Some developmental anomalies, such as NTDs, are to a large extent prevented by supplementation of folic acid before conception, but supplementation does not seem to prevent cardiovascular disease or cognitive decline. These diverging results can be elucidated by introduction of the PrOO concept, as MTHFR polymorphisms and inherent low folate levels induce both non-optimal maturation of the oocyte and unsuccessful DNA methylation and demethylation, i.e. epigenetic mutations. The PrOO concept is testable and predicts in a random population the following: (1) female carriers of specific genetic MTHFR variants exhibit more ovulatory disturbances and inherent subfecundity traits, (2) descendents from a carrier mother, when compared with those from a wild-type mother, are more frequently conceived in PrOO high-risk conditions and, thus, (3) disadvantaged in life expectancy. If so, some MTHFR polymorphisms represent a novel, genetically determined, PrOO high-risk conception category comparable to those which are environmentally and behaviorly influenced. These high-risk conditions may cause developmental anomalies and defective epigenetic reprogramming in progeny. The interaction between genetic and environmental factors is a plausible mechanism of multifactorial inheritance.Several conditions apparent at birth, e.g., neural tube defects (NTDs) and cardiac anomalies, are associated with polymorphisms in folate-related genes, such as the 677C → T polymorphism of the methylenetetrahydrofolate reductase (MTHFR) gene. Similar associations have been established for several constitutional chronic diseases in adulthood, such as schizophrenia, cardiovascular diseases, dementia, and even neoplasias in different organ systems. This spectrum of developmental anomalies and constitutional diseases may be linked to high-risk conceptions related to  Most theories related to the origin of adult diseases focus on genetic causes and direct environmental effects preceding disease onset by several years at most. The view that diseases in adulthood can partly be explained by conditions earlier in life or even before birth is gaining scientific support. Previously, we proposed non-optimal oocyte ripening or impaired oocyte maturation can be an important cause of developmental anomaly and disease later in adult life -4. The bab ovo for some congenital anomalies and constitutional diseases. Indeed, a genetic PrOO determinant emerges as an explanation for the diverging preventive effects of folate in NTDs versus adult diseases. We also discuss the relationship between low folate levels and unsuccessful DNA methylation patterns in the context of epigenetic mutations. Furthermore, testing strategies are proposed to establish the causality of the relation between MTHFR polymorphisms and PrOO conceptions in random populations.In this paper, we review current knowledge on MTHFR polymorphisms and folate levels as they relate to developmental anomalies at birth and selected constitutional disease in adulthood. We also recapitulate the ovopathy concept and posit that the 677C → T variant and inherent low folate levels are accompanied by low estrogenisation, and that this condition induces preovulatory overripeness ovopathy (PrOO). This leads to a high-risk conception mediated by genetic factors, analogous to the environmentally and behaviorally conditioned high-risk conceptions, and an origin Folate is an important B vitamin that plays a pivotal role in remethylation of homocysteine to methionine, which is essential for DNA-synthesis, DNA-repair, and DNA-imprinting processes . ReductiIt has become evident that the 677C → T variant as well as low-folate intake by the mother contribute to increased risks of NTDs and cardiac anomalies. The underlying pathogenic mechanism which causes this detrimental effect is not fully understood ,10. HoweAn additional enigma is the marginal association between MTHFR polymorphisms and several neoplasias with diverging incidences according to age ,28. A meth and 20th century as well as observations in human reproduction [optimally ripened oocytes (OptRO) are apparent during optimal conditions for reproduction. This coincides with optimal maternal age, adequate nutritional state, and post-pregnancy restoration of a regulatory ovulatory pattern, and with the seasonally-bound ovulation peaks, guaranteeing optimal embryo quality and favourable downstream effects on subsequent events, i.e., on life expectancy [This concept was initially derived from animal research in the 19oduction . Meioticpectancy . In factpectancy .preovulatory overripeness ovopathy (PrOO), and leads to fertilization of non-optimally matured oocytes [In contrast, abnormal estrogen concentrations cause deficient oocyte maturation which is the basis of  oocytes -4. The i oocytes -3.dose-response fallacy in reproductive studies described by Selevan and Lemasters [Excessive dysfunctional oocyte maturation results in poor quality oocytes and, after surpassing a certain threshold, in disproportionately increasing numbers of pathological conceptuses. Excess vanishing of pathological outcome will turn into excess preterm loss, and eventually in deficits of pathological births. This is called the emasters . In our The spectrum of developmental anomalies associated with MTHFR polymorphisms and/or folate deficiency appears analogous to the spectrum of anomalies related to PrOO induced by endocrine disturbances during the transitional stages of reproductive life and/or by divergent reproductive behavior, e.g. unusual maternal age or pregnancy interval  see Fig. Low foldose-response fallacy being inherent to the PrOO concept. Other findings are also in accordance with disproportionate levels of oocyte deterioration and a dose-response fallacy: a dose-specific reduced risk of progeny with cleft lip with or without cleft palate(CL/P) from mothers carrying either one or two copies of the 677C → T variant , a negative association for children with CL/P (RRs: 1.05 and 0.74) versus a positive one for cleft palate only , and a fourfold increased risk of orofacial clefts in mothers using folic acid [This genetically conditioned high-risk for PrOO explains several unexplained phenomena related to MTHFR variants as well as to low dietary folate. Both conditions are associated with increased fetal loss, intrauterine growth retardation, and heart defects in female mice , as welllic acid .The finding of MTHFR 677C → T mutations in the mother   is in geab ovo. In other words, preventive measures are effective only before conception but not afterward, and that threatened oocytes may be the culprit of specific constitutional diseases encountered long after birth.It should be noted that periconceptional multivitamin use including folic acid reduces the dose-response risk of preeclampsia and ovulatory infertility ,18. In Iab ovo for both developmental anomalies and constitutional disease is consistent with the environmental determinants related to non-optimally maturing oocytes [The conjecture of a common origin  oocytes -4. This  oocytes ,3,48,49  oocytes -54. AnalAs mentioned previously, adequate folate levels are a prerequisite not only for optimal oocyte maturation but also for remethylation of homocysteine to methionine – the key epigenetic contributor to gene activation and/or reprogramming . These eIncorrect DNA reprogamming results in epigenetic mutation, which does not follow the classic rules of Mendelian inheritance. Active transport of methionine happens in all animal and human oocytes and in early preimplantation embryos. DNA-methylation is concentrated at specific stages when developmental potency of cells changes, i.e., during the final phase of oocyte maturation. In contrast, DNA-demethylation occurs immediately after fertilization ,55,56. I12 and folate restricted diet during the periconceptional period, resulted in obesity, insulin resistance, hypertension, and altered immune responses when full grown [The combined effect of PrOO and epigenetic mutations evoked by MTHFR variants (and low folate levels) can explain many developmental puzzles in animals and humans. For example, epigenetic modifications in lambs derived from oocytes retrieved from ewes given a vitamin Bll grown . This wall grown . Additioll grown . Furtherll grown . A numbell grown , schizopll grown , and cerll grown ,65,66.MTHFR polymorphism status in females is hypothesized to entail a genetically determined propensity to PrOO, which may be likened to the high-risk conceptions elicited by environmental and behavioral conditions. The variant alleles operate either independently or in concert and may strengthen each other. Therefore, we predict that:1) Heterozygous and homozygous 677C → T carrier females suffer from subfecundity traits in a dose-response manner when compared to females with no mutation (CC). Apart from giving birth to affected progeny, they also experience more menstrual disorders, longer time to achieve pregnancy, longer interpregnancy intervals, and more pronounced reproductive seasonal variation.2) Offspring from homozygous (and to a lesser extent from heterozygous 677C → T carrier mothers), being genetically more susceptible to environmental triggers than mothers without this mutation, are more frequently conceived in high-risk conditions characterized by ovulatory disturbances. Typical PrOO characteristics are depicted in Figure 3) Descendents from homozygous (and to a lesser extent from heterozygous 677 → T carrier women) have reduced life expectancy compared to offspring from mothers without this polymorphism.dose-response fallacy will in particular occur at the extremes of maternal age, birth interval or at the seasonal transitions (Figure These effects may be obscured by early loss before birth as a consequence of preterm premature rupture of membranes and eclampsia associated with MTHFR polymorphisms ,38 or dus Figure  and 2c.conclusion, MTHFR polymorphisms and resulting low folate levels warrant consideration as factors inducing non-optimally matured oocytes before conception. They represent a novel, genetically determined, high-risk PrOO condition comparable to the endocrine disturbances elicited by environmental and behavioral conditions. Further study of the interaction between genetic and environmental factors may indentify mechanisms of multifactorial inheritance and explain many commonly associated enigmata in chronic constitutional diseases.In The authors declare that they have no competing interests.th of June 2007 at the Department of Epidemiology, Biostatistics, and Health Technology Assessment at the Radboud University Nijmegen, the Netherlands. The co-authors AV, MdH, and NR participated in the design of the study and the coordination for the manuscript preparation. They revised it critically for important intellectual content and interpretation of the arguments. All authors read and approved the final manuscript.The content of this manuscript was subject of the fare-well lecture of PHJ on 12


