Associations Between Genetic Variants In The Calcium Signaling Pathway Genes And Cutaneous Melanoma-specific Survival

Background: World widely,cutaneous melanoma(CM)is one of the most highly lethal skin cancers.However,how to discover more promising predictors and improve CM patients' survival remain a big challenge.Several clinicopathological variables were reported associated with CM survival,such as age,sex,tumor stage,Breslow thickness,ulceration and mitotic rate,but the improvement in accurately predicting patient prognosis is still limited.Molecular biomarkers play an important role in CM prognosis,as one of inheritable biomarker,gene polymorphisms can provide critical biological information for CM prognosis and individual treatment.Based on the knowledge of Human Genome Project(HGP)and Hap Map Project,and the development of high-throughput sequencing technology,now we can conduct genome-wide association analysis(Genome-wide Association Study,GWAS),and numerous SNPs have been identified associated with increased CM risks by GWAS.There are also limitations for GWAS,for example,some biologically functional SNPs may be missed when they are not ranking the top of the analysis;and it's hard to explain the molecular mechanisms behind those SNPs identified by GWAS.Pathway-based approach has been applied to understanding of the effects of genes and their biological pathways on CM development and progression.As a vital physiological signal,the calcium signaling is ubiquitously involved in nearly every aspect of cellular processes in human,including cell growth,proliferation,differentiation and even cell death.Accumulated studies have suggested that remodeling or deregulation of the calcium signaling pathway is a relevant hallmark of cancer,even the center control in carcinogenesis.In the present study,we performed a pathway-based approach to reanalysis two existing CM GWAS datasets to assess the associations between genetic variants in the calcium signaling pathway genes and CM-specific survival(CMSS).Thus,we may find out the key calcium signaling molecules that are likely to be promising biomarkers for CM development and prognosis,even a novel and prospective target for individual treatment.Objective: 1.To explore the associations between genetic variants in the calcium signaling pathway and CMSS;2.To identify the key molecules in the calcium signaling pathway that are associated with prognosis of CM patients;3.Discuss the underlying mechanisms that how these molecules affect CM patients' survival.Materials and Methods: Using data from a published genome-wide association study(GWAS)from The University of Texas M.D.Anderson Cancer Center(MDACC),we assessed the role of 41,377 common single nucleotide polymorphisms(SNPs)in 167 calcium signaling pathway genes in CMSS.Cox proportional hazards regression analysis and multiple test correction were conducted.We used another GWAS from Harvard University as validation dataset.In silico functional validation of the identified independent SNPs was performed using several online tools.Besides,e QTL(expression quantitative trait loci)analysis,genetic model analysis,combine analysis and stratified analysis were also performed.Receiver operating characteristic(ROC)curve and area under curve(AUC)were used to illustrate the ability of area under the curve in predicting CMSS.Results: 1.We extracted 41 377 SNPs in 167 calcium signaling pathway genes from the MDACC GWAS dataset.In the single-locus analysis,1,830 SNPs were significantly associated with CM-specific survival(CMSS)(P ? 0.050 and false-positive report probability ? 0.2),of which nine SNPs in four genes(CHRM3,PDE1 A,ITPR1 and RYR3)remained significant in the Harvard GWAS dataset(P ? 0.050).2.We identified three independent SNPs in three genes(PDE1A rs6750552 T>C,ITPR1 rs6785564 A>G and RYR3 rs2596191 C>A)had a predictive role in CMSS,with a meta-analysis derived hazards ratio(HR)of 1.52 [95% confidence interval(CI)= 1.19-1.94,P = 7.21×10-4)],0.49(0.33-0.73,3.94×10-4)and 0.67(0.53-0.86,0.0017),respectively.3.We found that PDE1 A rs6750552 C allele was associated with a poorer survival,while ITPR1 rs6785564 G allele and RYR3 rs2596191 A allele were associated with a better survival.In the MDACC GWAS dataset,the risk effect of PDE1 A rs6750552 C allele as well as protective effects of ITPR1 rs6785564 G and RYR3 rs2596191 A alleles on CM survival were statistically significant(trend test: P = 0.013,0.004,and 0.016,respectively);Similar results were observed in the Harvard GWAS dataset(trend test: P = 0.022,0.040,and 0.039,respectively).When we combined MDACC and Harvard datasets into one dataset,consistent results were observed(trend test: P = 0.007,0.019,and 0.003,respectively).4.We combined the protective genotypes of rs6750552 TT,rs6785564 AG+GG,rs2596191 CA+AA into one variable of the number of protective genotypes.The trend test demonstrated that an increased number of protective genotypes was associated with an improved survival in the MDACC dataset(P < 0.001),in the Harvard dataset(P = 0.0002)and in the combined dataset(P < 0.0001).Compared with those who had 0-1 protective genotype,patients with 2-3 protective genotypes had a significantly better survival(MDACC: HR = 0.49,95% CI = 0.31-0.76,and P = 0.002;Harvard: HR = 0.48,95% CI = 0.26-0.90,and P = 0.021;combined dataset: HR = 0.61,95% CI = 0.43-0.86,and P = 0.0049).5.We also observed significant correlations between the three SNPs and their corresponding gene m RNA expression level: rs2368253 C(in a high LD with rs6750552 C)was positively correlated with PDE1 A m RNA expression level,while rs6785564 G and rs2596191 A were negatively correlated with ITPR1 and RYR3 m RNA expression level,respectively.6.From the ROC curve,we observed a significantly improvement for these protective genotypes in combination with age and sex in prediction performance of the 5-year CMSS,compared with the model with age and sex only(AUC = 61.25% to 67.21%,P = 5.79×10-4).Conclusions: 1.Univariate analysis showed that age,sex,regional/distant metastasis,Breslow thickness,ulceration,and mitotic rate were significantly associated with CMSS in MDACC study;while only age was significantly associated with CMSS in the univariate analysis of Harvard study.2.PDE1 A rs6750552,ITPR1 rs6785564 and RYR3 rs2596191 in the calcium signaling pathway were independently associated with the survival of CM patients.3.PDE1 A rs6750552 TT,ITPR1 rs6785564 AG+GG? RYR3 rs2596191 CA+AA were recognized as protective genotypes.As the number of protective genotypes increased,hazards ratio for CM decreased significantly.4.The e QTL analysis showed that these genetic variants were also significantly associated with their corresponding gene m RNA expression levels.5.When these protective genotypes were included in the prediction model for CM prognosis,the prediction ability of the model was significantly improved.