Data for cardiovascular disease  and cancer  were obtained from the FinnGen consortium. A one standard deviation increase in genetically-predicted S-Ca concentration was associated with lower odds of longevity  and increased risk of cardiovascular disease . The associations were consistent in confirmatory analyses. There was no evidence supporting an association between genetically-predicted S-Ca and cancer, and no associations of genetically-predicted S-25(OH)D with the studied outcomes. Lifelong higher levels of S-Ca but not S-25(OH)D may shorten life expectancy and increase the risk of cardiovascular disease.Associations of serum calcium (S-Ca) and 25-hydroxyvitamin D (S-25(OH)D) concentrations with longevity, cardiovascular disease, and cancer are not clear. We conducted a Mendelian randomization study to examine the associations of S-Ca and S-25(OH)D with longevity and risk of cardiovascular disease and cancer. The primary genetic instruments for S-Ca and S-25(OH)D were obtained from genome-wide association meta-analyses that included 61,054 individuals for S-Ca and up to 79,366 individuals for S-25(OH)D. Genetic variants associated with S-Ca and S-25(OH)D in the UK Biobank were used as confirmatory instruments. We obtained summary-level data for associations of these instruments with individual survival later than the 90 Several other benefits and adverse effects of calcium5 and vitamin D7 have been suggested. Epidemiological evidence regarding the associations of these nutrients with all-cause and cardiovascular disease mortality is inconsistent and data on longevity are scarce14. The net benefit on health by calcium and vitamin D supplement use is accordingly unclear.Calcium and vitamin D supplements are frequently used for the prevention and treatment of osteoporosis despite weak and inconsistent evidence that they prevent fractures in community-dwelling women and men18. Whether regular calcium supplementation elevates S-Ca after several months of use is debatable19 but this question has great importance for the understanding of the effects of calcium supplementation and S-Ca on health outcomes since continued use for many years is recommended.Supplementation with calcium and vitamin D, alone or together, leads to increases in serum calcium (S-Ca) concentration with a peak 4 h after each ingestion and a more long-lasting elevation in serum 25-hydroxyvitamin D (S-25(OH)D), the marker metabolite for vitamin D status20. In this study, we first explored the long-term effects of calcium and calcium plus vitamin D supplementation on S-Ca concentrations in a meta-analysis. Then we used the MR design to assess the associations of genetically predicted lifelong small increases of S-Ca and S-25(OH)D concentrations with longevity as well as with risk of overall cardiovascular disease and cancer.The Mendelian randomization (MR) design can overcome residual confounding and other biases in epidemiological studies, thereby strengthening causal inference for an exposure–outcome association by leveraging genetic variants as instrumental variables for an exposureThere were 7 studies  included in the meta-analysis of calcium supplementation and 7 studies  included in the meta-analysis of calcium plus vitamin D supplementation. The interventions lasted from 6 months to 4 years. S-Ca concentrations were significantly higher in individuals who had been assigned supplementation with calcium alone or calcium plus vitamin D compared with those given placebo  was observed in the primary analyses for S-Ca and S-25(OH)D.Genetically predicted lifelong higher S-Ca was associated with lower odds of longevity, an association that was consistent in the two data sources Fig. . For oneAssociations of genetically predicted S-Ca and S-25(OH)D concentrations with cardiovascular disease and cancer are displayed in Fig. Our meta-analysis indicated that calcium and calcium plus vitamin D supplementation increased fasting S-Ca concentrations over 1–4 years of use. MR analyses showed that genetically predicted lifelong increases of S-Ca concentrations were associated with lower odds of longevity and with an increased risk of cardiovascular disease. There was no evidence supporting associations of S-Ca with cancer and of S-25(OH)D with longevity, cardiovascular disease, or cancer.17. S-Ca is homeostatically regulated and the effect of prolonged calcium supplementation on fasting S-Ca has not previously been comprehensively assessed. Here we conducted a meta-analysis to address this question and found that both calcium and calcium plus vitamin D supplementation led to increased fasting S-Ca after several years of supplement use. Mean fasting S-Ca was observed to be higher  in the group assigned calcium and vitamin D supplements than in the placebo group. This change in S-Ca corresponds well with the previously published short-term change in S-Ca measured 12 h after a dose of calcium carbonate and calcium citrate21, the two compounds most widely used for supplementation21. This change in S-Ca might imply a substantial shift in the proportion of individuals with very high concentrations. Hence, the moderate nadir mean change could have a considerable impact in high-risk individuals.Calcium supplementation (1000 mg dose) leads to an acute elevation of S-Ca with a peak of about 0.09 mmol/L increase after 4 h11. Similarly, a cohort study of 61,433 Swedish women with 19 years of follow-up found that women with a total calcium intake >1400 mg/day versus 600–1000 mg/day had a 40% increased risk of death from all causes and a 49% higher cardiovascular mortality22. In contrast, other observational studies whose participants had on average lower calcium intake found inverse associations of dietary calcium with death from all causes23 and cardiovascular disease23. With regard to supplemental calcium intake, two large-scale studies in older women had divergent conclusions that supported24 and opposed22 calcium supplement use in prolonging longevity. These inconsistent findings may potentially be related to residual confounding in the observational studies. The National Osteoporosis Foundation and American Society for Preventive Cardiology developed a guideline that total calcium intake <2500 mg/day should be considered safe from a standpoint of cardiovascular disease and cardiovascular and all-cause mortality25. Nonetheless, it was noted that the risk of death was 157% higher for individuals with daily calcium supplementation (≥500 mg) versus no treatment among women with a dietary calcium intake >1400 mg/day22. Our estimates are not directly comparable to those from previous studies as our results were scaled per SD increment of S-Ca, whereas the change in long-term S-Ca from high dietary or supplemental calcium intake may be smaller, as suggested by our meta-analysis of intervention studies of calcium supplementation.Our findings linking genetically higher S-Ca concentrations to lower life expectancy are consistent with some, but not all, traditional observational studies on calcium intake or circulating calcium in relation to mortality. A meta-analysis of eight cohort studies showed a 13% increased risk of all-cause mortality per one SD increase in S-Ca concentrations; however, this finding might be affected by confounding11 and a subsequent cohort study including 441,738 participants of the AMORIS database also showed a positive association between circulating calcium concentrations and risk of cardiovascular disease26. This association was confirmed in a large-scale study that included 16,718 women and its incorporation into a meta-analysis with 8 other studies on calcium supplements and cardiovascular events27. Two recent systematic reviews, however, suggested no effect of calcium supplements on cardiovascular disease risk25. With regard to individual cardiovascular diseases, genetically predicted S-Ca was associated with coronary artery disease and myocardial infarction28 but not with ischemic stroke29, heart failure30, or atrial fibrillation31 in previous MR studies. The elevated S-Ca may exert effects on vascular calcification, endothelial function, blood coagulation, and altered gene expression induced by signaling through the calcium-sensing receptor expressed in the endothelium and vascular smooth muscle cells32, thereby increasing the risk of cardiovascular disease. Calcium intake might be associated with certain specific cancers35, but data on the association of S-Ca with overall cancer risk are limited36.Our study further showed a possible causal association between elevated S-Ca and increased risk of cardiovascular disease, which may explain the observed association between S-Ca and longevity. A meta-analysis of 8 studies38. In addition, physical inactivity has been shown to increase S-Ca39, so an indirect detrimental effects of calcium loss from bone during immobilization could be a decrease in longevity and increase in risk of cardiovascular disease.Our study shows that the levels of S-Ca increase with dietary calcium supplement use. Menopause-related calcium loss from bone and osteoporotic calcium loss are also important in raising S-Ca levels40 and cardiovascular disease41. Nonetheless, evidence from randomized controlled trials does not confirm a reduced risk of all-cause mortality or cardiovascular disease with vitamin D supplementation42. Our MR findings confirm and extend the findings of the trials by showing that increased S-25(OH) concentrations are not associated with longevity or cardiovascular disease in the general population. Our study found no association between S-25(OH)D and cancer risk, in agreement with results of a meta-analysis of randomized controlled trials showing that vitamin D supplementation reduced total cancer mortality but not total cancer incidence43. Likewise, a recent large-scale randomized controlled trial revealed that vitamin D supplementation did not result in a lower incidence of invasive cancer42. A small proportion (~20%) of the population in the UK Biobank had S-25(OH)D levels in the deficiency range in the UK Biobank. Although no potential adverse effects of vitamin D supplementation have been reported in previous studies, the risk of hypercalcemia appears to increase with large supplemental doses of vitamin D taken for ≥1 year44. Given the potentially different effects of bioavailable and total 25(OH)D concentrations47 on disease and mortality, additional large clinical studies are warranted to disentangle these complex associations and potential threshold effects in both fair- and dark-skinned individuals.Findings on the associations of vitamin D with mortality and risk of cardiovascular disease are conflicting. Most observational studies have reported an inverse association of circulating S-25(OH)D concentrations with mortalityp value for the genome-wide association was retained. In addition, we calculated the F-statistic for all sets of instruments to assess the strength of the instrumental variables. These were all >10, indicating