| BackgroundUrolithiasis 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 ProvinceObjectiveCalcium 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.Methods1.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.Results1. 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.Conclusion1. 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 strategyObjectiveUrolithiasis 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.MethodsClinical 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.Results1. The basic incidence of urinary calculi in the local regionPatients 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 analysisThe 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 analysisPure 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 calculi22recurrence 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.Conclusion1. 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. |
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