KRAS Gene Polymorphisms and their Impact on Breast Cancer Risk in an Iranian Population

Single nucleotide polymorphisms (SNPs) in the let-7 miRNA binding site within the 3’ untranslated region (3’UTR) of KRAS appear related to the risk of cancer. The present case-control study was conducted with 244 BC patients and 204 healthy women to examine whether KRAS polymorphisms (rs61764370 T/G and rs712 G/T) are associated with breast cancer (BC) risk in an Iranian population. The polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method was used for genotyping of KRAS SNPs. Our results showed that the rs61764370 TG genotype (OR= 3.73; 95% CI =1.38-10.08; P=0.007) as well as the G allele OR= 3.56; 95% CI =1.33-9.53; P=0.008, respectively) increased the risk of BC. However, the KRAS rs712 TT vs GG+GT genotype in a recessive model was associated with a reduced risk of BC (OR= 0.56; 95% CI =0.38-0.84; P=0.006). In addition, the rs712 T allele decreased the risk of BC compared with the G allele (OR=0.75, 95%CI=0.58-0.97, P=0.031). However, we found no relationship among KRAS SNPs and clinicopathological characteristics of BC patients (P>0.05). Taken together, the present study provided evidence of relationships between KRAS polymorphisms and BC risk in a southeast Iranian population. Additional studies using larger sample sizes and diverse ethnicities are now warranted.     

Association Between Survivin Gene Polymorphisms and the Susceptibility to Colon Cancer Development in the Turkish Population

Background: Colon cancer is one of the most common cancers worldwide. Apoptosis is a necessaryphysiological process for cell elimination which is very important both cellular homeostasis and cell proliferationand differantiation. Dysregulation can lead to uncontrolled cell growth and tumor development. Survivin, amember of the IAP family, plays a key role in promotion of cell proliferation as well as inhibition of apoptosis incancer cells. The aim of this study was to investigate whether specific genetic polymorphisms of survivin could beassociated with colon cancer development and progression in a Turkish population. Our study is the first to ourknowledge to investigate the relationship between colon cancer risk and survivin gene polymorphisms. Materialsand Methods: The relation between colon cancer and survivin -31 G/C (rs9904341), -241 C/T (rs17878467) and-625 C/G (rs8073069) polymorphism in promotor site of survivin gene associated with apoptosis was investigatedusing the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Results:Individuals with -31C allele and CC genotype were found to have a higher risk of developing colon cancer(OR=13.4, p=0.01). The -241 CT genotype considerably increased the risk of colon cancer (OR=12.0, p=0.0001).However, there was no significant varaition of the survivin -625 C/G polymorphism among colon cancer patientsand controls in our study. Conclusions: This study provides the first evidence that survivin -31 G/C and -241C/T SNP significantly contribute to the risk of colon cancer in the Turkish population.     

XRCC4 c.1394G>T Single Nucleotide Polymorphisms and Breast Cancer Risk among Filipinos

Background: The identification of cancer-associated single nucleotide polymorphisms (SNP) and mutationgenes is a promising approach in recognizing individuals who are at risk of developing cancer. Hence, this studywas conducted to determine the association between XRCC4 c.1394G>T SNP and breast cancer development amongFilipinos. Methods: Genotyping for XRCC4 c.1394G>T SNP was performed on breast cancer patients (n=103) andtheir age- and sex- matched clinically healthy controls (n=103) by polymerase chain reaction – restriction fragmentlength polymorphism. Results: Significant difference in genotype (p=0.007) and allele (p=0.003) frequencies in XRCC4c.1394G>T was observed between the breast cancer cases and controls. Carriers of the XRCC4 c.1394 G>T genotypewere observed to have significantly higher risk of developing breast cancer compared to individuals with T/T genotype(OR=2.67, 95% CI: 1.36 – 5.25). XRCC4 c.1394G>T combined with passive smoking may also significantly increaserisk of breast cancer (OR=14.73; 95% CI= 9.88-18.86). Conclusion: XRCC4 c. 1394G>T may be associated withbreast cancer development among Filipinos.     