good strength of the genetic instruments we used. However, whether these SNPs reflect the levels of S-Ca and S-25(OH)D in the outcome populations could not be examined. The second assumption is that the SNPs used as instruments for the exposure D in this study) should not be associated with important confounders in the study population. Because of our reliance on summary-level genetic data, we were unable to examine this issue. The third assumption is that instruments in an MR study should influence the risk of outcomes via the exposure only, not via alternative pathways . We assessed this assumption using MR-Egger analysis and did not observe any indication of pleiotropy, except for S-25(OH)D proxied by SNPs from the UK Biobank in relation to longevity in the data from the GWAS conducted by Pilling et al. The associations also remained consistent in the MR-PRESSO analysis after removal of potential outliers. Nonetheless, we cannot completely rule out the possibility that observed associations were influenced by horizontal pleiotropy.There are three important assumptions of MR studies. The first is that the genetic instruments should be robustly associated with the exposure. In our study, we selected SNPs associated with S-Ca and S-25(OH)D at the genome-wide significance level. For SNPs with linkage disequilibrium, the SNP with the lowest There are several strengths of the present study. We examined the associations of lifelong higher S-Ca and S-25(OH)D with longevity in two independent outcome data sources with up to total of 123,694 individuals. The consistency of the results in primary and confirmatory analyses solidified the causal inference on the detrimental effect of high S-Ca on longevity. Although our analyses were confined to European populations, population structure was adjusted for in GWASs for exposures and outcomes. Thus, it is unlikely that population stratification bias affected our results, but we cannot generalize our findings to non-European populations with potentially different mean S-Ca and S-25(OH)D concentrations. We used summary-level data from large genetic consortia in combination with the UK Biobank study, thereby assuring high statistical power to detect weak associations.48. Thus, this bias is unlikely to be substantial. We detected an attenuated association between genetically predicted S-Ca levels and longevity in the supplementary analysis using SNPs from UK Biobank, which might reflect the compromised validity of certain instruments.Limitations of this summary-level MR study are that we could not assess possible non-linear associations and interaction effects of S-Ca and S-25(OH)D with other modifiable factors . Another limitation is that, in the longevity analysis, the inclusion of individuals lost to follow-up before the 60th percentile of age at death in controls introduces some misclassification since these individuals could have survived beyond the 60th percentile and potentially even beyond the age corresponding to the 90th survival percentile. However, the misclassification in controls was estimated to be small48, so the UK Biobank data provide useful confirmation of the findings in our main longevity analysis. Parental longevity similarly confirmed a GWAS of longevity in a previous study48. Of course, this is not surprising: parents share half of their genetic code (including longevity-associated genes) as well as possibly healthy lifestyles with their offspring.Longevity of subjects and parental lifespan are different, though related phenotypes. Nevertheless, there is strong genetic correlation between the twoIn summary, genetically proxied lifelong higher S-Ca but not S-25(OH)D was associated with an increased risk of cardiovascular disease and lower odds of longevity. These adverse impacts of high S-Ca concentrations may dominate over the positive effects since current evidence does not support the general use of such supplements for the prevention of fractures in healthy community-dwelling adults.49. We reviewed all included studies and extracted demographic features, dose and duration of supplementation, and post-treatment S-Ca information from all studies that reported S-Ca after intervention. Detailed methods are presented in Supplementary Methods Studies included in the present meta-analysis were identified from a published meta-analysis assessing the effect of calcium and calcium plus vitamin D supplementations on bone mineral density. That meta-analysis included 32 trials of calcium supplementation and 19 trials of supplementation with calcium plus vitamin D supplementationThe present MR study was based on summary-level data from published and publicly available GWASs regarding S-Ca and S-25(OH)D, longevity, cardiovascular disease, and cancer. Detailed information on the included studies and consortia is presented in Supplementary Table r2 < 0.01) SNPs associated with S-Ca at the genome-wide significant level (p < 5 × 10−8)50. Those SNPs were used as the primary set of genetic instruments for S-Ca in this study. A confirmatory set of 198 SNPs associated with S-Ca at p < 5 × 10−8 was obtained from the UK Biobank study that included 315,153 participants of European ancestry51. Detailed description of the instrument selection procedure for S-Ca in the UK Biobank is described in Supplementary Methods 53. One of these SNPs, rs117913124 with low frequency but a large effect on S-25(OH)D, was identified from a sample of 42,274 participants of European ancestry52, and the other six SNPs were selected from 31 cohorts with 79,366 participants of European ancestry53. We also used 143 SNPs as confirmatory instruments for S-25(OH)D from a recent study with 417,580 participants of European ancestry in the UK Biobank54. Detailed information on genetic instruments for S-Ca and S-25(OH)D is displayed in Table A meta-analysis of 28 GWASs of S-Ca that included up to 61,054 participants of European ancestry identified 7 independent . This phenotype, though conceptually different from longevity, has a strong genetic correlation with longevity48. In supplementary analyses, we examined the associations of S-Ca and S-25(OH)D with lifespan using data from a GWAS meta-analysis on parental lifespan in UK Biobank and LifeGen including over 1 million individuals56. Summary-level data for cardiovascular disease  and cancer  were obtained from the fifth wave of analyses of the FinnGen consortium57. Detailed data on involved cohorts, genotypes, endpoint definition, and association test in the FinnGen consortium are available on the FinnGen webpage. Diagnostic information for cardiovascular disease and cancer is presented in Supplementary Table Summary-level data for longevity were available from a discovery meta-analysis of 18 GWAS cohorts of longevity. Long-lived cases were 11,262 European-descent participants who lived to an age ≥90th survival percentile based on individual cohort life tables from census data from each country, by sex and birth cohort. Early mortality controls were 25,483 European descent individuals who died at or before the 60th percentile of the expected age at death or whose age at the last follow-up visit was ≤60th survival percentileI2 statistic was calculated to present heterogeneity among estimates from different studies. Funnel plot and Egger’s test were used to evaluate small study bias and publication bias. The meta-analysis was performed using “metan” package in Stata/SE 15.0.Both fixed- and random-effects inverse-variance weighted models were used to estimate the difference in means of S-Ca between arms with and without calcium or calcium plus vitamin D supplementation from the included studies. The 58. Estimates for S-Ca and S-25(OH)D in relation to longevity from the two data sources were combined using fixed-effects meta-analysis. Several sensitivity analyses with different assumptions and strengths, including the weighted median59, MR-Egger regression60, and MR-PRESSO61 methods, were used to examine the consistency of results and detect and correct for possible pleiotropy. Assuming ≥50% weight from valid instruments, the weighted median method can provide consistent MR estimates59. MR-Egger regression can detect pleiotropic effects and provide estimate after correction of pleiotropy, although it compromises statistical power60. The MR-PRESSO model can detect possible outliers and generate estimates after removal of outliers, thereby correcting for horizontal pleiotropy61. Cochrane’s Q was calculated in the inverse-variance weighted models to assess heterogeneity among estimates of used SNPs and the p value for the intercept in MR-Egger regression60 was used to examine possible pleiotropy (p < 0.05). ORs and the corresponding CIs were scaled to one between-person SD increase in genetically predicted S-Ca  and S-25(OH)D concentrations. All analyses were performed using the mrrobust package62 in Stata/SE 15.0 and the TwoSampleMR package63 in R Software 3.6.0.The random-effects inverse-variance weighted approach was employed in the primary statistical analysis in MR analysisSupplementary Information


SLCO1B1 gene on myopathy in patients with statin. Studies were searched using multiple databases and selected following inclusion criteria. Two reviewers independently performed data extraction and assessments for risk of bias. Fixed-or-random-effect was applied to pool allele frequency/effects. Mixed-effect logit model was used to pool genotypic effects using individual patient data. Heterogeneity and publication bias were explored. Fourteen studies were pooled for rs4149056; the minor C allele frequency were 15% in Caucasians and 14% in Asians. Six studies were pooled for rs2306283; the minor G allele frequency was 34% in Caucasian and 75% in Asians. Genotypic effects of rs4149056 polymorphism in Caucasians indicated that statin users who carried CC and TC genotypes had a significantly higher risk of myopathy than those who carried TT genotype, with a pooled odds ratio (OR) of 2.9  and 1.6 , respectively. For subgroup analysis, CC and TC genotypes also suggested a higher risk of myopathy in simvastatin users  and in atorvastatin users  than those who carried TT genotype. There was no significant association between rs2306283 polymorphism and myopathy in Caucasians and Asians. There was no evidence of publication bias for both polymorphisms.This meta-analysis was conducted to determine the genotypic effects of rs4149056 and rs2306283 polymorphism in  Hypercholesterolemia is a significant risk factor for cardiovascular diseases (CVDs) which is a vital cause of death in the world . The morSLCO1B1) [SLCO1B1 gene have been reportedly associated with statin and risk of myopathy in statin users, as it could influence the plasma concentration of statins [SLCO1B1 gene may potentially influence the pharmacokinetics and pharmacodynamics of statins [However, myopathy commonly occurred in statin users, leading to poor adherence , 7. Its SLCO1B1) . Two pol statins –18. Acco statins , 19. Bes statins , 