In contrast to the positive association found in three studies between maternal anaemia during pregnancy and childhood leukaemia, no such association was found in infant leukaemia . Maternal anaemia during pregnancy has been positively associated with childhood leukaemia in three studies (The human subject protection committees of each participating COG institution approved the study protocol. Eligibility criteria for cases included: (1) diagnosis with acute leukaemia in the first year of life between 1 January 1996 and 20 August 2002; (2) an English-speaking biological mother available for a telephone interview and (3) no Down syndrome. Controls were identified and contacted by random digit dialling (RDD) . Using RDD, a total of 430 households were identified with a child eligible as a control. Interviews were completed for 255 of the 430 eligible controls, an RDD response rate of 59%. Medical records were obtained for 236 case mothers and 215 control mothers.vs acute myeloid leukaemia (AML)) and the presence of MLL gene rearrangements (MLL+ vs MLL−). Potential confounders evaluated were maternal age at time of index child's birth, maternal race and education, household income, smoking during pregnancy, birth order of the child, birth weight and child gender.Statistical evaluation was performed using SAS version 9.1, with odds ratios (ORs) calculated using unconditional logistic regression, for all infant leukaemia cases and leukaemia subtypes defined by leukaemia type (acute lymphoblastic leukaemia (ALL) −1  classification, −1). Haemoglobin concentration was also analysed as both a categorical and continuous exposure.All mothers with an available haemoglobin value were included in the analysis. Anaemia was defined as a haemoglobin concentration of <11 g dl−1 that developed in the second or third trimester. First trimester anaemia is likely due to nutritional deficiency and results in poor foetal outcomes including low birth weight . Applying more stringent criteria for anaemia (haemoglobin <10 g dl−1), dividing the haemoglobin concentration into categories (mild vs severe anaemia), and evaluating haemoglobin concentration as a continuous variable, also did not affect the results (data not shown).Haemoglobin values were available for 229 cases and 207 controls; 48 case mothers were anaemic compared to 47 controls. We found no evidence that a maternal haemoglobin concentration of <11 g dlFurther evaluation was performed for the physiologic anaemia of pregnancy. A total of 178 case and 180 control mothers were included in this analysis. Of these cases, 115 were ALL (71 MLL+) and 63 were AML (21 MLL+).vs 1.7%, P=0.004). There was also a statistically significant difference in maternal age and birth order of the index child  and ALL (n=115), with ORs of 0.64 (95% CI 0.32–1.30) and 0.98 (95% CI 0.58–1.66), respectively. There was no association when cases were stratified by MLL status . There was also no association when the data were adjusted for maternal race and birth order. Other variables did not significantly alter the association and were not included in the final model.Overall, the unadjusted OR for physiologic anaemia of pregnancy and risk of infant leukaemia was 0.85  0.53–1.37) see . The ass−1 during pregnancy. Three previous studies found a positive association between maternal anaemia and childhood leukaemia. A case–control study of 153 cases of acute leukaemia diagnosed in children from birth to 14 years of age in Greece from 1993 to 1994 used maternal report of anaemia during pregnancy and found a positive association  , which was heavier than any other group in the analysis (We found no evidence for increased odds of infant leukaemia in the offspring of mothers with a haemoglobin concentration of <11 g dl39–4.86) . The morOur findings cannot confirm these studies as these covered all childhood leukaemias whereas our study concerned only infant leukaemia. It is possible the association seen previously does not exist for infant leukaemia. Also, in our North American patient population, prenatal care may differ from those in the earlier European studies. Our study subjects had essentially uniform use of prenatal vitamins, iron and folate supplementation.One weakness of our study is the lack of power. At 80% power, the study is only capable of detecting an OR of about 2.0 or greater. Subtler increases could be missed. A potential source of bias is the selective enrolment of eligible RDD identified controls (59% field response rate). There were significant differences between cases and controls with respect to maternal race, maternal age and birth order see . AlthougDespite the negative findings for this study, the plausible associations of maternal anaemia during pregnancy, high birth weight and childhood leukaemia remain an intriguing area for aetiology research. This study was inadequately powered to detect subtle associations and should be repeated with a larger sample size.


TNF-A) with gastric cancer risk. This meta-analysis reviews and summarises published evidence for these associations. Searching several databases yielded 24 independent studies that reported on the associations between TNF-A polymorphisms and gastric cancer risk. We analysed available data for the most commonly investigated polymorphisms: TNF-A –308G>A (23 studies), TNF-A –238G>A (9 studies), and TNF-A –857C>T (5 studies). Summary odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated in the random-effects model using the DerSimonian–Laird method. Q-statistic and I2-statistic were calculated to examine heterogeneity, and funnel plots were plotted to examine small study effects. The overall ORs (95% CIs) for AG and AA genotypes vs GG genotype for TNF-A –308 were 1.09 (0.94–1.27) and 1.49 (1.11–1.99), respectively. For TNF-A –238, the corresponding ORs (95% CIs) were 1.05 (0.84–1.33) and 1.25 (0.30–5.26), respectively. The overall ORs (95% CIs) for CT and TT genotypes (vs CC) for TNF-A –857 were 1.06 (0.89–1.27) and 1.57 (0.91–2.70), respectively. The statistically significant association between TNF-A –308GG and gastric cancer was limited to western populations. This association showed little heterogeneity (I2=0) and remained consistently strong when analyses were limited to anatomic and histologic subtypes of gastric cancer, or limited to studies in which genotype frequencies were in Hardy–Weinberg equilibrium, or limited to larger studies. These same subgroup analyses did not change results associated with other polymorphisms. In conclusion, TNF-A –308AA genotype was associated with a statistically significant increased risk of gastric cancer, whereas other studied polymorphisms were not. The association between TNF-A –857TT genotype and gastric cancer was near significant, and may become significant if more studies are published.Inflammation is one of the early phases in the development of gastric cancer. Therefore, several studies have examined the association of polymorphisms in tumour-necrosis factor-A gene ( Nature, which showed an increased risk of gastric cancer and its precursor lesions associated with proinflammatory polymorphisms in interleukin-1B (IL-1B) and IL-1RN genes  gene have been extensively studied in relation to gastric cancer. Three polymorphisms in TNF-A genes have been studied more than the other polymorphisms. TNF-A –308G>A is associated with an increased production of TNF-α  OR TNF) AND (‘Stomach Neoplasms’ [MeSH] OR (gastric cancer) OR (stomach cancer)) AND . The following terms were used in ISI Database search: (TS=(Interleukins) OR TS= OR TS=(Tumor Necrosis) OR TS=(TNF)) AND (TS=(Stomach Neoplasms) OR TS=(gastric cancer) OR TS=(stomach cancer)) AND  OR TS=(polymorphism) OR TS=(polymorphisms)). Other databases and search terms were MedCarib, LILACS, IMEMR, IndMed, and PAHO databases, searched for (gastric OR stomach) AND (cancer OR carcinoma OR neoplasms); IMSEAR database, searched for combinations of gastric or stomach with cancer or carcinoma or neoplasms; and J-EAST database, searched for combinations of gastric or stomach with cancer or carcinoma or neoplasms plus polymorphism. In addition, references of cited articles were reviewed.We conducted a comprehensive search by examining several databases for all papers that had been published on the association between Two of the authors reviewed results of each of the database searches to make sure that published papers are not missed. In addition, where overall data were missing, we contacted the authors for further information.TNF-A polymorphisms in relation to gastric cancer was found in a total of 29 articles  and TNF-A −238 (rs361525), numbers and percentages of GG, GA, and AA genotypes, and for TNF-A −857 (rs1799724), numbers and percentages of CC, CT, and TT genotypes were extracted by case status. For GG and GA vs GG genotypes (TNF-A −308 and −238) and for TT and TC vs CC genotypes (TNF-A −857), odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated. We performed similar calculations for AA vs a combination of GA and GG genotypes (TNF-A −308 and −238), and for TT vs a combination of TC and CC genotypes (TNF-A −857) to examine the applicability of recessive models. Likewise, we did similar analyses for a combination of AA and GA vs GG genotypes (TNF-A −308 and −238) and for combination of TT and TC vs CC genotypes (TNF-A −857) to examine whether dominant models apply. We used both random-effects models (DerSimonian–Laird method) and fixed-effects models (Mantel–Haenszel method) to calculate overall summary ORs and 95% CIs. Because these two methods yielded similar results, we chose only random-effects models  vs East Asian , and subgroup analyses were performed for each group. In all, 12 studies were from western and 12 studies were from East Asian countries.Some of the published studies found associations only with certain anatomical subsites  or histological subtypes  of gastric cancer. Therefore, we calculated summary ORs and 95% CIs for noncardia cancer, where genotype data were presented by anatomical location, and for intestinal-type cancer, where data on histology were available. We examined the association between α=0.05.We examined the effect of Hardy–Weinberg equilibrium (HWE) on the results of our meta-analysis by calculating summary ORs and 95% CIs for studies in which these alleles were in HWE among controls. The strategy was to exclude studies in which genotypes violated HWE at P for rank correlation and Egger's weighted regression method  using only larger studies.We plotted Begg's funnel plot to examine small study effects (Q-statistic for homogeneity (using Mantel–Haenszel weights) and the I2-statistic  and homozygous (AA vs GG) proinflammatory genotypes were 1.09 (0.94–1.27) and 1.49 (1.11–1.99), respectively, and the corresponding fixed-effect ORs (95% CIs) were 1.14 (1.02–1.27) and 1.51 (1.14–1.99), respectively. Recessive model was the best-fitting inheritance model for TNF-A −308. However, because GG genotype was much more frequent than GA and AA genotypes, comparing GA+GG vs AA genotype did not have a material effect on the test power. Therefore, the results are reported separately for GA vs GG and AA vs GG genotypes.Study-specific and summary ORs (95% CIs) are shown in vs diffuse histology types. Among these, summary ORs (95% CIs) for GA vs GG and AA vs GG genotypes were 1.17 (0.85–1.59) and 2.88 (1.74–4.77) for noncardia cancers and 0.98 (0.68–1.40) and 2.17 (1.17–4.03) for intestinal-type gastric cancers, respectively. When we limited our analysis to H. pylori-positive cases and controls, these