IFN-γ基因+874位点单核苷酸多态性与乳腺癌的相关性

 目的探讨IFN-γ基因+874位点单核苷酸多态性与乳腺癌的相关性。方法采用PCR技术对94例乳腺癌患者及96例正常女性IFN-γ基因+874位点A/T单核苷酸多态性(SNP)进行分析,将PCR产物进行克隆、测序。结果乳腺癌患者IFN-γ基因+874位点TT基因型频率为18.1%,显著高于正常人的8.3%,两者之间比较具有显著性差异(χ²=3.95,P=0.047);乳腺癌病例组T等位基因频率显著高于对照组(χ²=4.984,P=0.026)。进一步分析显示,乳腺癌病例组中不同年龄段基因型频率和等位基因频率相比,均无显著性差异。结论IFN-γ基因+874位点TT基因型可能与乳腺癌的发生相关,T等位基因可能为乳腺癌发生的遗传危险因素。     

Significant Association of the MDM2 T309G Polymorphism with Breast Cancer Risk in a Turkish Population

Background: Breast cancer is a leading cause of death in women worldwide. Genetic polymorphisms have beenreported to be important etiological factors. Murine double minute 2 (MDM2) T309G interacts with p53 and mutationsin p53 are present in approximately 50% of all cancers. However, it has been reported that effect of the polymorphismon breast cancer risk may vary in different populations. Here, we therefore investigated whether there is an associationbetween MDM2 T309G (rs2279744) polymorphism and breast cancer in a Turkish population. Materials and Methods:We analysed 110 patients with breast cancer and 138 matched? controls. For genotyping, polymerase chain reactionand restriction length fragment polymorphism methods were used. Results: A significant difference was observedbetween case and control groups with regard to the distribution of the MDM2 T309G polymorphism (p<0.05). Therewas a significantly higher frequency of the TT genotype in the control group (p=0.028; OR, 2.42; 95% CI, 1.09-5.37).However, we did not find any relationships among tumor grade and metastasis status and this polymorphism. Conclusion:This study indicates that the MDM2 T309G polymorphism GG genotype and the TG+GG combination may be riskfactors for breast cancer in our Turkish population.     

Association between IL-27 Gene Polymorphisms and Cancer Susceptibility in Asian Population: A Meta-Analysis

Background: Interleukin 27 (IL-27) has potent antitumor activity. Several epidemiological studies have designated that genetic variants of the IL-27 gene may contribute to various cancer susceptibility, but the data were inconclusive.  Objective: The current meta-analysis aimed to address the association between IL-27 rs153109, rs17855750, and rs181206 polymorphisms and the risk of cancer. Data Sources: Our team has selected eligible studies up to May 1, 2020, from several electronic databases, including Web of Science, PubMed, Scopus, and Google Scholar databases. Results: Our meta-analysis revealed that the carriers rs153109 A>G polymorphism in the IL-27 gene have higher risks of diseases in the heterozygous (OR=1.26, 95%CI=1.06-1.49, P=0.007, AG vs AA), homozygous (OR=1.18, 95%CI=1.01-1.37, p=0.33, GG vs AA), dominant (OR=1.25, 95%CI=1.07-1.47, P=0.006, AG+GG vs AA), and allele (OR=1.15, 95%CI=1.04-1.27, P=0.008, G vs A) genetic models. Stratified analysis by cancer type indicated that this variant was significantly associated with gastrointestinal cancer, colorectal cancer and breast cancer. The findings did not support an association between rs17855750 T>G, rs181206 T>C polymorphisms of IL-27 and cancer risk. Conclusion: the current study findings suggest that IL-27 rs153109 polymorphism significantly increased the risk of cancer susceptibility. Well-designed replication in a larger independent genetic association study with larger sample sizes in diverse ethnicities is required to verify the findings.     