20–22, SLCO1B1 gene and myopathy in statin users. They mainly focused on the SLCO1B1 (rs4149056) polymorphisms; only one MA also considered the rs2306283 polymorphisms in Caucasians [Recently, five meta-analyses (MA) –27, whicucasians  which coSLCO1B1 gene on myopathy in both Caucasian and Asian patients who received statin. In addition, a subgroup analysis was performed by statin types. Moreover, we also estimated both allele frequencies and mode of genetic effects of each polymorphism separately by ethnicity, as suggested by the guidelines of the Human Genome Epidemiology Network [Therefore, this MA was conducted to assess the effect of these two polymorphisms of  Network .http://www.crd.york.ac.uk/PROSPERO/display_record.php?ID=CRD42018105282). This review was conducted according to the guidelines of Preferred Reporting Items for Systematic Reviews and Meta-Analysis [This systematic review protocol was registered with PROSPERO, the International prospective register of systematic reviews  and outcome  domains, see Supplementary Table The studies were located from the MEDLINE via PubMed and Scopus databases up to April 2019, based on search terms of gene  had myopathy as the outcome. The studies were excluded if their data were insufficient for pooling and authors did not provide additional data after being contacted twice, as well as non-English studies. Any disagreements were resolved through consensus with the third author (TL).Two reviewers (ST and SS) independently selected studies by screening titles and abstracts. If a decision for eligibility could be made, the full articles were reviewed. Any types of studies were included if they met all of the following criteria: (1) studied in general adults (aged 18 years or older) who received statin regardless indications, (2) studied two polymorphisms  of the The primary outcome was myopathy defined by muscle weakness after statin use with/without confirming by CK higher rising three times, compared to the upper-normal limit .Data were extracted independently by two reviewers (ST and SS) focusing on five parts, i.e., general information, study characteristics, patient characteristics, genes and outcome, and data for pooling, see Table Two reviewers (ST and TL) independently assessed a risk of bias for genetic association studies  considerχ2) or exact test goodness-of-fit where appropriate. The studies were not included in pooling if they did not comply with HWE. Heterogeneity and its degree were assessed using Cochrane’s Q test and I2 statistic. If the heterogeneity was present , the sources of heterogeneity were explored by meta-regression or subgroup analysis by potential factors .Statistical analysis was performed following the method for meta-analysis of genetic association studies . HWE wasp value  < 0.1 or I2 ≥ 25%); otherwise, a fixed-effect model with inverse variance method was applied. For the per genotype approach, the OR1 (aa vs AA) and OR2 (Aa vs AA) were estimated using a mixed-effect logistic regression.A minor allele frequency (MAF) in the control groups was pooled and stratified by ethnicity. Gene effects were estimated using two approaches . For the per allele approach, ORs of minor allele a vs major allele A along with 95% confidence interval (CI) were estimated. The ORs were pooled using a random-effect model by DerSimonian and Laird method  if heterp value  < 0.05 was considered as statistically significant.A sensitivity analysis was determined by including and excluding studies that did not comply with HWE in the main pooling to see robustness of the results based on a method for meta-analysis of genetic association studies , 34. LasA total of 2427 and 184 studies were, respectively, located from Scopus and MEDLINE via PubMed, but only 15 studies were eligible, see Fig. The characteristics of 15 included studies were demonstrated in Table The results of bias assessment are shown in Supplementary Table Moreover, the risk of confounding bias might be presented in two studies that did not mention adjusting confounders. All studies demonstrated a significant association between SNPs and myopathy as a result of low risk of bias from selective outcome reporting. Ten of 15 studies (67%) assessed HWE.χ2 = 59.53, df = 10, p = < 0.001, I2 = 83.2%) with the pooled OR (C vs T) of 1.84 . Therefore, a random-effect model was used to pool ORs across studies.Fourteen studies investigated the association between rs4149056 and myopathy. Of these, 11 studies , 38–45 aχ2 = 0.73, df = 1, p = 0.392, I2 = 0%), with the pooled ORs (95% CI) of C vs T of 1.87 . A fixed-effect model was used to pool ORs across studies. The results suggested that statin users who carried minor C allele had ~1.8 times higher risk of myopathy than those who carried T allele in Caucasians and Asians. Sensitivity analysis was not performed, because all included studies were complied with HWE  and were included in the main pooling.The pooled frequency of C allele in two Asian studies was 0.24  and 0.14  in case and control groups, respectively. However, the allele effect was homogeneous (n = 1433 vs 2878 for myopathy vs control groups). Heterogeneity was present for both OR1  and OR2 . A mixed-effect logit model was used to pool OR1 and OR2 across studies, which yielded OR1 and OR2 of 2.9  and 1.6 , respectively  and 2.0 , see Fig. 1 and OR2 in Caucasian with statin users, indicating no evidence of asymmetry of funnels for both ORs, see Supplementary Table The potential sources of heterogeneity were explored by fitting age, female gender, duration of therapy, and the statins type in a meta-regression model  and 0.75 , respectively. The ORs (G vs A) were 1.00  in Caucasians and 1.17  in Asians, see Supplementary Table n = 998 vs 1487 for myopathy vs control groups) and two Asian studies (n = 199 vs 956 for myopathy vs control groups), see Supplementary Table 1 (GG vs AA) and OR2 (AG vs AA) in statin users among Caucasians were 0.8  and 1.0  with moderate heterogeneity I2 of 25.6% and I2 = 63.9%, respectively  [SLCO1B1 gene have been reported that they could influence statin and the risk of myopathy association [SLCO1B1 gene is able to induce variations in plasma concentrations of statin through the influx transporter OATP1B1, it could influence the pharmacokinetic and pharmacodynamic profiles of statins [This meta-analysis of genetic association studies was conducted to assess the genetic effects of single nucleotide polymorphisms (rs4149056 and rs2306283) of the  604843) . Two polociation –18. Beca statins , 52.Fourteen studies were included for rs4149056, and six studies for rs2306283 in the main pooling. The frequency of the minor allele for each polymorphism was pooled and the magnitude of genotype effects was determined by ethnicity. The analysis was performed to include additional studies provided from previous evidence and complied with the guidelines of the Human Genome Epidemiology Network , 53, 54.SLCO1B1 genotype in statin-associated myopathy and suggested that this association may be more robust for atorvastatin and simvastatin. There were some reasons suggesting that individuals who received atorvastatin may have higher risk of myopathy than those who took simvastatin. It may explain why patients who obtained higher equivalent doses of statins or more potent statins, in terms of lipid-lowering ability, may have an increasing tendency for myopathy risk. Consequently, included studies on atorvastatin used dosage between 20 and 80 mg daily, which was classified as a moderate to high-intensity therapy. On the other hand, included studies on simvastatin mostly used 10–40 mg daily, which was a low to moderate-intensity therapy. Nevertheless, we did not report a specific dosage for any statin because of insufficient data for pooling from the original studies.Our findings further supported the role of the p < 0.001), and simvastatin users 2.2  and atorvastatin users 2.1 . Our finding is similar to Xiang et al. [In addition, we performed TC + CC vs TT of rs4149056 polymorphism among ten studies in Caucasians. The results showed that individuals who carried TC + CC genotype also had a higher risk of myopathy when compared with TT genotype in statin users, with a pooled OR of 1.9  or the data were not sufficient for pooling.SLCO1B1 gene and myopathy among other types of statin to confirm that the effects could be a class effect or specific types of statin only. Future research including analysis cost-effectiveness study on the SLCO1B1 gene is also required. It may be useful for assessing the value of money of pharmacogenetic testing before prescribing statin, preventing the adverse events, and improving patient’s adherence considering that pharmacogenetic testing plays an extensive role on precision and personalized medicine.Given that all types of statins were substrates of OATP1B1 transporter, we would need more genetic association studies of the This study provided an understanding of the roles of genetic and drug response. Our findings may be considered an extension of traditional approaches to treat a disease, which is useful for physicians, as they could select a drug treatment or intervention based on a patient’s molecular profile. This could lead to a reduction of harmful adverse effects, ensuring an effective outcome as well as containing costs from a “trial-and-error” approach to disease treatment. Moreover, policy-makers could make evidence-based decisions for the selection of drugs or alternative drugs on a list of Thai National List of Essential Medicines for patients through the genetic information added to the clinical and economic evidence.In summary, this meta-analysis provides a robust association in Caucasians indicating that individuals who carried the minor C allele, with CC or TC genotypes of rs4149056 polymorphism may particularly have higher risks of myopathy among various statin users and especially in those who received simvastatin and atorvastatin, compared with those who carried the TT genotype. No significant association between rs2306283 polymorphism and myopathy was caused by various statin users was found in Asian and Caucasian populations.Supplementary information