ORs (95% CIs) were 0.66 (0.33–1.30) and 3.23 (0.10–99.4), respectively. The summary ORs (95% CIs) for GA vs GG and AA vs GG genotypes were 1.14 (0.95–1.37) and 1.74 (1.21–2.51) for western countries, and 1.02 (0.78–1.34) and 1.14 (0.70–1.84) for eastern countries, respectively.TNF-A −308 genotypes among controls were not in HWE (vs GG and AA vs GG genotypes were 1.08 (0.91–1.28) and 1.73 (1.22–2.44), respectively.In three studies, the distributions of t in HWE . LimitinTNF-A −308 and gastric cancer. This figure shows logarithm of OR (Y axis) vs its standard error (SE) (X axis). Smaller studies have larger SEs, therefore points showing these studies are in the right-hand side of the graph. For GA vs GG genotypes, there was evidence for bias using Egger's weighted regression method (P for bias=0.03) and using the method of Begg and Mazumdar (P=0.07). There was no evidence of bias for AA vs GG genotypes using either Egger's method (P for bias=0.44) or Begg's method (P=0.87). We excluded small studies, defined as those having an SE>0.5. After these exclusion, the summary OR (95% CI) changed to 1.13 (0.97–1.32) for GA vs GG, and 1.68 (1.08–2.61) for AA vs GG.Q- and I2-statistics for the overall and subgroup analyses. For the overall analysis, the Q-statistic was significant (P=0.02) and I2 (34%) showed a moderate variation for GA vs GG genotypes. For AA vs GG genotypes, the Q-statistic was not significant (P=0.74) and I2 was equal to zero. Subgroup analyses showed similar patterns as overall analyses. While for most subgroup analyses, the P-value for GA vs GG genotypes was significant or remained close to significance level and I2 showed moderate heterogeneity among studies, P for heterogeneity was not significant for AA vs GG genotypes  were 1.05 (0.84–1.33) for GA vs GG genotypes and 1.25 (0.30–5.26) for AA vs GG genotypes. The fixed-effect ORs (95% CIs) were 1.04 (0.83–1.31) and 0.87 (0.41–1.87), respectively. When we investigated the inheritance models, TNF-A −238 showed neither recessive nor dominant models (data not shown). Therefore, the results are reported separately as GA vs GG and AA vs GG genotypes.For noncardia cancers, the ORs (95% CIs) were 1.04 (0.71–1.52) for GA genotype and 1.38 (0.01–157.0) for AA genotype. Limiting the results to intestinal-type cancers, the ORs (95% CIs) were 1.40 (0.93–2.10) for GA genotype and 0.13 (0.01–2.20) for AA genotype. For this last comparison, one of the three studies was excluded because of the null values for AA genotype frequency among both cases and controls. For studies from western countries, summary ORs (95% CIs) for GA and AA genotypes were 1.05 (0.78–1.41) and 1.25 (0.01–171.9), respectively. For the studies from East Asian countries, these corresponding ORs (95% CIs) were 1.04 (0.69–1.58) and 1.28 (0.39–4.21), respectively.TNF-A −238 genotype in control subjects were not in HWE  or Egger's weighted regression method . For GA genotype, after excluding studies with an SE>0.5, the summary OR (95% CI) was 1.04 (0.82–1.33). For AA genotype, all of the studies had an SE>0.5.Begg's funnel plot for the association between Q-statistic was nonsignificant for the overall association (P=0.55) and subgroup analyses, and I2-statistic showed little heterogeneity among studies  and also for noncardia, intestinal-type, and western populations subgroups, where I2-statistic showed moderate-to-high heterogeneity.For GA genotype, the  studies . For AA TNF-A −857T carrying genotypes (TC and TT vs CC) and gastric cancer risk. For all gastric cancers, the random-effect overall ORs (95% CIs) for TC vs CC and TT vs CC genotypes were 1.06 (0.89–1.27) and 1.57 (0.91–2.70), respectively. The fixed-effect ORs (95% CIs) were 1.06 (0.89–1.27) and 1.54 (0.98–2.43), respectively. Like TNF-A −238, TNF-A −857 showed neither recessive nor dominant model. Therefore, the results are reported separately for TC vs CC and TT vs CC genotypes.TNF-A −857 and noncardia cancers. This study was also the only study from western countries; the ORs (95% CIs) for TC and TT genotypes in this study were 0.94 (0.63–1.41) and 0.75 (0.17–3.38), respectively. For studies from eastern countries, summary ORs (95% CIs) for TC and TT genotypes were 1.09 (0.90–1.33) and 1.73 (0.95–3.14), respectively.Only one study reported the associations between TNF-A −857 genotypes among controls were not in HWE  or Egger's weighted regression method . For TC vs CC genotypes, none of the studies had an SE>0.5. For TT vs CC genotypes, after excluding two studies with SEs>0.5, the summary OR (95% CI) changed to 1.66 (0.81–3.42).Begg's funnel plot for the association between Q-statistic was nonsignificant for the overall associations and also for the subgroup analyses. Furthermore, I2-statistic showed little to moderate heterogeneity among studies  and a penta-allelic variable number tandem-repeat polymorphism and allele 2 (IL-1RN*2). So far, at least three meta-analyses of the associations between these polymorphisms and gastric cancer risk have been published  and gastric cancer risk. However, there was a statistically significant increased risk associated with AA genotype using both fixed-effects and random-effects models. This association was limited to studies from western countries, and no association was found in studies from East Asian countries. Previous studies have suggested that frequencies of genetic markers often shows high variations among various ethnic and racial groups . However, unlike what has been shown for most previous associations  were 1.29 and 1.65, respectively. The studies that participated in this Consortium were all from western countries.No SNPs have been consistently associated with gastric cancer risk . HoweverTNF-A −308AA genotype did not suggest a dominant effect for any single study. We used funnel plots and two formal statistical methods (Egger's weighted regression method and the rank correlation method of Begg and Mazumdar) to detect bias. In general, smaller studies, that is, those with higher SEs, had lower ORs, and there was some evidence for publication bias using both formal methods. However, excluding smaller studies did not materially change the results. In 20 out of 23 studies, distribution of TNF-A −308 among controls was in HWE. Limiting the analyses to these 20 studies, the results remained essentially unchanged. The overall heterogeneity between the studies was very low, as indicated by the I2 value of zero.The forest plots for TNF-A −308 polymorphism to noncardia gastric cancers or intestinal-type cancers made little difference. The summary ORs for the AG genotype remained close to null and were nonsignificant, whereas those for the GG genotype remained statistically significantly above one.Since subgroup analyses are often limited by selective reporting of significant subgroup results, such analyses generally need to be interpreted with caution. However, in this report, limiting the results of TNF-A −238G>A polymorphism and gastric cancer risk. Unlike that for TNF-A −308G>A polymorphism, a clear biologic role for this polymorphism has not been found (We found nine studies that examined the association between TNF-A −857C>T polymorphisms and gastric cancer. No evidence was found for the association between CT (vs CC) genotype and gastric cancer. Almost all studies showed null associations, and there was little evidence for heterogeneity. The overall association with the TT genotype, however, was near significant. The overall pattern and magnitude of association is similar to that found for TNF-A −308G>A polymorphism, but further studies are needed to examine whether gastric cancer is significantly associated with this polymorphism. The studies showed some heterogeneity, with three showing strong positive associations, one showing no association, and one showing an inverse association. We believe that the currently available data do not provide conclusive evidence for the presence of an association, or lack thereof, between TNF-A −857TT genotype and gastric cancer.We found only five studies that had examined the association between TNF-A polymorphisms are unlikely to be associated with these conditions. Because TNF locus is on chromosome 6 (and not sex chromosomes), the distribution of this polymorphism is not associated with sex. Therefore, in theory, matching for sex should not affect the results.Inappropriate selection of controls is a major source of bias in case–control studies. However, control groups for most of the studies used in this meta-analysis were selected from among healthy volunteers or blood donors, and Strengths of this meta-analysis are including 24 published studies with a large number of cases and controls, presenting data on several relevant methodologic aspects of these studies, subgroup analyses according to predefined criteria, and using other methods to examine the robustness of the summary statistics. We acknowledge that this meta-analysis also has limitations. Combining observational studies conducted in different populations with various qualities of design to obtain summary ORs and 95% CIs can sometimes be misleading , and sumTNF-A −308AA genotype was moderately associated with an increased risk of gastric cancer. TNF-A –308AG, TNF-A −238AA or AG, and TNF-A −857CT or TT were not statistically significantly associated with gastric cancer risk. It is possible that, with increasing the number of studies, TNF-A −857TT may also be associated with an increased risk of gastric cancer.In summary, this systematic review found that


Sir,et al in this issue. The need of a comprehensive combined analysis of multiple germline polymorphisms in genes involved in the folate metabolism and expression of these genes and their proteins in view of corresponding expression data is demanded by the authors for prediction of efficacy and toxicity of 5-fluorouracil (5-FU)-based chemotherapy. We agree to this statement. Our group conducted a combined analysis of polymorphisms of TS and MTHFR (folate pathway) as well as polymorphisms of GSTP1, ERCC1 and ERCC2, enzymes with potential impact on cisplatin efficacy  remain easily accessible and cost-effective at any time of treatment. Despite, an existing controversially discussion about the value of host In conclusion, we agree with the demand for a comprehensive combined analysis in cancer pharmacogenetic studies including several relevant and reliable factors. So far, the process of identifying the most important factors is still ongoing and reveals controversial results. Maybe even more important, there are currently no sufficient statistical models to incorporate the different impact of frequency and functionality of a single polymorphism for the prediction value of the whole analysis. For most polymorphisms, its functional significance is not even clear. Especially before that background, we have to incorporate as much information in our pharmacogenetic analyses as possible and perform true comprehensive studies.n=175), If we focus on TS polymorphisms, we note that there are several divergent observations being published so far. In a study by The TS example shows that we will constantly learn in pharmacogenetics and that it needs large and comprehensive studies to find the ‘truth’ in order to develop robust tests for the clinical setting.