MDM2 启动子区309 位点基因多态性与乳腺癌风险的关系*

目的:采用病例- 对照研究检测MDM2 启动子区309 位点T>G 单核苷酸多态（SNP 309）在中国女性人群中的频率分布,分析其与中国女性乳腺癌发病风险的关系。方法:提取病例组698 例原发性乳腺癌患者及对照组525 例健康人的外周血单核细胞DNA,采用聚合酶链反应- 限制性片段长度多态性（PCR-RFLP ）分析法,检测MDM2 启动子区309 位点基因多态性,确定此位点三种基因型,即T/T、T/G、G/G 基因型。统计分析病例组和对照组人群MDM2 SNP 309 各基因型频率分布,及各基因型与乳腺癌发病风险的相关性。结果:在研究的病例组与对照组整体人群中,经年龄、月经状态、家族史及生育史等因素校正后,与MDM2 SNP 309 T/T基因型比较,T/G 型及G/G 型与乳腺癌的发病风险无显著相关性（T/G,adjusted OR= 1.2,95%CI:0.8～1.6,P=0.30;G/G,adjusted OR= 1.0,95%CI:0.7～1.5,P=0.88）。 进一步分层分析后显示:在绝经后人群中,与T/T基因型比较,T/G 基因型及G/G 基因型显著增加乳腺癌的发病风险（T/G,adjusted OR= 1.8,95%CI:1.2～3.0,P=0.011;G/G,adjusted OR= 1.9,95%CI:1.2～3.3,P=0.014）。 提示绝经后人群携带T/G 型、G/G 型者比携带T/T基因型者患乳腺癌的风险分别升高约1.8、1.9 倍。在绝经前人群中,各基因型与乳腺癌的发病风险无显著相关性（P>0.05）。 结论:MDM2 启动子309 位点突变型G 等位基因携带者显著增加绝经后女性乳腺癌的发病风险。      

Association of Functional Polymorphisms of the XRCC4 Gene with the Risk of Breast Cancer: A Meta-analysis

Objective: X-ray cross-complementing group 4 (XRCC4) is a major repair gene for DNA double-strand breaks(DSB) in the non-homologous end-joining (NHEJ) pathway. Several potentially functional polymorphisms of theXRCC4 gene have been implicated in breast cancer risk, but individually published studies showed inconclusiveresults. The aim of this meta-analysis was to investigate the association between XRCC4 polymorphisms and therisk of breast cancer. Methods: The MEDLINE, EMBASE, Web of science and CBM databases were searchedfor all relevant articles published up to June 20, 2012. Potential associations were assessed with comparisons ofthe total mutation rate (TMR), complete mutation rate (CMR) and partial mutation rate (PMR) in cases andcontrols. Statistical analyses were performed using RevMan 5.1.6 and STATA 12.0 software. Results: Five studieswere included with a total of 5,165 breast cancer cases and 4,839 healthy controls. Meta-analysis results showedthat mutations of rs2075686 (C>T) and rs6869366 (G>T) in the XRCC4 gene were associated with increasedrisk of breast cancer, while rs2075685 (G>T) and rs10057194 (A>G) might decrease the risk of breast cancer.However, rs1805377 (A>G), rs1056503 (G>T), rs28360317 (ins>del) and rs3734091 (A>G) polymorphisms ofXRCC4 gene did not appear to have an influence on breast cancer susceptibility. Conclusion: Results from thecurrent meta-analysis suggest that the rs2075685 (G>T) and rs6869366 (G>T) polymorphisms of the XRCC4 genemight increase the risk of breast cancer, whereas rs2075685 (G>T) and rs10057194 (A>G) might be protectivefactors.     

Polymorphisms in the KDR and POSTN Genes: Association with Breast Cancer Susceptibility and Prognosis

Angiogenesis is an important step in the development of cancer. Vascular endothelial growth factor is a major regulator of breast cancer angiogenesis, the effects of which are transmitted through the kinase domain receptor (KDR). Up-regulation of KDR by periostin (POSTN) induces angiogenesis. We screened the KDR and the POSTN genes for published single nucleotide polymorphisms (SNPs) and chose two SNPs in each gene for further analyses. We carried out a case–control study consisting of 412 familial and 912 unselected breast cancer cases together with ethnically and geographically selected controls. Genotype, haplotype and genotype combination analyses were carried out to evaluate their effect on susceptibility to and prognosis of breast cancer. A haplotype in the POSTN gene was associated with an increased risk even after correction for multiple comparisons. Nominal associations between the SNPs and prognostic indicators were also observed. Tumors of the KDR 472His allele carriers were less often progesterone receptor negative according to both genotype and haplotype analyses (OR 0.61, 95%CI 0.40–0.92 and OR 0.60, 95%CI 0.40–0.91, respectively). The POSTN -33G allele carriers had more often high grade and estrogen receptor negative tumors (OR 1.75, 95%CI 1.02–3.01 and OR 1.70, 95%CI 1.04–2.78, respectively). The overall and cancer specific survival after 15 years of follow-up was more than 75%, and it did not depend on the genotype. Although a major effect of the SNPs in the KDR and the POSTN genes on breast cancer susceptibility and prognosis was excluded, the effect of the POSTN C-33G SNP on prognosis needs further characterization.     