Linear dose-response analysis indicated that each 10% increase in UPF consumption  was associated with a 15% higher risk of T2DM  among adults. Non-linear dose-response analysis demonstrated a positive linear association between UPF consumption and T2DM  among adults. (4) Conclusions: A higher intake of UPF was significantly associated with an increased risk of T2DM. However, underlying mechanisms remain unknown and future experimental studies are warranted.(1) Background: Recent individual studies have demonstrated that consumption of ultra-processed food (UPF) may be related to type two diabetes mellitus (T2DM). We aimed to synthesize the results from these individual studies by conducting an updated systematic review and meta-analysis of observational studies evaluating the association between UPF consumption and the risk of T2DM. (2) Methods: A systematic search was conducted using ISI Web of Science, PubMed/MEDLINE and Scopus electronic databases from inception up to August 2021. Data were extracted from five studies . Risk ratios (RR) of pooled results were estimated using a random-effects model. (3) Results: Our results revealed that higher UPF consumption was significantly associated with an increased risk of T2DM (RR = 1.74; 95% CI: 1.36, 2.22; I The growing incidence of chronic non-communicable diseases such as type two diabetes mellitus T2DM) is the major healthcare concern worldwide . AccordiDM is then = 1). Since then, several individual studies have assessed this association. Therefore, the present study aimed to build on previous work by synthesizing the findings of observational studies investigating the association between UPF intake and the risk of T2DM.In the last half-century, the processing of foodstuffs has substantially evolved . The NOVThe current systematic review and meta-analysis were conducted based on the 2020 PRISMA guidelines . The stuA systematic search was conducted using the following electronic databases: ISI Web of Science, PubMed/MEDLINE and Scopus, from database inception up to 10 August 2021 without restrictions for language. Search terms were a combination of free-text terms and controlled vocabulary related to UPF and diabetes, including the following that were applicable to a search via MELINE: ((“fast foods” [All Fields] OR “fast foods” [MeSH Terms] OR “ultra processed food *” [All Fields] OR “ultraprocessed food *” [All Fields] OR “ultra processed food *” [All Fields] OR “processed food *” [All Fields] OR “ultra-processed” [All Fields] OR “ultraprocessed” [All Fields] OR “ultra-processed” [All Fields] OR “NOVA” [All Fields] OR “nova food classif *” [All Fields] OR “nova food *” [All Fields] OR “nova food classif *” [All Fields] OR “NOVA food classification system” [All Fields]) AND )  undertaken in adults (≥18 years) that reported on the association between UPF consumption and the risk of T2DM, and provided effect estimates in the form of hazard ratio (HR), relative risk (RR), or odds ratios (OR) with 95% Confidence Interval (95% CI). Studies conducted in children and adolescents (<18 years), reviews, conference letters, notes, reports, short surveys and case reports were excluded. The population, intervention, comparator and outcome (PICO) description can be observed in The assessment of titles and abstracts and the full-text review process for studies retrieved through our search strategy were undertaken separately by two researchers (S.M. and H.M.), with any disagreements about the inclusion and exclusion of selected studies decided by consensus or discussion. A standardized method was applied to the inclusion and exclusion criteria, which took into consideration the setting, population and evaluated exposure(s) and outcome(s) of individual studies.A standardized method was also applied to the data extraction process undertaken independently by two researchers (S.M. and H.M.) through Microsoft Office Excel 2013 . The following parameters were extracted: (a) the first author’s name; (b) year of publication; (c) country and setting of the study; (d) number of participants, (e) age, (f) gender; (h) follow-up duration in cohort studies; (i) methods for evaluating exposure; (j) study main findings; and (k) covariates applied for adjustments in the multivariable analyses. Any discrepancies and disagreements about data extraction were determined by consensus or discussion with a third researcher (A.J.).Two researchers (S.M. and H.M.) independently evaluated the quality of each study by applying the Newcastle–Ottawa Scale (NOS) . The NOS2) statistics. The I2 value was calculated as ([Q-df])/Q × 100%, Q being the χ2 value and df the corresponding degrees of freedom. The heterogeneity was considered significant where the Q statistics were significant (p < 0.01) or I2 > 50%; more specifically, low, moderate, high and extreme heterogeneity was defined according to the I2 statistics cut-offs of <25%, 25–50%, 50–75% and ˃75%, respectively. Sensitivity analysis was carried out by removing each study and recalculating the pooled effect estimates . Publication bias was assessed by the visual inspection of funnel plots, formal testing by the Egger’s regression asymmetry and Begg’s rank correlation tests [p < 0.05.We conducted statistical analyses with STATA version 14.0  and SPSS version 25.0 . The OR and its 95% CI were assumed as the effect size. The effect estimates reported by the original studies and considered for inclusion in our meta-analyses included OR and HR (and their 95% CI); HR was considered equal to RR . The synon tests  and resuWe also conducted a dose-response meta-analysis to estimate the RRs for each 10% increment in UPF intake, according to the method introduced by Greenland and Orsini ,28. For Our search strategy retrieved 4083 studies and, after removing duplicates records, 2754 studies remained. The titles and abstracts of these studies were assessed and 2745 records were subsequently excluded based on our inclusion criteria. Eight full-text articles were reviewed and three of these studies were omitted due to the following reasons: two studies were conducted on women with gestational diabetes ,31 and o2 = 68.9%; p < 0.001)  among adults  among adults.The results revealed that UPF consumption was associated with an increased risk of T2DM  . Linear g adults . MoreoveSensitivity analysis showed that the study results were not affected by any single study.p = 0.007). However, Begg’s rank correlation tests (p = 0.463) result did not show publication bias among studies.As illustrated in This study demonstrated that higher UPF intake was associated with an increased risk of T2DM. Additionally, increasing the intake of processed foods by 10% leads to a 15% increase in the risk of T2DM. A linear positive association was found between UPF intake and the risk of T2DM. The results of each study included in our meta-analyses were in line with our findings; they each showed that increased UPF intake was associated with an increased odds of the prevalence or incidence of T2DM. Two cohort studies conducted in Netherlands and Brazil reported a 10% increase in UPF consumption was associated with a 33% and 13% enhanced risk of developing T2DM, respectively. The other two cohort studies conducted in Spain and the United Kingdom showed that participants who consumed a higher versus a lower amount of UPF had a 53% and 44% higher risk of developing T2DM ,22. The Our study adds to the body of literature showing that particular dietary factors such as those common to UPF may foster certain aberrant health states, including diabetes. These dietary factors mainly include low nutritional quality, which more specifically consists of higher intakes of sodium, energy, fat, sugar, lower amounts of fiber, protein, essential vitamins and minerals. Indeed, our findings are consistent with a recent meta-analysis of observational studies that showed individuals with diabetes had higher sodium status compared to individuals without diabetes . The autThe typically scarce levels and limited types of dietary fiber in UPF may also play a role in the observed association between UPF intake and T2DM. Dietary fiber is often recommended as adjunctive nutritional therapy in diabetes management. This is because dietary fiber lowers postprandial hyperglycemia and increases satiety by delaying digestion and absorption of carbohydrates and improving blood lipids, body mass and inflammation . In addiAnother possible mechanism relevant to the dietary properties of UPF that may partly explain the association between UPF intake and T2DM is the added sugar content of UPF. A study conducted in the United States showed that almost 90% of the added sugar consumed by people is obtained from UPF . It is wWhile nutrition research has historically targeted the role of dietary calorie intake and macro and micronutrients in T2DM, the relevance of the NOVA food classification is underscored when considering non-nutritional factors related to food processing. These include incorporating artificial additives into UPF and the manifestation and/or migration of chemical compounds that may occur due to the production process and the encasing of formulations with packaging made from synthetic materials, respectively. For example, food additives such as carrageenan as a thickener and stabilizer can interfere with insulin signaling and may cultivate insulin resistance . UPF is 2 > 50% for our meta-analyses, was the between-study difference in expressing UPF intake as the exposure variable. For example, UPF intake was expressed as a continuous-only variable in two studies (10% increments) [While the high quality of studies included in our review, as per the NOS that was applied, may indicate reliable evidence, results from our meta-analyses should be viewed considering the following limitations. Few original studies were included in our syntheses, where inadequate estimation of the between-study variance remains possible . In addirements) ,20 comparements) ,22 and qrements) ). UPF inrements)  or weighrements) ,19,22, wThe current systematic review and dose-response meta-analysis was conducted to elucidate whether the UPF consumption has a potential association with the risk of T2DM. Observational studies undertaken in adults that met inclusion criteria were included in the final analysis. In summary, our outcomes suggested that higher UPF consumption may be significantly associated with an increased risk of T2DM. In addition, the linear dose-response analysis showed that each 10% increase in UPF consumption  was associated with a 15% higher risk of T2DM among adults. Non-linear dose-response analysis also demonstrated a positive linear association between UPF consumption and T2DM among adults. In terms of directions for future research, further studies could investigate the following points. Current tools available for estimating UPF intakes are subjective , rather limited in scope, with a majority evaluating only one dimension . To more precisely estimate the actual burden of UPF intakes, new tools should be adapted or progressed to evaluate all UPF consumption dimensions, namely food class, UPF foods specific components, their effects on health and specific processes or additives. The new technology-based dietary assessment methods (web-based and mobile device applications) may help more precise UPF intakes evaluation. However, underlying mechanisms remain unknown. In future studies, evaluation of associated variables including lifestyle, demographic, genetic background, socioeconomic and clinical factors, as well as differences in treatment, can accelerate the finding of possible mechanisms. Due to identified limitations, to better understand the association between UPF consumption and the risk of T2DM, more prospective cohort studies with further extended follow-up periods are necessary.