Lung cancer is the leading cause of cancer deaths worldwide. New diagnostics are needed to detect early stage lung cancer because it may be cured with surgery. However, most cases are diagnosed too late for curative surgery. Here we present a comprehensive clinical biomarker study of lung cancer and the first large-scale clinical application of a new aptamer-based proteomic technology to discover blood protein biomarkers in disease.We conducted a multi-center case-control study in archived serum samples from 1,326 subjects from four independent studies of non-small cell lung cancer (NSCLC) in long-term tobacco-exposed populations. Sera were collected and processed under uniform protocols. Case sera were collected from 291 patients within 8 weeks of the first biopsy-proven lung cancer and prior to tumor removal by surgery. Control sera were collected from 1,035 asymptomatic study participants with ≥10 pack-years of cigarette smoking. We measured 813 proteins in each sample with a new aptamer-based proteomic technology, identified 44 candidate biomarkers, and developed a 12-protein panel  that discriminates NSCLC from controls with 91% sensitivity and 84% specificity in cross-validated training and 89% sensitivity and 83% specificity in a separate verification set, with similar performance for early and late stage NSCLC.This study is a significant advance in clinical proteomics in an area of high unmet clinical need. Our analysis exceeds the breadth and dynamic range of proteome interrogated of previously published clinical studies of broad serum proteome profiling platforms including mass spectrometry, antibody arrays, and autoantibody arrays. The sensitivity and specificity of our 12-biomarker panel improves upon published protein and gene expression panels. Separate verification of classifier performance provides evidence against over-fitting and is encouraging for the next development phase, independent validation. This careful study provides a solid foundation to develop tests sorely needed to identify early stage lung cancer. Lung cancer is the leading cause of cancer deaths, because ∼84% of cases are diagnosed at an advanced stage Over the past decade the clinical utility of low-dose CT has been evaluated Other types of biomarkers have also been sought To enable biomarker discovery, we developed a new proteomic technology that is based on a new generation of aptamer protein binding reagents and has potentially broad application All samples were collected from study participants after obtaining written informed consent under clinical research protocols approved by the following institutional review boards: The University of Pittsburgh Institutional Review Board (Pitt); The New York University School of Medicine Institutional Review Board (NYU); The Roswell Park Cancer Institute Institutional Review Board (RP); and The Cape Cod Healthcare Institutional Review Board (BS).The objectives of this study were to discover biomarkers that discriminate NSCLC from smokers with ≥10 years of cigarette smoking history, to train and cross-validate a multi-biomarker classifier of NSCLC to meet pre-specified performance criteria, and to verify the performance of this classifier with a separate set of blinded samples. The overall design of the study is shown in The sample cohort comprised 1,326 serum samples obtained from four independent biorepositories: New York University (NYU) All serum specimens were collected following uniform protocols recommended by the National Cancer Institute's Early Detection Research Network In order to prevent potential bias, this study followed a strict standard operating procedure for sample de-identification and blinding, such that all physical samples and data records were identified exclusively by a unique, unidentifiable barcode number and the key was stored in a secure database accessible only to designated responsible administrators. All sample aliquots run in this study were stored in identical tubes identified only by assigned barcode. The sample blinding code was broken only according to the pre-specified analysis plan for the purposes of classifier training with the training set and classifier verification with the verification set. For the verification sample set, a unique blinding key was generated and provided exclusively to a third party reader (K.C.), unaffiliated with the study centers or SomaLogic, to score and report the final verification results.Serum samples were analyzed on our proteomic discovery platform as described in Gold et al Biomarkers were selected with a strategy designed to identify analytes with the highest performance in classifying NSCLC cases from controls across all study sites and that were least affected by preanalytical variables. In the first step of this analysis, we eliminated analytes that exhibited unexpected variation compared to internal controls, due to, for example, sample instability. In this process, we chose a set of analytes that performed well in a total of six naïve Bayes (NB) classifier training analyses. First we divided the training set into two distinct populations to control for possible biological variability between them: (1) all cases and controls with benign nodules identified by CT; and (2) all cases and all other smoker controls (nodule status unknown). For each population, we compared cases to controls in three NB training analyses designed to control for potential preanalytical variability between study sites. The three NB analyses started with a unique set of potential biomarkers based on the following criteria: (1) cases versus controls KS≥0.3 for all comparisons within each of the four study sites; (2) cases versus controls KS≥0.3 for comparing all sites combined; (3) both criteria one and two were met. For each analysis, we used a greedy forward search algorithm to select subsets of potential biomarkers, build NB classifiers (see below), and scored their performance for classifying lung cancer and controls using the training set. In this process, this meta-heuristic approach efficiently searches classifier space to identify potential biomarkers that perform best in classification. We used a simple measure of diagnostic performance of classifiers, the numerical sum of sensitivity + specificity, and measured the frequency with which potential biomarkers were selected by the greedy algorithm for inclusion in classifier panels with sensitivity + specificity ≥1.7. This step produced a set of potential biomarkers for each of the six parallel analyses. We selected the final set of biomarkers as the union of these six sets.The KS statistic is a non-parametric measure of the difference between two distributions. The two-sample KS Statistic is: The naïve Bayes classifier assumes independence between the samples, and models the distributions of the training classes to make predictions 2 (Eq. 1).We constructed Bayesian classifiers using sets of potential biomarkers identified as described above. We used a parametric model to capture the underlying protein distribution for a given state. The simplest parametric model for the probability density function (pdf) for a single protein is a normal distribution, completely described by a mean u and variance σMany protein distributions were observed as normal with respect to the logarithm of the concentration. The numeric cdfs can be fit to a normal distribution in log concentrations x (Eq. 2).The models fit the data well. More complex models of the probability distribution functions may be used when warranted but the simple model provided a good description of our data.To combine multiple markers, we used a multivariate normal distribution to model the probability density function (pdf) for each class. For n markers, the multivariate pdf is given by the following equation (Eq. 3).x is an n-component vector of protein levels, µ is an n-component vector of mean protein levels, Σ is the n x n covariance matrix and |Σ| and Σ−1 are its determinant and inverse. In its simplest form, we can assume a diagonal representation for Σ. Such an approximation leads to a naïve Bayes model, which assumes independence between the markers. In this work, we exclusively use the naïve Bayes model for constructing classifiers. The parameter values for µ and Σ used in the naïve Bayes classification were obtained from nonlinear regression analysis as described above.where The addition of subsequent markers with good KS distances will, in general, improve the classification performance if the subsequently added markers are independent of the first marker. We searched for optimal marker panels with a “greedy” algorithm, which is any algorithm that follows the problem solving meta-heuristic of making the locally optimal choice at each stage with the hope of finding the global optimum. We used the sensitivity (fraction of true positives) plus specificity (fraction of true negatives) as a classifier score. The algorithm approach used here is described as follows. All single analyte classifiers were generated from a table of potential biomarkers and added to a list. Next, all possible additions of a second analyte to each of the stored single analyte classifiers were performed, saving a predetermined number  of the best scoring pairs on a new list. All possible three marker classifiers are explored using this new list of the best two-marker classifiers, again saving the best thousand of these. This process continues until the score either plateaus or begins to deteriorate as additional markers are added.We analyzed 1,326 serum samples from four independent biorepositories: New York University (NYU) Samples