Association between Genetic Polymorphisms of miR-1307, miR- 1269, miR-3117 and Breast Cancer Risk in a Sample of South East Iranian Women

Introduction: MicroRNAs (miRNAs) play an essential role in the susceptibility and development of cancer cells. Objective: Examining the dependency of breast cancer risk with genetic polymorphisms of miR-1307, miR-1269, and miR-3117 in a sample of Iranian women (southeast region). Methods: The case-control study consisted of 520 individuals (260 diagnosed BC patients, 260 healthy individuals). The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used for genotyping of miR-1307 rs7911488, miR-1269 rs73239138, and miR-3117 (rs4655646 and rs7512692) polymorphisms. Results and Conclusion: This study provided evidence that miR-1307 rs7911488 polymorphism significantly reduced the risk of BC in heterozygous AG genotype, as well as dominant (AG+GG) genotype and G allele. A significant correlation was found between dominant (AA+AG) genotype, the A allele and protection against BC due to miR-1269 rs73239138 in the sample of study. In contrast, our findings suggested that AG genotype and G allele of miR-3117 rs4655646 polymorphism could increase BC’s susceptibility among the southeastern Iranian females. The miR-3117 rs7512692 variant also increased the risk of BC in codominant, dominant and recessive models, as well as the T allele. The possible dependency of miR-1307, miR-1269, and miR-3117 variants with patients’ clinicopathological characteristics and BC was also studied. It was concluded that there is a correlation between miR-3117 rs7512692 variant and tumor grade (p=0.031); also, a correlation between miR-1269 rs73239138 variant and progesterone receptor status (p=0.006). The current investigation revealed that miR-1307, miR-1269, and miR-3117 polymorphisms might play a crucial role in the Iranian population’s vulnerability to BC.     

Lack of Association Between LIG4 Gene Polymorphisms and the Risk of Breast Cancer: A HuGE Review and Meta-analysis

Objective: Non-homologous end joining (NHEJ) is one of the pathways of repair of DNA double-strandbreaks. A number of genes involved in NHEJ have been implicated as breast cancer susceptibility genes such asLIG4. However, some studies have generated conflicting results. The aim of this Human Genome Epidemiology(HuGE) review and meta-analysis was to investigate association between LIG4 gene polymorphisms in the NHEJpathway and breast cancer risk. Methods: Studies focusing on the relationship between LIG4 gene polymorphismsand susceptibility to breast cancer were selected from the Pubmed, Cochrane library, Embase, Web of Science,Springerlink, CNKI and CBM databases. Data were extracted by two independent reviewers and the meta-analysiswas performed with Review Manager Version 5.1.6 and STATA Version 12.0 software, calculating odds ratios(ORs) with 95% confidence intervals (95%CIs). Results: According to the inclusion criteria, we final includedseven studies with a total of 10,321 breast cancer cases and 10,160 healthy controls in the meta-analysis. Theresults showed no association between LIG4 gene polymorphisms (rs1805386 T>C, rs1805389 C>T, rs1805388C>T and rs2232641 A>G) and breast cancer risk, suggesting that the mutant situation of these SNPs neitherincreased nor decreased the risk for breast cancer. In the subgroup analysis by Hardy-Weinberg equilibrium(HWE) and ethnicity, we also found no associations between the variants of LIG4 gene and breast cancer riskamong HWE, non-HWE, Caucasians, Asians and Africans. Conclusion: This meta-analysis suggests that thereis a lack of any association between LIG4 gene polymorphisms and the risk of breast cancer.     