The use of statins is a potential protective factor against the development of hepatocellular carcinoma. Therefore, we conducted a meta-analysis to evaluate the contribution of statins to the risk of hepatocellular carcinoma.  We searched for PubMed and EMBASE through January 2021.  Thirty-two studies  reporting 56,838 cases of hepatocellular carcinoma in 4,963,518 persons were included. Statin users were less likely to develop hepatocellular carcinoma than nonusers . Stratified analysis showed that statins reduced the risk of hepatocellular carcinoma in Asian and Western populations . Besides, statins have protective effects against hepatocellular carcinoma after hepatitis B virus  and hepatitis C virus infections . Statins have protective effects on people with chronic liver disease  and on the general population . Lipophilic statins can prevent hepatocellular carcinoma , while hydrophilic statins cannot . The single-drug analyses showed that simvastatin , atorvastatin , rosuvastatin , lovastatin , and pitavastatin  had significant benefits. Further studies have shown that those in the high-dose group experienced better effects in preventing hepatocellular carcinoma . Further research found that the combined use of aspirin did not increase the chemoprevention effect of liver cancer . In addition, the preventive effect of statins improved with the extension of follow-up time .  Our meta-analysis shows that the use of statins is associated with a lower risk of liver cancer. Liver cancer is one of the most common cancers in the world, imposing substantial burdens on patients and society , 2. TradAccordingly, our group conducted this meta-analysis to evaluate the relationship between statin use and the risk of liver cancer; this updated meta-analysis has the benefits of including recently published studies and more subgroup analyses.The meta-analysis is reported based on the Meta-analysis Of Observational Studies in Epidemiology (MOOSE) checklist , followiRandomized controlled trials (RCT), cohort studies, nested case-control studies, and case-control studies were included in this meta-analysis. The patients included were adults (18 years or older) who were ultimately diagnosed with liver cancer (diagnosed using any recognized diagnostic criteria), regardless of sex or race. In addition, the interventions were defined as any dose, route of administration, and duration of statin therapy . The control group could be a placebo or a nonstatin therapy.Studies were excluded if they were animal experiments, reviews, meta-analyses, editorials, and published letters.The risk of HCC in statin users was assessed first. We use the odds ratio (OR) to evaluate the risk of HCC. When the OR was less than 1, the use of statins had a protective effect against the occurrence of HCC. To explain the potential heterogeneity, experimental design, experimental location, etiology, presence of chronic liver disease, type of statins, and dosage of statins were investigated.Two electronic databases containing articles published in English, namely, PubMed and EMBASE, were thoroughly searched to identify the studies that could be included in the analysis. Based on the guidance in the Cochrane handbook, we developed detailed strategies, including “statin” [MeSH term] or “statin” or “atorvastatin” or “fluvastatin” or “cerivastatin” or “lovastatin” or “pravastatin” or “rosuvastatin” or “simvastatin” or “pitavastatin” or “HMG-CoA reductase inhibitors” and “hepatocellular carcinoma” [MeSH terms] or “liver neoplasms” [MeSH term] or “liver cancer” or “HCC.” The reference lists of the studies included and related reviews were manually checked to identify other citations. To ensure that the qualified studies were correctly identified, two researchers independently read each report, and all the researchers resolved their differences by reaching consensus. The flowchart summarizing the study selection process is shown in Data were extracted from all eligible studies by 2 independent researchers (Yikai Wang and Wenjun Wang). The following data were collected from each study: study design, country of origin of the study population, time period of the study, number of people included, outcome events, and adjusted confounding factors.Data on the following confounding HCC risk factors were extracted from each study: age, sex, race, socioeconomic status, body mass index (BMI), diabetes (DM), the presence of cirrhosis, hepatitis B virus (HBV) infection, hepatitis C virus (HCV) infection, presence of alcoholic liver disease, and the use of angiotensin-converting enzyme inhibitors/nonsteroidal anti-inflammatory drugs/aspirin/metformin/proton pump inhibitors (PPIs)/nonstatin lipid-lowering drugs and lifestyle habits Tables  and 2.We used the Cochrane intervention system evaluation manual V.5.1.0 to evaluate the quality of RCTs. The risk of bias tool covers seven aspects: random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, selective reporting, and other bias. In the end, we classified each RCT into one of three levels: high risk, low risk, and unclear risk of bias. The results of the risk assessment are shown in The quality of observational research was assessed using the Newcastle–Ottawa scale, which is one of the most commonly used rating systems for evaluating the quality of observational research in meta-analyses . The scaP < 0.05 to indicate statistical significance, and all statistical tests were two-sided. For statistical analysis, we chose the commonly used software Review Manager . To obtain a more generalizable result, we used the random-effects model for the meta-analysis of the OR data because all potential heterogeneities were included in the construction of model [We considered of model . By analI2 and Cochran's Q tests to evaluate heterogeneity, as these are often used to measure inconsistency among different studies. If the I2 value was >50% or P < 0.10, the heterogeneity was considered significant [We used the nificant . We usedWe selected 638 and 1,453 articles from PubMed and EMBASE, respectively. After applying the inclusion criteria and deleting duplicate studies, 30 studies were finally selected for inclusion in the meta-analysis , 22–49. The characteristics of these studies are shown . NineteeNine articles were from Asia, including two from China (one from Taiwan and one from Hong Kong), five from Korea, and two from Japan. Twenty-two articles were from Western countries, including eleven from the United States, four from the United Kingdom, three from Switzerland, one from Denmark, and one from Italy. One RCT was a multicenter study from Europe, Australia, and South America. Taking into account the differences among ethnicities, we conducted a subgroup analysis in the follow-up analysis. The study population variables used for adjustment were age, sex, etiology of the potential liver disease, other complications, and the use of other drugs.The median Newcastle–Ottawa scale score was seven ; eighteen of the thirty studies were considered to be of high quality. The methodological quality of all studies is described . Most stQ test P < 0.00001, I2 = 99%). The shape of the funnel plot indicated that there was a low significant publication bias (P=0.064)  .Based on the predetermined assumptions, we conducted a hierarchical analysis according to the study design . An analAccording to the subgroup analysis stratified by geographic location, the use of statins significantly reduced the risk of HCC by 46% in Asian populations  and by 40%  in Western populations . Unfortunately, these two subgroup analyses did not explain the significant heterogeneity in the overall analysis.I2 = 65%). Moreover, HCV-positive patients also experienced clear preventive effects , and no heterogeneity was found.Next, the protective effect of statins against liver cancer caused by HBV and HCV infections was evaluated. It was confirmed that the use of statins in HBV-positive patients had a significant preventive effect against HCC . However, the protective effect of statins in the general population is slightly lower than that of patients with chronic liver disease .I2 = 23%). However, we did not find an association between hydrophilic statin drugs (pravastatin and rosuvastatin) and a reduction in the risk of liver cancer . Single-drug analyses showed that simvastatin , atorvastatin , rosuvastatin , lovastatin , and pitavastatin  had significant benefits, with moderate heterogeneity. However, we found no benefit of fluvastatin , pravastatin , or cerivastatin .Lipophilic statins  were associated with a significant reduction in the incidence of HCC  and 0.38 (95% CI: 0.28–0.50), respectively, in patients receiving cumulative defined daily doses (CDDDs) less than or greater than 365 .Next, we further explored the effect of coadministration of aspirin on the effect of statins on the prevention of hepatocellular carcinoma. Concomitant use of aspirin did not significantly improve the chemopreventive effect of statins . In addition, aspirin alone has no chemopreventive effect on the occurrence of liver cancer .In addition, we discussed the incidence of liver cancer in the population within 10 years of follow-up and those with more than 10 years of follow-up, respectively. Interestingly, we found a lower incidence of liver cancer in the population over 10 years .I2 = 94%).Sensitivity analyses were conducted based on study design and quality to identify the source of the heterogeneity in observational studies . The cheI2 = 44%), while Western research has significant heterogeneity (I2 = 92%). Therefore, this explains to a certain extent that the overall heterogeneity of the article comes from high-quality Western observational research  reductase inhibitors that have long been used for the treatment of dyslipidemia and cardiovascular diseases. In recent years, there have been an increasing number of experimental studies on statins as potential anticancer drugs. A number of studies have shown that statins have immunomodulatory properties, antiproliferative properties that regulate cell cycle regulatory proteins, proapoptotic properties, and antiinvasive properties , 55–57. β, tumor necrosis factor, reactive oxygen species (ROS), and other molecules that mediate inflammatory damage, thereby further inducing hepatocyte apoptosis [α and IL-6 and significantly reduce ROS production and metalloproteinase activity, thereby reducing liver inflammation [In addition, chronic liver damage is also an essential factor affecting the progression of HCC . Liver ipoptosis . Howeverammation , 63. Relammation .The activation of MYC is very important in the tumorigenesis of liver cancer, and its inactivation can lead to the continuous regression of HCC , 66. In Statins can induce cell apoptosis, but the underlying pathway has not been fully confirmed. Statins may block the cell cycle in the G0/G1 phase by interfering with the Ras/Raf/MEK/ERK signaling pathways or regulating cyclin-dependent kinases