P < 0.001), whereas the levels of delta-5 desaturase , delta-6 desaturase (P = 0.001), elongase (P = 0.16) and 15-lipoxygenase-2  are higher in donor  prostates, and 2) Contrary to the observation in the normal tissues, significantly high levels of only 15-LO-1; whereas low levels of delta-6 desaturase, elongase, delta-5 desaturase and 15-LO-2 respectively, were observed in PCa tissues. Although the cyclooxygenase (COX)-1 and COX-2 mRNA levels were high in PCa, no significant differences were observed when compared in donor tissues. Our study underscores the importance of promising dietary intervention agents such as the omega-3 fatty acids as substrate competitors of LA/AA, aimed primarily at high 15-LO-1 and COX-2 as the molecular targets in PCa initiation and/or progression.One of the major components in high-fat diets (Western diet) is the omega -6 polyunsaturated fatty acid (PUFA) called linoleic acid (LA). Linoleic acid is the precursor for arachidonic acid (AA). These fatty acids are metabolized to an array of eicosanoids and prostaglandins depending upon the enzymes in the pathway. Aberrant expression of the catabolic enzymes such as cyclooxygenases (COX-1 and/or -2) or lipoxygenases  that convert PUFA either AA and/or LA to bioactive lipid metabolites appear to significantly contribute to the development of PCa. However, PUFA and its cellular interactions in PCa are poorly understood. We therefore examined the mRNA levels of key enzymes involved in the LA and AA pathways in 18 human donor  prostates compared to 60 prostate tumors using the Affymetrix U95Av2 chips. This comparative  study showed that: 1) the level of 15-LO-1 expression (the key enzyme in the LA pathway) is low ( P-value to 0.05 divided by the number of comparisons). However, the analyses conducted in the present study do not evaluate the prognostic ability of these genes, they merely evaluate whether they are differentially expressed. Our data on donors spanned from ages 5 to 60 and all tumor patients are older than 40. Therefore, in order to examine whether or not the differential gene expression between donors and patients is affected by age specific differences in their prostates, we segmented the donors into different age groups and compared only the 40 to 60 year old donors with tumors of the same age group. Although the number of cases in the study were small, the expression pattern observed in this age matched analysis is identical (data not shown) to that when all donors are included suggesting that potential age related differences in donor prostates do not contribute to the results of the donor versus tumor analysis. This comparative  study showed that: 1) the level of 15-LO-1 expression (the key enzyme in the LA pathway) is low (P < 0.001), whereas the levels of delta-5 desaturase , delta-6 desaturase (P = 0.001), elongase (P = 0.16) and 15-lipoxygenase-2  are higher in donor  prostates, and 2) However in PCa tissues, significantly high levels of only 15-LO-1; whereas low levels of delta-6 desaturase, elongase, delta-5 desaturase and 15-LO-2 respectively, were observed. Although the cyclooxygenase (COX)-1 and COX-2 mRNA levels were high in PCa, no significant differences were observed when compared in donor tissues. This study suggests that an increase in the n-6 to n-3 PUFA ratio in diet when the n-6 enzymes such as 15-LO-1 and COX-2 are aberrantly expressed can promote PCa, and the need for designing promising early dietary intervention agents such as the omega-3 fatty acids as substrate competitors of LA/AA, aimed primarily at high 15-LO-1 and COX-2 as the molecular targets  remains the second most commonly diagnosed cancer in American men, with over 230,000 new cases predicted for 2005 alone . Project13-S-HODE, 13-S-hydroxyoctadecadienoic acid; 15-LO, 15-lipoxygenase; COX, cyclooxygenase; LA, linoleic acid; AA, arachidonic acid; PG, prostaglandin; PCa, prostatic carcinoma; AA, arachidonic acid; COX, cyclooxygenase; LO, lipoxygenase; HETE, hydroxyeicosatetraenoic acid.The author(s) declare that they have no competing interests.UK conceived the study, carried out the data query analysis and drafted the manuscript. YL and DL performed the statistical analysis. UC carried out the data retrieval and query analysis. RD participated in its design. All authors read and approved the final manuscript.