Interleukin-10 Gene Promoter Polymorphisms and Risk of Gastric Cancer in a Chinese Population: Single Nucleotide and Haplotype Analyses

Objectives: Interleukin (IL) -10 is a potent cytokine with a dual ability to immunosuppress or immunostimulate.We aimed to explore the association of IL10 promoter polymorphisms with risk of gastric cancer (GC) in a Hanpopulation in Southwestern China. Methods: We enrolled 308 pairs of GC and control subjects from four hospitalsand a community between October 2010 and August 2011 in a 1:1 matched case-control design. Demographicinformation was collected using a designed questionnaire. IL10-592 A>C and IL10-1082 A>G polymorphismswere determined by Sequenom MassARRAY analysis. Results: Patients with GC reported statistically higherproportions of family history of cancer (29.9% versus 10.7%, P<0.01) and alcohol drinking (54.6% versus 43.2%,P<0.01) than did controls. Similar results were observed in comparison between non-cardia GC patients andcontrols (PC and IL10-1082 A>G were not associated withoverall GC risk (adjusted OR, 0.94, 95% CI, 0.66-1.33; adjusted OR, 1.00, 95% CI, 0.62-1.60). Sub-analysisshowed that the IL10-592 AC/CC variant genotype was associated with decreased non-cardia GC risk (adjustedOR, 0.58; 95% CI, 0.36-0.95). No association was found between any of the IL10 haplotypes established fromtwo polymorphisms and risk of non-cardia GC. Conclusions: In conclusion, our data do not link the two SNPs ofIL10-592 and IL10-1082 with overall GC risk. We demonstrate that IL10-592 polymorphism is associated withprotective effect against non-cardia GC. Our findings may offer insight into risk associated with the developmentof GC in this region.     

Evaluating the Possible Association between PD-1 (Rs11568821, Rs2227981, Rs2227982) and PD-L1 (Rs4143815, Rs2890658) Polymorphisms and Susceptibility to Breast Cancer in a Sample of Southeast Iranian Women

Introduction: Programmed cell death-1 (PD-1) and its ligands (PD-L1 and PD-L2) play a critical role as a  regulator of immune-system cells, including T cell, natural killer T (NKT), monocytes, dendritic cells (DC), and B cells. Objective: This study aimed to find a possible association between PD-1 (rs11568821, rs2227981, rs2227982), and PD-L1 (rs4143815, rs2890658) variants and Breast Cancer (BC) risk in a sample of southeast Iranian women. Method: The case-control study consisted of 520 individuals, including 260 histologically confirmed BC patients and 260 non-cancer age-matching healthy women as the control group. The Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and Tetra-Primer Amplification Refractory Mutation System-Polymerase Chain Reaction (T-ARMS-PCR) methods were used for genotyping of PD-1 (rs11568821, rs2227981, rs2227982), and PD-L1 (rs4143815, rs2890658) polymorphisms. Results and Conclusion: Our findings indicated that the PD-L1 rs4143815 (G/C) variant meaningfully reduced the risk of BC. However, the PD-L1 rs2890658 variant increased the BC risk in the AC genotype as well as the A allele. Furthermore, we could not find a meaningful association between PD-1 rs11568821, PD-1 rs2227981, PD-1 rs2227982, and BC. Our team examined the possible association between variants and clinicopathological characteristics, including age, size of tumour, lymph node, histology, grade of tumour, estrogen and progesterone receptors status as well as human growth factor receptor 2 (HER2). Our findings demonstrated that PD-L1 rs4143815, PD-L1 rs2890658, PD-1 rs2227982 had a significant association with age. Additionally, we found a significant relation between PD-1 rs2227982 variant and tumour size. Statistical analyzes of PD-1 rs2227981 and PD-1 rs11568821 variants showed a meaningful relation between tumour grade and tumour stage (p=0.006), respectively.     

Association between Mismatch Repair Gene MSH3 codons 1036 and 222 Polymorphisms and Sporadic Prostate Cancer in the Iranian Population

The mismatch repair system (MMR) is a post-replicative DNA repair mechanism whose defects can lead to cancer. The MSH3 protein is an essential component of the system. We postulated that MSH3 gene polymorphisms might therefore be associated with prostate cancer (PC). We studied MSH3 codon 222 and MSH3 codon 1036 polymorphisms in a group of Iranian sporadic PC patients. A total of 60 controls and 18 patients were assessed using the polymerase chain reaction and single strand conformational polymorphism. For comparing the genotypefrequencies of patients and controls the chi-square test was applied. The obtained result indicated that there was significantly association between G/A genotype of MSH3 codon 222 and G/G genotype of MSH3 codon 1036 withan increased PC risk (P=0.012 and P=0.02 respectively). Our results demonstrated that MSH3 codon 222 and MSH3 codon 1036 polymorphisms may be risk factors for sporadic prostate cancer in the Iranian population.     