and their inhibitors . It is wIn addition, Raf-MEK-ERK and PI3K-AKT-mTOR pathways are necessary for cancer cell survival. Statin-induced isoprene can not only reduce the activation of these two channels but also inhibit HCV replication . TherefoThe chemopreventive connection between statins and liver cancer is obvious among Asians but not among Western populations, based on the data from high-quality observational studies. We can speculate that the difference may be related to the different etiologies of liver cancer in the two regions.Eighty percent of HCC cases occur in East Asia and sub-Saharan Africa . We knowHBV participates in the occurrence and development of HCC through direct and indirect mechanisms, and the HBV protein HBx plays a major role. Compared with liver cancer caused by other risk factors, HBV-related liver cancer has a higher chromosomal mutation rate, p53 gene mutation rate, and inactivation mutation rate; fetal liver/liver progenitor cell genes are often overexpressed, and the Wnt/b-catenin pathway is often activated, but the mutation rate of activated b-catenin is low . SimilarIn Asian populations, statins mainly work by antagonizing the carcinogenic effects of HBV, while in Western populations, the mechanism is unclear. The results of our analysis also confirmed the difference in this benefit between Eastern and Western populations and the possible source of the difference. When we analyzed the subgroups of HBV-related liver cancer and HCV-related liver cancer, the results showed that statins had protective effects against both types of liver cancer, especially HBV-related liver cancer . IncreasFurther analysis showed that the chemopreventive effect of statins is more significant in patients with chronic liver disease than in the general population. Patients with chronic liver disease are at high risk of HCC. The chemopreventive effect of statins is more likely to yield positive results in people with chronic liver disease.After further analysis of the dosage of statins, it was found that higher cumulative doses had greater chemopreventive effects than lower doses of statins .P values were all less than 0.001. Simon et al. [In a cohort study of HCV-positive patients, Tsan et al. confirmed the chemopreventive effect of statins against liver cancer . Comparen et al.  used a sn et al.  also shon et al. . The AHRn et al. .Our research showed that lipophilic statins had a chemopreventive effect against liver cancer, while hydrophilic statins did not . As has Our study showed that coadministration of aspirin did not significantly improve the preventive effect of statins on liver cancer , and aspirin alone had no preventive effect on liver cancer . This conclusion contradicts previous studies. Studies by Luca et al. have shown that low-dose aspirin has a preventive effect on a variety of malignancies, including hepatocellular carcinoma . The reaInterestingly, we analyzed the relationship between follow-up time and HCC incidence, and the results were different from our assumptions. According to the general assumption, with the increase of follow-up time, the incidence of liver cancer will also increase. However, our analysis showed that the incidence of liver cancer was lower in the population with a follow-up period of more than 10 years than in the population with a follow-up period of fewer than 10 years . Such results demonstrate the effect of statins in preventing liver cancer from another aspect because with the increase of follow-up time, the time of taking statins will theoretically increase, thus improving its prevention effect of liver cancer, and the incidence of liver cancer will decrease accordingly.Alcohol is a recognized carcinogen and is closely related to the occurrence of liver cancer . TherefoThis analysis mainly included observational studies. The number of people included in observational studies accounted for 96.99% of the total number of patients, and a significant chemopreventive effect was observed in observational studies . RCTs accounted for a relatively small proportion of the included studies, and no obvious chemopreventive effect was observed .We should consider the results of the RCTs included in the context. Most of the RCTs involved Western population, and the different etiologies of liver cancer may have weakened the chemopreventive effect of statins. Moreover, long-term RCTs with large populations are difficult to conduct, and there was a risk of loss to follow-up. This should also be considered.In addition, the results of this study are significantly different from those of Yi et al. . ResearcThis study included more than 4.9 million participants, which is the largest study we have known so far. Compared with the previous meta-analysis, we focused on the relationship between the use of statins and the risk of liver cancer. Due to the inclusion of a large number of studies, the stability and persuasiveness of the results have increased. In addition, this study conducted a comprehensive discussion on potential confounding factors and demonstrated in detail the study design, study area, research quality, virus type, population characteristics, and cumulative defined daily dose. These arguments explain to a certain extent that the overall heterogeneity of the article comes from high-quality Western observational research and provide an improved direction for subsequent research design.In addition to the limitations formerly mentioned, other limitations deserve further consideration. First, the incidence of cardiovascular events in people with chronic liver disease is correspondingly higher , so theyOverall, this meta-analysis showed that the chemopreventive effect of statins on liver cancer is more pronounced in Asian populations than in other populations. Due to the differences in the etiology of liver cancer between Eastern and Western populations, we should carefully judge the benefits of statins in Western populations. At present, the relevant large-scale RCTs have mainly been performed in Western countries. In the future, it is necessary to carry out RCTs in areas with high burdens of liver cancer (such as East Asia). The chemopreventive effects of statins require their long-term use, and the burden imposed by the cumulative cost may have a certain passive influence on their clinical use. However, in areas with high burdens of liver cancer, the preventive use of statins should be considered.


Background: Fluoropyrimidine is widely used owing to its clinical efficacy, however, patients with dihydropyrimidine dehydrogenase (DPD) deficiency can experience fluoropyrimidine-associated toxicity. The dihydropyrimidine dehydrogenase (DPYD) gene encodes DPD, and studies suggest that DPYD polymorphisms can result in DPD deficiency. Since there is not a complete consistency of how much the risk of complication is elevated, we aimed to conduct a systematic literature review and a meta-analysis to provide the risk of fluoropyrimidine-associated toxicity in patients with DPYD rs1801160 polymorphism. Methods: We searched for qualifying studies published before October 2021 from PubMed, Web of Science, and EMBASE based on Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to evaluate the strength of the association between rs1801160 polymorphism and toxicities. A sensitivity analysis using the leave-one-out method was performed on the overall toxicity. Results: The pooled OR for overall toxicity in the patients with A allele was elevated 1.73 times higher than those with the GG genotype (95% CI 1.44–2.07). Sensitivity analysis yielded similar results, showing the robustness of the result. Subjects with variants showed a 2.37-fold increased hematological toxicity (95% CI 1.48–3.81); especially a 1.87-fold increased neutropenia compared to patients with wildtype (95% CI 1.49–2.34). Patients with A allele revealed 1.22 times higher gastrointestinal toxicity compared to those with GG genotype (95% CI 0.93–1.61), and among gastrointestinal toxicity, the risk of diarrhea was elevated 1.43 times higher in those with variants than patients with wildtype (95% CI 1.12–1.83). Conclusions: rs1801160 polymorphism is associated with elevated fluoropyrimidine-associated toxicity. Therefore, rs1801160 can be a potential candidate for DPD deficiency screening prior to fluoropyrimidine-based regimen. For several decades, cancer has remained one of the most devastating diseases inflicted on mankind, undoubtedly being one of the leading causes of death. It is expected that by 2040, the number of cancer-related deaths would exceed a stunning 16 million cases , the exiDPYD) encodes the dihydropyrimidine dehydrogenase (DPD) enzyme, which plays a vital role in the metabolic catabolism of fluoropyrimidine [DPYD gene may cause DPD deficiency [DPYD gene are known to affect DPD activity, specific variants associated with fluoropyrimidine-induced toxicity are still being investigated.The dihydropyrimidine dehydrogenase gene , is known to be associated with severe toxicity accompanying fluoropyrimidine-based regimens. Previous researches have shown the association between rs1801160 polymorphism and the aforementioned drug-induced complications; however, the results across studies failed to show consistency. Therefore, the present study aimed to investigate the possible association between rs1801160 polymorphism and fluoropyrimidine-induced toxicity through a systematic literature review and meta-analysis. rs1801160 of Two investigators independently performed a systematic search for all studies published before 18 October 2021 using PubMed, Web of Science, and EMBASE. The following search terms were included: (DPYD OR DPD OR (dihydropyrimidine dehydrogenase)) AND  AND (toxicity OR adverse OR side-effects OR (adverse effects)). Once duplicates were removed, initial screening through the titles and abstracts were performed to pinpoint potential studies for analysis. Subsequently, a full-text review was carried out to finalize studies to include in this study according to the eligibility criteria.Studies were included if they (1) were randomized controlled trials (RCT) or cohort studies; (2) included patients receiving fluoropyrimidine-based regimens; (3) evaluated the association of rs1801160 SNP with toxicity; (4) included applicable data on genotype in both cases and controls; or (5) published in English. Studies were excluded if they were (1) reviews, comments, letters, updates, news, editorials, conference or meeting abstracts, case reports, or case series; (2) in vitro or animal studies; (3) unable to extract genotype; or (5) unable to provide appropriate data.The following information was collected from each study: the last name of the first author, year of publication, the number of patients, country, the mean of participants’ age, percentage of female subjects, cancer type, treatment regimen, the definition of toxicity, and genotype.The methodologic quality of the selected studies was evaluated using