were randomly distributed into segregated sets for classifier training and verification  with no We measured the quantity of 813 proteins in each of the 1,326 samples with our proteomic discovery platform To select biomarkers we performed a systematic analysis that narrowed the potential biomarker field for algorithm training to increase the probability of true discovery, yet still cast a relatively broad net. We used a naïve Bayes (NB) method to systematically assess potential biomarker performance with pre-specified criteria. We applied the NB method to subsets of the training data to broaden our cast for potential biomarkers see . The resTo develop a potential diagnostic to distinguish NSCLC from controls, we trained NB classifiers starting with the 44 potential biomarkers we identified using a “greedy” forward search algorithm and ten-fold stratified cross validation, starting with three biomarkers and adding one more at each step. We assessed classifier performance with pre-specified performance criteria . We consThe twelve biomarkers are shown in To determine whether our classification results were affected either by age, smoking status, or smoking history, which are the demographics with significant differences between the case and control populations , we compPreanalytical variability underlies common failures to translate candidate biomarkers into clinically useful tests The primary findings of this study are 44 potential lung cancer biomarkers that discriminate stages I-III NSCLC cases from at-risk heavy smoker controls that can be combined into classifier panels that meet and exceed pre-specified performance criteria. The results of this study are novel in the following: (1) most of the proteins identified in this study have not been identified previously as serum lung cancer biomarkers; (2) we have identified novel protein biomarker panels that distinguish lung cancer cases from appropriate controls with high sensitivity and specificity in an independent, blinded verification set; and (3) this study achieves a new level of evidentiary standard in clinical proteomic biomarker studies as a result of a large sample size, a study design to control preanalytical variability, and the unique capability of this proteomic technology to interrogate the circulating proteome quantitatively with a breadth, sensitivity, and dynamic range unmatched by other broad serum profiling platforms The best overall performing classifier used 12 of the 44 biomarkers and achieved 91% sensitivity and 84% specificity in cross-validated training and similar performance of 89% sensitivity and 83% specificity in blinded validation. These results provide evidence that these biomarkers are valid and that the classifier was not over-fit to the training data. This performance and the biological plausibility (following) of the 12 biomarkers are encouraging for the next phase of development – validation in an independent clinical study.The 12 biomarkers identified in this study  encompasSix of the 12 proteins, CD30 ligand, endostatin, HSP90, MIP-4, pleiotrophin, PRKCI, and YES were observed up-regulated in lung cancer in this study, consistent with their proposed biological roles in proliferation, invasion, or host inflammatory and immune response to the tumor. CD30 ligand is a member of the TNF ligand superfamily, which stimulates T-cell growth. Up-regulation of this protein correlates with proliferation in hematological malignancies The chaperone HSP90α is important for the stability of and function of a wide range of oncoproteins, including BCR-ABL, ERBB2, EGFR, BRAF, and AKT, among others, and inhibitors of this protein are now in oncology clinical trials, including NSCLC We observed decreased levels of proteins in the serum of lung cancer patients compared to controls, including cadherin-1, LRIG3, sL-selectin, SCRsR, ERBB1 and RGM-C. Lower circulating levels of many of these proteins are associated with relief of inhibition of growth and invasion. For example, cadherin-1 is critical for cell adhesion and indirectly affects transcriptional regulation circuits through β-catenin Some of the proteins described in this study are the soluble domains of membrane receptors, and the function of the circulating form of these proteins may oppose their membrane-bound counterparts. Turner et al. The limitations of this study include the following. We did not test cases prior to clinically apparent disease. We did not demonstrate organ-specificity and many of the markers are known to be elevated in other cancers. However, the markers will be used in combination and in the proper diagnostic context, such as with imaging, smoking history, and symptoms. We did not validate our findings in an independent set of clinical samples. Our multi-center study was designed to minimize the effects of potential preanalytical variability, which is mitigated, but not eliminated by this study. All of these limitations will be addressed in the next phase of development, which is enabled by the positive results of this study.The biomarkers that we discovered have several potential clinical applications. The first application is early detection of lung cancer in long-term smokers when it may be cured by surgery. Our results are a significant improvement on the performance of other recently published lung cancer biomarker studies aimed at early diagnosis A second potential application is a test for diagnosing lung cancer in subjects with suspicious lung nodules identified by CT, which could help mitigate the problem of morbidity and cost associated with surgical interventions. CT screening reveals suspicious nodules in ∼40% of long-term smokers Based on the discoveries reported here, we have initiated clinical validation studies of populations at risk for lung cancer. Our goal is to develop a clinical blood test to enable an earlier diagnosis. This study is the first to be published in a sequence of successful biomarker discovery studies that we have already completed in different cancers and demonstrates the power of our proteomic technology to discover robust biomarkers in important diseases. This general approach can also be applied to discover biomarkers for many more conditions including infectious, inherited, neurological, and metabolic diseases.


Eriobotrya japonica (Thunb.) Lindl.) cultivars/selections grown in Dörtyol, Hatay, Turkey were investigated in 2008. The cultivars/selections included ‘Baduna 5’, Güzelyurt 1, ‘Hafif Çukurgöbek’, ‘Ottaviani,’ and Type 1. The characteristics evaluated included fruit weight, width, length, seed number and weight, flesh/seed ratio, total soluble solids (TSS), pH, acidity, total phenolic (TP) content, and total antioxidant capacity (TAC), determined by the ferric reducing antioxidant power (FRAP) assay. The analyses were conducted by three replicates, with 30 fruits in each replicate. The results indicated that there were significant differences among the cultivars, for all the traits tested. For example, ‘Hafif Çukurgöbek’ and ‘Ottaviani’ had smaller fruits than others, although ‘Hafif Çukurgöbek’ had heavier seeds. The flesh/seed ratio was the highest in Type 1, while ‘Hafif Çukurgöbek’ had the highest pH and high soluble solids. ‘Baduna 5’ and ‘Hafif Çukurgöbek’ had the highest acidity. The TP ranged from 129 (‘Baduna 5’) to 578 (‘Hafif Çukurgöbek’) mg gallic acid equivalent (GAE)/kg fresh fruit (fw). ‘Hafif Çukurgöbek’ also had the highest FRAP mean /kg fw). The results suggest that loquat cultivars have a variable range of TP content and a relatively high total antioxidant capacity, which is crucial for human health.Several fruit characteristics of five loquat ( Eriobotrya japonica Lindl. Rosaceae, Maloideae) is an evergreen fruit species grown in the subtropical zone, especially in the citrus-growing regions.[The loquat  was measured with a scale sensitive to 0.01 g . Flesh/seed ratio was calculated from these values. Fruit size determinations were made using a digital compass . Soluble solids were determined with a hand-held refractometer  and pH was determined by a pH-meter. Acidity (expressed as % citric acid) was determined by titrating with 0.1 N NaOH to an endpoint of pH 8.10. The soluble solid/acidity ratio was calculated. The seed number was also determined in each fruit.For the TP and TAC analyses, harvested fruit samples were frozen and stored at −20°C until analyzed. After thawing to room temperature, triplicate 100 g lots of loquat fruits from each cultivar were homogenized in a blender. All triplicates were screened for their total phenolic contents and antioxidant capacity following a single extraction procedure. For thisThe sample total phenolic contents were measured according to Singleton and Rossi with sliFor obtaining the antioxidant capacity, the FRAP (ferric reducing antioxidant power) method was conducted according to Benzie and Strain. To conduData were analyzed using SAS software and procedures. Means anThe results indicated that there were significant differences among cultivars/selections for all the variables tested . For exaStatistically significant differences were seen for TP . TP rangThe relationships between TP and TAC and among these traits with other pomological characteristics were investigated by correlation analysis . TP and 22In this study, we did not determine the carotenoid content and its effect on TAC. More detailed studies are needed to assess the contribution of loquat pigments to TAC. However, our results suggest that loquat cultivars have a variable range of TP content and relatively high total antioxidant capacity, which is crucial for human health.