CAV-1基因多态性与散发乳腺癌易感性的相关性分析

[目的]探讨CAV-1基因多态性与散发乳腺癌的相关性。[方法]采用病例对照研究,纳入经病理确诊的135例女性乳腺癌患者作为实验组,166例女性健康体检者为对照组。通过竞争性等位基因特异性PCR法对研究对象基因位点进行分型;采用χ2检验比较CAV-1各SNP基因型及等位基因频率在两组中的分布差异;非条件Logistic回归分析CAV-1基因多态性与乳腺癌易感性的关联。[结果]在共显性模型、显性模型及等位基因模型下rs3807987及rs7804372位点多态性与乳腺癌易感性密切相关。rs3807987:相对于GG基因型,AG、AA基因携带者(AG/AA基因型)均增加乳腺癌的发病风险(P<0.05),OR值分别为2.110(95%CI:1.270~3.505)、1.968(95%CI:1.205~3.216)。rs7804372位点:相对于TT基因型,AT、AA基因携带者(AT/AA基因型)均增加乳腺癌的发病风险(P<0.05),OR值分别为2.088(95%CI:1.285~3.392)、2.059(95%CI:1.293~3.280)。rs12672038位点:在共显性模型、显性模型、等位基因模型均未见rs12672038位点多态性分布与乳腺癌发病风险之间存在相关性。[结论]CAV-1基因rs3807987与rs7804372多态性与乳腺癌易感性相关。     

Effects of p53 Codon 72 and MDM2 SNP309 Polymorphisms on Gastric Cancer Risk among the Iranian Population

Background: Development of gastric cancer (GC) is a multistep process that requires alterations in the expression of oncogenes and tumor suppressor genes, occurring over several decades. The p53 tumor suppressor protein is involved in cell-cycle control, apoptosis and DNA repair. One of the most important regulators of p53 is MDM2, which acts as a negative regulator in the p53 pathway. Based on the key role of p53 and MDM2 in tumor suppression, polymorphisms that cause change in their function might affect cancer risk. We therefore elevated associations of the polymorphisms of p53 (R72P) and MDM2 (SNP309) with GC in Iran. Materials and Methods: A total of 104 patients with gastric cancer and 100 controls were recruited. Genomic DNA was extracted from fresh gastric samples. Genotyping of the p53 and MDM2 genes was performed using allele specific PCR(AS-PCR). Results: There was no significant difference between the p53 codon 72 polymorphism distribution in control and patient groups (p=0.54), but the G allele of MDM2 was found to be over-represented in patients (p=0. 01, Odds Ratio=2. 08, 95% Confidence Interval= 1.37-4.34). Conclusions: The p53 R72P seems not to be a potential risk factor for development of GC among Iranian patients, but our data suggest that MDM2 SNP309 might modify the risk related to GC.     

Single Nucleotide Polymorphisms in the u-PA Gene are Relatedto Susceptibility to Oral Tongue Squamous Cell Carcinoma inthe Northern Chinese Han Population

Aim: The purpose of this study was to determine whether susceptibility to oral tongue squamous cellcarcinoma (OSCC) is related to polymorphisms in the u-PA gene. Methods: We examined the rs2227564 C/Tand rs2227562 G/A single nucleotide polymorphisms (SNPs) in 196 OSCC patients and 201 age- and gendermatchedcontrols via direct sequencing and PCR-RFLP methods. Results: Significant differences were found inallelic and genotypic distributions of the rs2227564 and rs2227562 loci when comparing cases and controls. Inaddition, logistic analyses indicated that the rs2227564 C/T genotype was related to a 1.52-fold increased riskof developing OSCC (adjusted OR=1.521, 95%CI: 1.144~2.022, P=0.004). Linkage disequilibrium analysis wasconducted and no association between the two loci was found (D’=0.031, r2=0.000). Conclusions: Our findingsprovide evidence that the rs2227564 C/T SNP in the u-PA gene is associated with the development of OSCC.