the Newcastle–Ottawa Scale (NOS). The NOS contains three components , and each study can attain a maximum score of 9.2 was used for heterogeneity evaluation among studies. When heterogeneity was high (I2 > 50%), the random-effects model was applied. If I2 was less than or equal to 50%, the fixed-effects model was applied. The odds ratio (OR) with 95% confidence intervals (CIs) was calculated to evaluate the association between toxicity and polymorphism. Ip-value less than 0.05 was considered statistically significant. Statistical analyses and publication bias were performed using Review Manager (RevMan) version 5.4  and RStudio software . This meta-analysis was written based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.To identify publication bias, both Egger’s and Begg’s regression tests of the funnel plot were generated. A n = 507), Web of Science (n = 625), and EMBASE (n = 172). After excluding duplicates (n = 473) and irrelevant studies (n = 768), 63 records were selected for full-text review, and 57 records were excluded due to the following reasons: not related to toxicity outcome (n = 17), not related to polymorphisms of rs18001160 (n = 24), pharmacokinetic studies (n = 6), case reports (n = 3), not involving fluoropyrimidine-based regimens (n = 4), or unable to extract data (n = 3). Ultimately, six studies were included in this meta-analysis [A flow diagram of the literature search and selection process is depicted in es n = 76, 63 recoanalysis ,11,12,13The baseline characteristics of the included studies are displayed in DPYD gene and the risk for overall fluoropyrimidine-associated toxicity. Overall meta-analysis with six studies indicated that the rs18001160 polymorphism was associated with increased toxicity . Since there was no heterogeneity between the included studies (I2 = 30%), the fixed-effects model was used to calculate the effect size. The funnel plot was symmetrical . The sensitivity analysis showed similar results, suggesting that no one study predominantly affected the pooled results . Neither Egger’s test nor Begg’s test showed significant publication bias . The sensitivity analysis yielded similar results, indicating that the results were robust. Specific types of toxicity, including gastrointestinal toxicity, hematological toxicity, neutropenia, and diarrhea, showed the same trend with ORs of 1.22, 2.37, 1.87, and 1.43, respectively.The main finding of this study is that subjects with the A allele of rs1801160 showed an approximately 1.72-times elevated risk of fluoropyrimidine-associated overall toxicity compared to those with the GG genotype  through hepatic metabolism ; this istoxicity . The prevalence of partial and complete DPD deficiency range from 3 to 15% and 0.1 to 0.5%, respectively . CurrentDPYD gene which can affect DPD activity [DPYD and fluoropyrimidine-associated toxicity. However, the risk of overall complications varied from 1.16-fold to 3.64-fold among studies. Li et al. conducted a meta-analysis in 2014 on several SNPs, including rs1801160; however, only 2 studies with 628 subjects were included for the analysis. Our meta-analysis updated those results, using 6 studies including more recent data from a total of 5331 subjects. rs1801160 is a missense SNP in the coding region of the activity . The minactivity ,24. ManyThis study showed that patients with the A allele had an approximately 2.4- and 1.9-fold increased risk of hematological toxicity and neutropenia, respectively, compared to those with the GG genotype. Hematological toxicity is one of the reasons for chemotherapy discontinuation , while nThis meta-analysis bears several limitations. First, all studies included in this meta-analysis were conducted in the European population. As the allele frequencies of the variants in Europeans (5%) are different from the variants in Asians (2%), further investigation should be assessed to generalize the present results to other ethnic groups. Second, due to limited data availability, critical toxicities including cardiotoxicity could not be analyzed. Third, some confounding factors that could affect the risk of toxicities could not be adjusted for. In general, cancer patients generally take more than one class of medication; hence, co-medications may have affected the study results. Furthermore, while tests did not show evidence for publication bias, the possibility cannot be ruled out due to the low sample size. Nevertheless, this study provided a candidate SNP for screening to prevent fluoropyrimidine-induced complications through an improved estimate of the effect size of the association. As a systematic review and meta-analysis on the rs1801160 polymorphism and fluoropyrimidine-associated toxicity, this analysis may provide clinical evidence for future considerations in the treatment and management of fluoropyrimidine-based regimens.


This meta-analysis was aimed to estimate the diagnostic performance of volatile organic compounds (VOCs) as a potential novel tool to screen for the neoplasm of the digestive system. An integrated literature search was performed by two independent investigators to identify all relevant studies investigating VOCs in diagnosing neoplasm of the digestive system from inception to 7th December 2020. STATA and Revman software were used for data analysis. The methodological quality of each study was assessed using the Quality Assessment of Diagnostic Accuracy Studies tool. A bivariate mixed model was used and meta-regression and subgroup analysis were performed to identify possible sources of heterogeneity. A total of 36 studies comprised of 1712 cases of neoplasm and 3215 controls were included in our meta-analysis. Bivariate analysis showed a pooled sensitivity of 0.87  0.83–0.90), specificity of 0.86 (95% CI 0.82–0.89), a positive likelihood ratio of 6.18 (95% CI 4.68–8.17), and a negative likelihood ratio of 0.15 (95% CI 0.12–0.20). The diagnostic odds ratio and the area under the summary ROC curve for diagnosing neoplasm of the digestive system were 40.61 (95% CI 24.77–66.57) and 0.93 (95% CI 0.90–0.95), respectively. Our analyses revealed that VOCs analysis could be considered as a potential novel tool to screen for malignant diseases of the digestive system. Thus, early detection and timely treatment are integral for a favorable prognosis and long-term survival. At present, tumor markers have been widely used for tumor screening and diagnosis, monitoring treatment response, and surveillance of tumor recurrence after treatment. In clinical practice, markers commonly used to screen and diagnose tumors of the digestive system are mostly derived from blood, including carbohydrate antigen , alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), pepsinogen, etc. However, the sensitivity and specificity of a single tumor marker are suboptimal, and it is often necessary to combine several tumor markers for the screening and early diagnosis of tumors5. Of course, there are also circulating tumor DNA and methylated genes from blood, urine or feces that are still being evaluated. Although these have shown good performance in the diagnosis of tumors of the digestive system, they have not been widely used in clinical practice due to immaturity in technology and high cost. Nevertheless, endoscopic examination with biopsy remains the gold standard for the diagnosis of gastric, esophageal, and colorectal cancers, while endoscopic ultrasonography and other imaging examinations can be used for the diagnosis of liver and pancreatic cancer. However, endoscopic screening and diagnostic approaches have limitations, including patient discomfort and potential major complications and harms. Therefore, an effective noninvasive screening tool for malignancies of the digestive system malignancies is desired.The incidence of the digestive system neoplasm is rising yearly worldwide. These tumors including colorectal cancer, stomach cancer, liver cancer, etc. have been associated high risk of morbidity and mortality based on the Global Cancer Statistics 20186. Contrary to the conventional biomarker studies subjected to the health status of the individuals, the analysis of VOCs reflects the fingerprint characteristics of individuals that represent an instance of individualized medicine7. Moreover, a variety of sensor technologies are now being applied in analyzing the patterns of VOCs, such as eNoses and nanomaterials. As technology advances and clinical research progresses, VOCs analysis does not only enable early detection of cancer, but also the monitoring of the response to cancer therapy and detection of disease recurrence early when secondary treatments are most effective8.Breath analysis is recognized as a simple and non-invasive method for screening and monitoring pathology or disease. Nevertheless, studies have shown that volatile organic compounds (VOCs) from urine also demonstrated good performance in the screening of cancer, in particular, special phenotypes of VOCs in urine could be used for the screening of prostate and gastrointestinal cancers11. Therefore, it is vital to explore the differences in VOCs released by varying cancer types to identify specific representative VOCs to each tumor that can be used as a diagnostic tool. For instance, the study by Kumar et al.12 has shown significantly lower concentrations of several specific volatile compounds in non-tumor individuals than those with tumors.VOCs are organic compounds with relatively low molecular weight and high vapor pressure. Cancer-originated VOCs have been frequently detected in feces, urine, blood, skin, sweat, and gases exhaled from cancer patients. These have been produced by tumor cells, which can reflect the disease14. These specific VOCs have been considered to be the results of an imbalance between the systemic manifestations of oxidative stress, metabolic abnormalities, or reactive oxygen and the ability of the biological system to detoxify or repair damages17. In particular, the gut microbiome plays a key role in diseases of the digestive system, including colorectal cancer and inflammatory bowel disease. Changes in the composition of gut bacterial flora affect the fermentation products and the forms of volatile organic compounds (VOCs). Given that different diseases are associated with specific metabolomics that reflects cell metabolism, providing a new way of thinking for the diagnosis of diseases. In fact, in inflammatory bowel disease, changes in the gut microbiome are associated with colonic inflammation and can influence VOCs production. Also, some bacterial communities have been implicated in the development of colorectal cancer21. At the cellular level, changes in the production of VOCs have been associated with oxidation of polyunsaturated acids in the cell membrane as a result of genetic and/or protein mutations within tumor cells and the increased relative incidence of reactive oxygen species within cancer cells23. These cancer-related volatile organic compounds