Several polymorphisms within the renin-angiotensin system cluster of genes have been associated with the advent of coronary artery disease (CAD) or related pathologies. We investigated the distribution of 5 of these polymorphisms in order to find any association with CAD development and distinguish if any of the biochemical and behavioural factors interact with genetic polymorphisms in the advent of the disease.ACE I/D (rs4340), ACE A11860G (rs4343), AT1R A1166C (rs5186), AGT T174M (rs4762) and AGT M235T (rs699) gene polymorphisms were PCR-RFLP analysed in 298 CAD patients and 510 controls from Portugal. Several biochemical and behavioural markers were obtained.ACE I/D DD and ACE11860 GG genotypes are risk factors for CAD in this population. The simultaneous presence of ACE I/D I and ACE11860 A alleles corresponds to a significant trend towards a decrease in CAD incidence. We found several synergistic effects between the studied polymorphisms and classical risk factors such as hypertension, obesity, diabetes and dyslipidaemia: the presence of the DD genotype of ACE I/D  increases the odds of developing CAD when associated to each one of these classical risk factors, particularly when considering the male and early onset CAD subgroup analysis; AGT235 TT also increases the CAD risk in the presence of hypertension and dyslipidaemia, and AT1R1166 interacts positively with hypertension, smoking and obesity.ACE polymorphisms were shown to play a major role in individual susceptibility to develop CAD. There is also a clear interaction between RAS predisposing genes and some biochemical/environmental risk factors in CAD onset, demonstrating a significant enhancement of classical markers particularly by ACE I/D and ACE11860. ACE) gene and mutations at the angiotensin II AT1 receptor. Several mutations at the angiotensinogen (AGT) gene have also been studied as candidates in essential hypertension [Cardiovascular diseases represent today the main cause of death in human adults in western populations. A reasonable number of studies have focused in testing specific genetic markers among groups of ischemic coronary artery disease (CAD) patients and control groups, aiming to find a correlation between these gene polymorphisms and disease. The renin-angiotensin system (RAS) has been shown to play a key role in the regulation of blood pressure and influence the cardiovascular system , and sevrtension ,3 or myortension .ACE for instance has been found to be linked to MI [AT1R A1166C polymorphism has also been subjected to opposite reports as to its role in CAD, particularly when it is related to the patient's geographic origin: it was found to be significantly associated with CAD in Caucasians [AGT M235T polymorphism has been shown to be positively related to essential hypertension [Albeit all these and other markers that have been identified in latter years as possible risk factors for several pathologies associated to CAD, a considerable number of opposing results also exist. The DD genotype of ed to MI ,6 but coed to MI ,8. Thereed to MI . The AT1ucasians ,10 but nrtension  and MI [rtension  but othertension ,14 and fIn this report we have studied the distribution of genotypic and allelic frequencies at 5 loci, in two groups of individuals from Madeira Island , an island settled mainly by the Portuguese. A group of patients with a known CAD history and a control group with no CAD related pathologies were selected. The main focus was to analyse these genetic polymorphisms, alone or combined in haplotypes, and clarify their potential association with CAD related conditions. Knowing that in most cases CAD has a multifactorial basis, involving a number of genes and environmental factors interacting to determine whether or not the disease will develop, we also tried to determine a possible interaction between the five gene polymorphisms and several well known CAD linked factors.2; dyslipidaemia was considered for plasmatic values of total cholesterol ≥ 200 mg/dl, triglycerides ≥ 150 mg/dl, LDL ≥ 130 mg/dl and HDL ≤ 40 mg/dl. Table The total population of this study consisted of 808 Caucasian individuals (aged between 18 and 70 years old) divided in two groups: 510 subjects without a history of CAD, MI, or stroke – excluded after a medical examination and interview – randomly selected from the electoral rows, who participated as controls, plus 298 individuals recruited from patients admitted to the Cardiology Care Unit of the Main Hospital of Funchal . Patients' recruitment satisfied the following criteria: stable coronary disease suggested by clinical analysis and proved by angiographic exam  or occurrence of MI as defined by the WHO criteria. This investigation is in conformity with the principles outlined in the Declaration of Helsinki and was approved by the Hospital's Ethics Committee; all subjects gave their informed consent. Cases and controls filled in a questionnaire about their personal histories – age, sex, essential hypertension, diabetes mellitus, smoking habits, overweight, sedentary habits, alcohol ingestion and family medical history – and provided blood samples for genotype analysis and biochemical measurements. The presence of traditional risk factors in both groups was determined using the criteria standardized by the European Society of Cardiology: an hypertensive condition was attributed when systolic blood pressure values were ≥ 139 mm Hg and/or diastolic blood pressure values ≥ 89 mm Hg in at least two separate measurements or when being medicated against hypertension; subjects were considered smokers when consuming more than five cigarettes per day or non-smokers when never smoked or had stopped smoking at least one year before sample collection; obesity was defined for BMI values ≥ 30 kg/mACE I/D gene alleles (D and I) were identified by PCR amplification as previously described [AT1R A1166C [AGT M235T [AGT T174M [ACE A11860G [Genomic DNA was extracted from an 80 μl aliquot of whole blood using standard phenol/chloroform methodologies with ethanol precipitation. The escribed . In ordeescribed . The othGT M235T , AGT T17GT T174M  and ACE  A11860G . Amplifi2 tests. Associated probabilities (P) were calculated applying Fisher's exact test adjusted for multiple comparisons of associated genotypes. To test the significance of association between genotypes at pairs of loci in each sample we used a log-likelihood ratio G-statistic as implemented in Genepop. The relative odds ratio (OR) and 95% confidence interval of relative CAD risk for any of the genetic polymorphisms and biochemical and behaviour markers, was assayed by logistic regressions using the SPSS package. To analyse possible positive or negative interactions between classical risk factors of CAD and genetic polymorphisms we used a 4 × 2 table approach and epiInfo 3.4.3 to calculate ORs, respective 95% confidence intervals and two-tailed p values, as well as synergy measures in additive (SI) and multiplicative models (SIM) [00 is assumed to be 1); OR10 refers to the relative risk for disease among people without the susceptibility genotype for disease but exposed to the environmental risk factor relative to those with neither the susceptibility genotype nor exposure; OR01 refers to the relative risk among people with the susceptibility genotype who are not exposed to the risk factor relative to those with neither the susceptibility genotype nor exposure; OR11 is the ratio of disease risk among exposed people with susceptibility genotype to diseased risk among unexposed people without the susceptibility genotype. These ORs were then used in the calculation of synergy indexes: SI = (OR11-1)/(OR10+OR01-2), SIM = OR11/(OR10 × OR01) [11-OR10-OR01+1 [11 [In order to analyze the relative influence of genotypes and biochemical parameters among gender, the two groups (controls and patients) were each subdivided into male and female subgroups. Further grouping according to age (45 or less years old) was performed, aiming to analyse the association between genetics and early onset CAD. Individuals aged ≥ 45 years were not included in this sub-analysis because no data about the age of CAD onset was obtained. Basic genetic parameters such as allele and genotype frequencies at each locus, proportion of individual heterozygous samples (direct count heterozygosity as well as the unbiased estimate) and population differentiation were calculated using Genepop v3.1d  and Arlels (SIM) -26. It w × OR01) ,26; the ACE I/D DD polymorphism  to be independently related to CAD.As expected, both the control and patient group showed differences in the biochemical markers and other conventional risk factors analysed Table . SystoliACE loci show statistically different distributions for both the overall analysis and male subgroup. Only the group of patients is in Hardy-Weinberg equilibrium at each locus and overall as the control group is not at HWE at locus ACE I/D . As expected, mutations at the AGT and ACE loci show strong significant linkage disequilibrium within each gene (P < 0.0001) for both groups, even after Bonferroni correction. The group of patients also shows significant genotypic disequilibrium at AGT235/AT1R (P < 0.03). An exact G-test for population differentiation gave an overall value of 0.00238 in which the loci contributing significantly to the differentiation between the two samples are both ACE polymorphisms . Thus taking in consideration all loci combined, both populations are significantly different concerning the genic differentiation .Table ACE I/D and ACE11860 loci . This is also reflected on the distribution of genotypes across ACE I/D which is highly significant in a G-like test (P < 0.003).Male patient and control groups show significant differences in allele content at both ACE polymorphisms and CAD development we obtained significant results when testing ACE I/D DD and ACE11860 GG, while ACE I/D ID genotype showed to decrease CAD risk . Considering all genes involved in the RAS system, only five out of nineteen obtained combinations yielded significant associations with CAD  as being 1.0, the OR estimating the effect of joint exposure to hypertension and ACE I/D DD, AGT235 TT or AT1R CC was significantly higher than the ORs estimating the effect of each factor in the absence of the other. The synergy index in early onset CAD analysis (ACE I/D DD) was above 9, indicating a departure from an additive relation. In this group the proportion of CAD attributable to the interaction of hypertension and ACE I/D DD was as high as 85%. The genotypes AT1R CC in the overall analysis, or ACE I/D DD in all subgroups, interact with smoking habit to develop CAD, showing more than a multiplicative