age,≤19years old group is lowest for only1.51%, and the30-60years group is highest, total for64.12%.
2. The incidences of urinary tract stones in various parts and component analysis
The incidences of urinary tract stones in various parts are as follows:711cases in upper urinary tract,150cases in lower urinary tract, the upper urinary tract to lower urinary tract radio is4.74:1. The sex ratio of the general is2.21:1, the incidence of female bladder calculi is lower than male, and the sex ratio is7.31:1. While that of urethral calculi is2.61:1, closing to the general incidence. However, the constituent ratio of female renal calculi is higher than other, the sex ratio is1.42:1. There are17cases of urethral calculi, only found in male.
The upper urinary tract stones were more frequently found than the lower urinary tract stones, renal calculi accounts for46.92%, ureteral calculi accounts for35.66%, bladder calculi accounts for15.45%, urethral calculi accounts for1.97%. The combination of dahlliter and calcium oxalate stones(44.72%) is the most common combination calculi of all the parts of urinary tracts, the secondary component is pure calcium oxalate stones(COS)(31.24%). Infection stones, which are mainly consisted of ammonium magnesium phosphate hexahydrate, are mostly found in the bladder calculi and renal calculi (respectively3.48%and3.25%), uric acid is also mainly found in the bladder calculi and renal calculi (respectively5.69%and3.14%).
3. The general urinary calculi component analysis
Pure stones are found in41.00%of the stones and two-components mixed stones are49.94%, the above and three-components mixed stones are9.06%. The pure stones are mainly consisted of COS(31.24%), the mixed stones are mainly consisted of COS and dahllite (44.72%). Other mixed stones were listed from more proportion to less as follows:ammonium magnesium phosphate hexahydrate+dahllite+COS7.67%, uric acid(UA)+COS2.44%, ammonium magnesium phosphate hexahydrate+ammonium urate+dahllite1.39%, ammonium urate+dahllite+COS1.05%, Calcium hydrogen phosphate dihydrate+COS0.93%,L-cystine+COS0.46%, L-cystine+dahllite0.35%. Other pure stones were listed from more proportion to less as follows:UA5.34%, ammonium urate1.97%, dahllite1.74%, Calcium hydrogen phosphate dehydrate0.70%.
The most dominant composition of urinary stones was calcium oxalate (88.50%), followed by carbonate apatite (55.87%), uric acid or ammonium urate (12.20%), ammonium magnesium phosphate hexahydrate(9.06%), calcium hydrophosphate dihydrate(1.63%), and cystine (0.81%). For male, the most dominant composition of urinary stones was calcium oxalate (88.03%), followed by carbonate apatite (54.47%), uric acid or ammonium urate (14.00%), ammonium magnesium phosphate hexahydrate(6.75%), calcium hydrophosphate dihydrate(1.69%), and cystine (0.84%). For female, the most dominant composition of urinary stones was calcium oxalate (89.55%), followed by carbonate apatite (58.96%), ammonium magnesium phosphate hexahydrate(14.18%),uric acid or ammonium urate (8.21%), calcium hydrophosphate dihydrate(1.49%), and cystine (0.75%). Urinary stone composition is different in terms of gender, the detection rate of uric acid stones is higher in male than female (14.00%vs.8.21%, P=0.016), inversely, the detection rate of Infection stones was lower in male than female (6.75%vsl4.18%, P=0.000). While there are no difference in terms of gender for COS, dahllite, L-cystine, calcium hydrophosphate dihydrate.
4. Component analysis of recurrence calculi
22recurrence calculi were studied with the component analysis. All of them were consisted of mixed stones. Infection stone has the highest recurrence rate, there were10cases (45.45%)of mixed stones with ammonium magnesium phosphate hexahydrate+dahllite+COS, and8cases (36.37%) of mixed stones with COS+dahllite, and2cases of mixed stones with cystine+dahllite,2cases of mixed stones with UA+COS.
Conclusion
1. The basic incidence of urinary calculi in the local region:
The incidence of male urolithiasis is higher than female, the incidence of urinary tract stones gradually increases with age,≤19years old group is lowest, and the30-60years group is highest. The incidence of rural urolithiasis is higher than urban, The incidence of upper urinary tract is higher than lower urinary tract.
2. The combination of dahlliter and calcium oxalate stones is the most common combination calculi of all the parts of urinary tracts, the secondary component is pure calcium oxalate stones (COS). Uric acid and infection stones, which are mainly consist of ammonium magnesium phosphate hexahydrate, are mostly found in the bladder calculi and renal calculi.
3. Majority of the stones are mixed stones with2or3components. The pure stones are mainly consisted of COS, the mixed stones are mainly consisted of COS and dahllite. The most dominant composition of urinary stones was calcium oxalate, followed by carbonate apatite, uric acid or ammonium urate, ammonium magnesium phosphate hexahydrate, calcium hydrophosphate dihydrate, and cystine.
4. Urinary stone composition is different in terms of gender, the detection rate of uric acid stones is higher in male than female, inversely, and the detection rate of Infection stones was lower in male than female.
5. Infection stones, which are mainly consist of ammonium magnesium phosphate hexahydrate had the highest recurrence rate, followed by the mixed stones with COS and dahllite.
6. The dominant stone composition in inhabitants of Southwestern Fujian province is calcium oxalate, which significantly higher than other regions of China. Chemical composition analysis of urinary stones is very important in providing information to the etiology, treatment and prevention of urinary stones in the local region.
引文
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