are released from the affected tissues into the feces or bloodstream and eventually excreted either through breath or through urine or feces14. Therefore, VOCs, as a comparatively new and non-invasive biomarker, provides a promising and attractive option for the screening of tumors in the digestive system.VOCs reflect changes in pathology and metabolic processes26. However, these studies have some shortcomings, with some only analyzing VOCs in exhaled gas, while others had too a small sample size. In our meta-analysis, VOCs from all sources and tumors of the digestive tract were summarized and analyzed. This would ascertain the diagnostic efficacy of VOCs for tumors of the digestive system.Several studies have shown that VOCs analysis is valuable in the diagnosis of tumors of the digestive systemth December 2020. Search terms were as follows: volatile organic compounds or VOCs and esophageal neoplasm or esophageal cancer or gastric cancer or gastric neoplasm or liver cancer or liver neoplasm or hepatic neoplasm or pancreas cancer or pancreas neoplasm or colorectal cancer or colorectal neoplasm. All relevant literature was also screened for other possible studies.A comprehensive, systematic electronic literature search of PubMed and Web of Science was conducted to identify all relevant papers from inception to 7All articles were reviewed by two independent investigators according to the inclusion and exclusion criteria. The inclusion criteria were: (1) VOCs were used to detect patients with neoplasm of the digestive system; (2) the control group consisted of patients with benign diseases of the digestive system and/or healthy individuals; (3) patients did not undergo any therapy; (4) extracted data could be used to measure true-positive (tp), false-positive (fp), false-negative (fn), and true-negative (tn) values. Studies/articles were excluded for the following criteria: (1) studies not within the field of our research; (2) non-human or animal study; (3) non-English literature; (4) non-diagnostic study; (5) studies on the neoplasm of not the digestive system; (6) data from the study were insufficient to constitute 2 × 2 tables; (7) review articles, editorials, case reports, conference proceedings, or letters.For each study included in our meta-analysis, the following data were extracted: first author, country, year of publication, number of participants, mean age of participants, percentage of males, cancer type, the source of VOCs, analytical platforms, and the control group. Also, each investigator registered and counted the numbers of tp, fp, tn, and fn. , false positive (fp), true negative (tn) and false negative (fn) according to the degree of matching between the results obtained by pathology and VOCs analysis. Sensitivity was calculated as tp/(tp + fn), while specificity was calculated as tn/(tn + fp). Correspondingly, four basic data could be extracted from the known sensitivity and specificity. Any disagreements between the investigators were assessed by a third reviewer and resolved by mutual agreement.27 was used, which was an evidence-based quality assessment tool for systematic reviews of diagnostic accuracy studies that included four domains: patient selection, index test, reference standard, and flow and timing.To assess the quality of the included studies, the Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2) toolP-value of the Cochran-Q test and I-square statistic28. Studies were considered homogenous if P > 0.05 and I2 ≤ 50%, and we performed only pooled sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic odds ratio (DOR). If P < 0.05 or I2 ≥ 50%, indicating statistically significant heterogeneity between the studies, further meta-regression and subgroup analysis were carried out to determine the possible sources of heterogeneity.Heterogeneities between the studies were evaluated by measuring the P-value of < 0.05 was considered statistically significant.Sensitivity, specificity, accuracy, positive and negative predictive values, PLR, NLR, and DOR of VOCs in the diagnosis of digestive system neoplasm were obtained from the individual study, and forest plots were used to calculate and graphically display pooling of the data. The SROC curve was displayed to obtain the optimal diagnostic efficiency for VOCs, and the area under the summary ROC curve (AUC) was calculated. The publication bias was presented by using the Deeks’ funnel plot asymmetry test. Meta-analysis was performed using STATA15.0  with the MIDAS module, while RevMan 5.4  was used to evaluate the quality of the included studies. A The initial search of PubMed and Web of Science databases yielded 589 articles. Of these, 168 were excluded due to repetitive publications, and 350 articles were excluded based on the inclusion/exclusion criteria after reading the titles and abstracts: 263 did not associate VOCs as tools for the diagnosis of neoplasm of the digestive system; 46 were review articles; 28 were conference abstracts; 2 were letters; 10 were not based on human studies; 1 was non-English literature. A total of 35 articles were excluded after reading the full text: 14 were not of diagnostic research; 5 were research of non-digestive tract neoplasm; 16 articles contained insufficient data to form 2 × 2 tables. Finally, a total of 36 studies were included in our meta-analysis  in exhaled breaths, 8 (from 7 studies) in urine, 1 in blood, 2 (from 1 study) in saliva, 1 in bowel gas, and 1 in bile.The 36There were 2 datasets that used the electronic nose as the analytical platform, 16 datasets (from 15 studies) used gas chromatography coupled with mass spectrometry (GC–MS), 5 datasets used field asymmetric ion mobility spectrometer (FAIMS), 8 datasets (from 7 studies) used selected ion flow tube mass spectrometer (SIFT-MS), and 8 studies used other analytical platforms: 2 datasets used silicon nanowire field-effect transistor (SINW-FET), 1 dataset used proton transfer reaction mass spectrometer (PTR-MS), 1 dataset used ion–molecule reaction mass spectrometry (IMR-MS), 1 dataset used single-photon ionization mass spectrometry (SPI-MS), 1 dataset used field asymmetric ion mobility spectrometer–mass spectrometry (FAIMS–MS) and 2 datasets used sensor  and 0.86 (95% CI 0.82–0.89), respectively Fig.  and AUC 2 = 71.04%, P = 0.00), as well as the pooled results of sensitivity  , indicating great discriminatory test capability. Furthermore, the likelihood ratio and post-test probability indicated the risk of tumors of the digestive system when tested positive or negative. Our analysis revealed a PLR of 6, indicating that patients with digestive system tumors were six times more likely to test positive than healthy individuals. Coupled with the NLR of 0.15, VOCs analysis represented a promising method to diagnose the neoplasm of the digestive system.To identify potential sources of heterogeneity, univariate meta-regression and meta-analysis were performed, which revealed that factors including race, sample size, source of VOCs, analytical platforms, and types of the control group might be sources of heterogeneity in our meta-analysis. Further analysis demonstrated higher specificity for VOCs in the screening of tumors in the digestive system tumors of the Asians than the others (Europeans and Americans), suggesting that the performance of VOCs in cancer screening may vary according to race or ethnicity. Also, we found that analysis of VOCs from the exhaled breath performed better than other sources of VOCs. However, the sample size of studies investigating other sources of VOCs was smaller compared with those of VOCs from exhaled breath. For the analytical platform, the use of MS (mass spectrometry) to analyze VOCs had the highest sensitivity and specificity compared with other platforms. However, the sample size of studies utilizing MS was also much larger than that of other analytical platforms. In our study, the performance of VOCs as a screening tool for different tumors was not analyzed, given that our objective was to explore VOCs analysis as a rapid and non-invasive screening tool for tumors of the digestive system, with its applicability as a primary population screening rather than as a diagnostic tool for individual tumor types.There were several reasons why this meta-analysis did not conduct threshold effect analysis: (1) different studies used different instruments for analyzing organic matter; (2) different instruments detected different types of organic matter; (3) different studies had different ways of setting the threshold value of organic compounds. Therefore, it was difficult to analyze the diagnostic ability of specific organic compounds for tumors. However, this did not affect the results that VOCs have good diagnostic ability in the digestive system.66. Besides, the combination of CA199 with CA242 has demonstrated a sensitivity of 0.90 and a specificity of 0.7667. From our analyses, the sensitivity (0.90) and specificity (0.91) of VOCs for gastric cancer, and the sensitivity (0.84) and specificity (0.82) of VOCs for colorectal cancer exhibited superior performance to conventional biomarkers in screening for neoplasm.Compared with other tumor markers currently applied in clinical practice, VOCs have demonstrated superior diagnostic ability in screening for gastric cancer and colorectal cancer. Serum pepsinogen is a widely used biomarker with good diagnostic efficacy for gastric cancer screening, with a meta-analysis demonstrating a sensitivity of 0.69 and a specificity of 0.73, while the sensitivity and specificity could be improved further to 0.70 and 0.79 respectively when applying the ratio of PG I to PG II concentrationsThere were limitations to our study. The included studies were mostly of case–control and cross-sectional nature, with few prospective longitudinal studies to associate VOCs with disease severity. Second, only articles in the English language had been selected, which invariably resulted in selection bias. Third, the cancer types, and analytical platforms of tumor-associated VOCs also varied between the studies. Therefore, we did not analyze the performance of varying VOCs in screening for different tumors of the digestive system.Therefore, further studies are warranted to not only innovate and combine advanced technologies in the sampling, detection, and analysis of VOCs but also to standardize these methods. Moreover, it is vital to explore the differences of VOCs between varying types and stages of tumors. Clinical trials would then be required for the validation of results before widespread clinical application.The pooled results of our meta-analysis have confirmed the difference in the VOCs analysis between patients with tumors of the digestive system and healthy individuals, which sheds light on VOCs analysis as a promising novel screening tool for early detection of tumors.Supplementary Information 1.Supplementary Figure S1.Supplementary Figure S2.Supplementary Figure S3.Supplementary Figure S4.