effect in females . The risk provided by obesity was found to be positively reinforced by AT1R CC in the whole population analysis and ACE I/D DD in all subgroups, particularly in females  and ≥ 45 subgroup . Performing the same analysis regarding diabetic individuals, significant results were found for ACE I/D DD carriers in the whole population analysis and a strong enhancement was found in females , but not in males . None of the 2 polymorphisms presented in table AT1R CC and diabetes were CAD patients. The join presence of dyslipidaemia and ACE I/D DD interacts significantly in CAD onset, except in females . This combination was shown to be accountable for 89% of the disease in the = 45 years subgroup . All 12 individuals with AT1R CC genotype and dyslipidaemia were found to be CAD patients, even though no significant results were obtained due to statistical constraint.The analysis of the possible positive/negative association between genotypes and classical risk factors is expressed in Tables ACE, AT1R and AGT gene polymorphisms with coronary events of several degrees, related or not with MI and essential hypertension. Up to know there is no consistent genetic pattern that may link a given haplotype to the risk of developing a CAD related syndrome or at least to make an individual more prone to be affected. All 5 gene polymorphisms surveyed here promote phenotypic variants on known mechanisms leading to changes in the biochemical status of an individual. For example, the ACE I/D variant is linked to ACE activity [AGT M235T to different angiotensinogen plasma levels [Previous studies have focused on the association of any of the activity  and AGT a levels .ACE gene seem to be linked to CAD and even these are apparently male-linked because no such association was visible in females. The association of ACE I/D DD genotype with CAD in men but not in women, has been reported previously in several studies [ACE gene, the control subpopulation was not, due to an excess of heterozygotes ID. Similar results were previously reported [By evaluating the distribution of RAS gene polymorphisms in a series of patients undergoing coronary angiography, the present study has shown that only mutations at  studies ,28. Whilreported ,29 and mACE gene has been shown to be associated with both CAD and MI [ACE I/D or ACE11860 genotypes may predispose an individual to the development of MI remains unclear. ACE is responsible for the conversion of angiotensin I to the peptide precursor angiotensin II, which has been implicated in the pathogenesis of atherosclerosis [ACE polymorphisms did not influence the development of MI or other manifestations of CAD [ACE gene polymorphisms and CAD or MI have been restricted to relatively small subgroups. Even more, in some studies, the presence or absence of CAD was not determined by angiography and might even have used false-negative control populations. These findings also stress the necessity of considering ethnic factors in the assessment of genetic risk identifiers.The deletion polymorphism of the D and MI ,30,31. Tclerosis ,33. In cs of CAD ,35. TherAGTs and AT1R polymorphisms and CAD is in agreement with other studies [AGT variants and angiotensinogen levels. Nevertheless, our study revealed a significant influence of the A allele and AA genotype of AT1R upon an increase in carotid-femoral PWV values, which may be regarded as a risk factor for CAD (results not shown).The total lack of association between the 2  studies ,36 that ACE I/D I and ACE11860 A alleles in ACE haplotypes corresponds to the lowest risk of developing CAD in the whole population, male and female subgroups, expressed by ORs lower than 1. When analysing the influence of RAS haplotypes in the development of CAD, it is clear that the major influence comes from both ACE polymorphisms. Yet again, the simultaneous presence of ACE I/D I and ACE11860 A alleles, corresponds to a significant trend towards a decrease in CAD, both in male subgroup and overall analysis. The combined set of RAS alleles ACE11860 G/ACE I/D D/AGT174 T/AGT235 T/AT1R A was the only one found to significantly increase CAD risk in the whole population analysis. No significant association was found in females, showing once again the interest of separating this type of data between sexes.The presence of the ACE11860 G/ACE I/D D/AGT174 T/AGT235 T/AT1R A. Here we must point the impossibility to further subdivide this group according to sex, due to small sample size, as a limitation to perform a more thorough analysis. One should be aware that in almost all case-control studies, particularly those involving haplotype analysis, problems related to multiple comparisons , the potential influence of genetic and environmental factors not considered, limited sample size for subgroup analysis and the possible inclusion of patients with silent CAD in the control group should be taken into account. The fact that the control and CAD patients groups are not sex and age matched may also be regarded as a constraint in the study design.Premature CAD is known to have a particularly strong genetic component. Previous data have suggested that genetic factors are more likely to affect young rather than old people . We condACE polymorphisms have shown to be in strong linkage disequilibrium, we chose not to show the results of this analysis for ACE11860 GG. They are very alike those presented in table ACE I/D DD genotype around 30% for the whole population and male subgroup analysis. In the early onset CAD analysis, this proportion goes up to 85%. The vascular risk factors that might be related to serum ACE activity are not yet fully understood. Nevertheless, a previous study has shown a correlation between male sex and history of hypertension with serum ACE activity [AGT235 TT or AT1R CC as the risk genotypes . Smoking may increase ACE levels by means of a nicotine enhancement of ACE gene expression [ACE I/D DD join presence, which is in accordance with a previous study [ACE gene expression, but also in other RAS intervenients, such as AGT or AT1R [AGT235 TT or dyslipidaemia and AT1R CC, even though all 12 dyslipidaemic individuals carrying AT1R CC were CAD patients. A similar study, analysing AGT235 T allele synergistic effect with several risk factors, found a positive association with hypercholesterolemia but neither with hypertension nor smoking [ACE I/D polymorphism and Type 2 diabetes has been rather controversial. Some studies have affirmed a clear association between DD genotype and the disease in Caucasians [The complex aetiology around cardiovascular disorders and the multiple environmental conditionings are most of the times not evaluated together. In most cases, CAD has a multifactorial genetic basis, involving a number of genes and environmental factors interacting to determine whether or not the disease will develop. Therefore, the inherited genes generally predispose to a greater or lesser extent of CAD, but it is the environmental factors  interacting with the individual's genotype that determine whether or not CAD will develop. We performed further analysis in order to determine whether the simultaneous presence of genetic polymorphisms and well established risk factors – hypertension, smoking habit, obesity, diabetes and dyslipidaemia – would enhance the effect in CAD onset of these last. Some interesting results were obtained, particularly in the subgroups analysis. As both activity , which mpression . A largepression  found a us study . Moreove or AT1R . Neverth smoking . The assucasians . We founACE I/D and ACE11860 A/G – interact synergistically with them in CAD onset. Taking into account that CAD is a multifactorial disorder driven by numerous environmental, behavioural and genetic components interacting together, our results seem to corroborate the hypothesis that RAS gene polymorphisms may indeed enhance the influence of traditional risk factors in CAD development. The sample used in this study is also a novelty because it is based on the population of Madeira Island that until the beginning of the 20th century was a relatively isolated island. To our knowledge this analysis on such a population had not yet been performed. Madeiran inhabitants with a history of a small founder population, long lasting isolation and population bottleneck represent an exceptional resource in the identification of genes involved in the pathogenesis of multifactorial diseases.The most relevant findings of this research are based on the influence of RAS gene polymorphisms evaluated together with the presence of the main classical risk factors for CAD, showing how at least two polymorphisms – The authors declare that they have no competing interests.AIF performed this work as part of her PhD thesis, under guidance of AB and AC. AB, IM and RPR conceived the idea for the study. Lab and clinical data were performed under AB, IM and AC guidance. All authors performed data analysis, interpretation and discussion of results and also read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Platelet aggregation may contribute to the pathogenesis of systemic sclerosis: following activation, platelets release significant amounts of serotonin – which promotes vasoconstriction and fibrosis, and further enhances aggregation. The C+1354T polymorphism in the exonic region of the serotonin 2A receptor gene determining the His452Tyr substitution was associated with blunted intracellular responses after serotonin stimulation, and may have a role in susceptibility to scleroderma.452Tyr substitution of the serotonin 2A receptor gene, and associations were sought with scleroderma and its main clinical features. The functional relevance of the His452Tyr substitution was also assessed by evaluating the aggregation of platelet-rich plasma from His452/His452 and His452/Tyr452 healthy individuals after stimulation with adenosine diphosphate ± serotonin.One hundred and fifteen consecutive systemic sclerosis patients and 140 well-matched healthy control individuals were genotyped by sequence-specific primer-PCR for the HisP < 0.001, chi-square test and 1,000-fold permutation test) and its carriage reduced the risk for systemic sclerosis . Platelets from His452/Tyr452 healthy subjects more weakly responded to serotonin stimulation compared with platelets from His452/His452 individuals .The T allele of the C+1354T polymorphism was underrepresented in scleroderma patients compared with control individuals (5.2% versus 12.4%, 452Tyr substitution may influence susceptibility to systemic sclerosis by altering platelet aggregation in response to serotonin.The His Systemic sclerosis (SSc) is a complex connective tissue disease characterised by fibrosis of the skin and internal organs, widespread vasculopathy and abnormalities of the immune system . Whilst Amongst the vascular alterations described in SSc patients, perturbations in platelet homeostasis have long been recognised . Platele1 to 5-HTR7) [2A) that accounts for most of the detrimental profibrotic, vasoconstrictive, mitogenic and proaggregating activities of 5-HT [2A gene is located at 13q14-q21, and several single nucleotide polymorphisms (SNPs) have been identified within this region, a few of which determine amino acid substitutions and may thus be relevant from a functional point of view [2A gene  that determines a His452Tyr substitution in the C-terminal region of the receptor [452Tyr substitution [The 5-HT functions are mediated by a superfamily of seven related G-coupled receptors (5-HTR 5-HTR7) , but it  of 5-HT -14. The  of view . Amongstreceptor . This rereceptor  and may titution , and may2A gene and SSc, and we then further clarified its functional role by evaluating platelet aggregation in response to the costimulation with ADP and 5-HT [2A gene.In the present study we first explored a possible association between the C+1354T SNP of the 5-HTRand 5-HT  in healt2 cells  using a standardised technique [One hundred and fifteen consecutive unrelated Italian SSc patients referred to our outpatient clinic were included. All of the patients fulfilled the classification criteria proposed by the American College of Rheumatology , and werechnique . ExtractOne hundred and forty healthy ethnically matched, sex-matched and age-matched subjects were also included as control individuals .All of the patients and all of the controls gave their written consent for the present research. The protocols of the study as well as of the functional study described below were approved by the ethic committee of our institution, and are in compliance with the Declaration of Helsinki.2A gene was amplified using PCR. In brief, a set of primers was designed to encompass the C+1354T polymorphic site in the 5HTR2A gene . The PCR reaction was performed using 100 ng genomic DNA, 10 pM each primer, 1.5 mM MgCl2, 2.5 mM dNTPs and 1 U Euro Taq  in a final volume of 25 μl. PCR amplification was initiated at 96°C for 5 minutes and was performed for 40 cycles, each consisting of 30 seconds at 96°C, 45 seconds at 58°C and 45 seconds at 72°C. A final elongation step of 5 minutes at 72°C was added.Ten millilitres of blood were collected into tubes containing sodium citrate. Genomic DNA was isolated with the DNA Isolation Kit for Mammalian Blood . To detect the C+1354T SNP, the 5HTRAll of the PCR products were sequenced. Prior to sequencing, the unincorporated dNTPs and primer were removed by ExoSAP-IT  at 37°C for 15 minutes, after which the enzymes were deactivated by incubation at 80°C for 15 minutes. Samples were sequenced in both directions on an Applied Biosystems 3100 Genetic Analyzer using the Big-Dye Terminator Cycle Sequencing Reaction Kit . The cycling conditions were 25 cycles of denaturation at 96°C for 10 seconds, annealing at 50°C for 5 seconds and extension at 60°C for 4 minutes.452His/452Tyr and 452Tyr/452Tyr healthy nonsmoker individuals were obtained for the functional study; none of these subjects was receiving steroids or antiaggregation therapy. Whole blood, anticoagulated with sodium citrate , was immediately centrifuged at 130 × g for 15 minutes in order to obtain platelet-rich plasma. A subsequent centrifugation at 1,050 × g for 15 minutes allowed one to obtain platelet-poor plasma, used to set the 100% light transmission of the instrument. Then 250 μl platelet-rich plasma was warmed at 37°C for 3 minutes, and the agonist was added. Platelet aggregation was performed on a Chrono-Log Aggregometer  with ADP  in a plain Tyrode's solution containing 2 mM CaCl2, 1 mM MgCl2, 0.1% dextrose, 0.35% BSA, 0.05 U/ml apyrase, pH 7.35, at a final concentration of 1 μM, or with ADP + 5-HT (Sigma-Aldrich Corp.) both at a 1 μM final concentration. Platelet aggregation was recorded for 3 minutes and the maximum light transmission in this period was measured. The response to 5-HT was then calculated as the fold increase in 5-HT-induced aggregation with respect to the aggregation observed after stimulation with ADP alone.Blood samples from In the present work the decision was made not to replicate the functional study in SSc patients. This decision was mainly due to different reasons. Firstly, all of our patients were being treated with a combination of drugs that may alter platelet function , and thus it would have been unethical to interrupt their ongoing therapy. Secondly, SSc platelets show an activated phenotype that that would have been a significant confounding factor in the analysis of platelet function -7.The distribution of the C+1354T genotypes was tested for Hardy–Weinberg equilibrium with the goodness-of-fit chi-squared test both in patients and in control individuals.The distribution of the C+1354T genotypes and alleles between control individuals and SSc subjects was tested by the chi-squared test or Fisher's exact test when necessary. Odds ratios and their relative 95% confidence intervals were also calculated from 2 × 2 contingency tables. Statistical significance was also evaluated using a 1,000-fold permutation test.Genetic-association studies might be flawed by the possibility of false-positive results, even in the presence of statistically significant findings – that is, according to the definition of Wacholder and colleagues , the falP value while π was set from 0.001 up to relatively high values (0.5 – 1), given that only five SNPs within the 5-HTR2A gene determine amino acid substitutions and may thus be functionally relevant, and the C+1354T SNP indeed alters platelet function in vitro  was calculated by the PS Program , and it The FPRP values for the present study were then reported in a table with the corresponding π and odds ratio values; FPRP values < 0.5 are then highlighted. These values are considered adequate in small exploratory studies on genetic associations  – given 452His/452His and 452His/452Tyr healthy subjects was compared by the nonparametric Kolmogorov–Smirnov test and was then verified by linear regression. As platelet-induced aggregation displays a ceiling effect, the magnitude of the dependent variable (5-HT-to-ADP aggregation ratio) may be correlated with the variance of baseline (ADP-induced) aggregation, thus violating the assumption of homoscedasticity [The 5-HT-to-ADP aggregation rate in asticity . Linear P < 0.05 was considered significant.All of the statistical procedures were carried out with the SPSS version 15.0 software . Continuous values are expressed as the mean ± standard deviation – except for skewed distributions (skewness <-2 or skewness >2), where the median and interquartile range are reported.The demographic and clinical characteristics of the patients are reported in Table 452His/452Tyr heterozygosity) and the TT genotypes (452Tyr/452Tyr homozygosity) of the C+1354T SNP were underrepresented in SSc patients compared with control individuals . Similarly, a decreased frequency of the T allele was observed in SSc patients . All of the results were confirmed after the 1,000-fold permutation test . Overall, 452His/452Tyr and 452Tyr/452Tyr individuals had a threefold reduction in the risk for SSc compared with 452His/452His individuals .As reported in Table 452His/452Tyr substitution are reported in Table The FPRP values of the association between SSc and the 452Tyr substitution was not associated with any of the following: the disease subset, the anticorpal status, age at the onset of the disease, past history of digital ulcers, the forced viral capacity, the diffusing capacity for carbon monoxide or the right systolic ventricular pressure.Robust associations with clinical variables were difficult to calculate owing to the low prevalence of the rarer allele of the C+1354T SNP in SSc patients; however, from an exploratory point of view, the His452His/452His homozygous subjects, seven 452His/452Tyr heterozygous subjects) had poor aggregating responses after stimulation with ADP alone . Aggregating responses were markedly increased after costimulation with 5-HT and ADP  with respect to the stimulation observed with ADP alone . The functional significance of the His452Tyr substitution was better assessed by linear regression analysis, weighted for ADP-induced platelet aggregation; by this procedure, it was confirmed at a highly significant level that the His452Tyr substitution of the 5-HTR2A dampened platelet aggregation in response to 5-HT . The regression equation of the final model with adjusted R2 = 0.98 had an almost perfect fit: 5-HT-to-ADP-response ratio (fold increase) = 4.995 - (0.019 × ADP-induced aggregation) - (1.148 × gender) - (1.192 × His452Tyr substitution) .The ADP-induced baseline aggregation, age, platelet count and gender distribution did not differ between 2A gene in a population of Italian SSc patients.Owing to the evidence of involvement of vasculopathy in the pathogenesis of SSc ,3, much 2A gene [452His/452Tyr heterozygotes compared with 452His/452His homozygotes, as indicated by our ex vivo study , regardless of its effect on 5-HT-induced platelet aggregation. Indeed, Hazelwood and Sanders-Bush also described a reduced intracellular signalling capacity in murine fibroblasts expressing the His452Tyr substitution after stimulation with 5-HT [The 5-HT concentrations are increased in plasma samples from SSc  as a conith 5-HT .452Tyr substitution, however, makes our findings modestly prone to false positive results. The significance of our results can also be gauged considering the FPRP values we obtained under different scenarios  we observed in a population of Italian SSc patients. The C+1354T SNP is therefore worth inclusion in a panel of candidate genes for future association studies in the SSc field.We provide evidence that the His2A: serotonin 2A receptor; PCR: polymerase chain reaction; SSc: systemic sclerosis; SNP: single nucleotide polymorphism.BSA: bovine serum albumin; ELISA: enzyme-linked immunosorbent assay; FPRP: false-positive report probability; 5-HT: serotonin; 5-HTRThe authors declare that they have no competing interests.LB was responsible for the study design, manuscript preparation, analysis and interpretation of data, and statistical analysis. MC participated in collection of data, interpretation of data and genetic analysis. MM performed genetic analysis. FC was responsible for manuscript preparation and interpretation of data. AA and GM performed the platelet functional study. RS was responsible for the study design and fundraising.