HFE Human genome nomenclature committee ID:4886), and mutations in HFE are a cause of the iron mismetabolism disease, hemochromatosis. Several reports have tested the association of HFE variants with neurodegenerative diseases, such as AD and PD with conflicting results.Pathological brain iron deposition has been implicated as a source of neurotoxic reactive oxygen species in Alzheimer (AD) and Parkinson diseases (PD). Iron metabolism is associated with the gene hemochromatosis  and 132 PD patients. Additionally, a series of 115 healthy age-matched controls was also screened.Genotypes were analysed for the two most common variants of A statistically significant association was found in the PD group when compared to controls, showing that the presence of the C282Y variant allele may confer higher risk for developing the disease.HFE may be a risk factor for PD, but not for AD in the Portuguese population.Taken together these results suggest that the common variants in  HFE on chromosome 6p21.3. The most common mutation, C282Y, was initially found in a subset of patients with hereditary hemochromatosis, in a total of 83% of all individuals. A second mutation, H63D, was also described, although the clinical effects of this modification are clearly more limited. However, about 1 to 2 percent of individuals with compound heterozygous HFE mutations appear to be at risk for hemochromatosis [Classic Hemochromatosis is an autosomal recessive disorder that is associated with a deregulation of the iron metabolism . ClinicaHFE mutations have been associated with different stages of dementia (Braak stages) and increased oxidative stress, thus a study including MCI patients is of relevance [Alzheimer's disease is the most common late-onset neurodegenerative disorder. While several studies have tried to unveil the precise mechanisms underlying the etiology of typical sporadic AD, these remain largely unknown. Nonetheless, several studies have reported that oxidative stress may be implicated in the pathogenesis of this condition ,5. Oxidaelevance . HoweverParkinson's disease is the second most common form of neurodegenerative disease, characterized clinically by resting tremor, muscular rigidity, bradykinesia, and postural instability. Post-mortem examinations of PD brains and magnetic resonance imaging of PD patients have revealed increased iron contents in the substantia nigra . The cauHFE variants on the onset of PD and AD have been contradictory [HFE mutations are a risk factor for the development of these diseases, we conducted a genetic screening for the most common HFE mutations in two series of patients and in a healthy control group.Previous studies assessing the effect of adictory ,10-12. TA total of 428 individuals were screened for two mutations in the HFE gene: C282Y (rs1800562) and H63D (rs1799945).The first series of patients integrated AD and MCI patients. The diagnosis of probable Alzheimer's disease was made in accordance with criteria defined by the Diagnostic and Statistical Manual, revision 4 (DSM-IV)  and the A total of 132 PD patients were selected according to the United Kingdom Parkinson's Disease Society Brain Bank Clinical Diagnostic Criteria (UK PDS Brain Bank) . PatientThe control group included 115 healthy controls with a mean age of 70,7 ± 10,3 years, 38 males and 77 females. All subjects were examined by a neurologist and were free of any clinical signs or symptoms of neurodegeneration. This group comprised mainly spouses of patients and caregivers that were accompanying patients to the clinic.All individuals included in this study are Caucasian with an apparent Portuguese ancestry. The study was submitted to the Ethics board of the University Hospital of Coimbra and all the subjects involved gave their informed consent.HFE mutations C282Y and H63D using the polymerase chain reaction (PCR) technique with subsequent restriction and gel electrophoresis, as previously described [APOE genotypes were assessed by a PCR-based methodology, as previously described [Genomic DNA was isolated from whole blood by means of standard procedures and the samples were genotyped for the escribed . Similarescribed .t-test. Kaplan-Meier (KM) survival analysis was used to analyze the effects of the HFE mutations on the age of AD, PD and MCI onset. The log-rank test was employed to determine whether genotype-specific survival functions were significantly different from one another. All tests were interpreted at the 0,05 level of significance. All statistical analyses were performed with the SPSS package, version 10.0 .Observed genotype distributions were compared with those expected by cross-tabulation and analyzed using Chi-square and Fisher Exact-tests. Means of quantitative variables were compared using Student's To test the association between the presence of the C282Y and H63D mutations and the development of AD or PD, we screened these series of patients and a series of healthy controls. The genotypes in these cohorts were at or near Hardy-Weinberg equilibrium.There was no significant difference in genotype or allele frequencies in the AD or MCI groups, compared to controls. Analysis of the genotypes in the PD series revealed a significant overrepresentation of 282Y carriers and of the 282Y allele compared to controls p = 0.01) .HFE do not contribute significantly to the risk of developing AD or MCI, in the Portuguese population. These data are consistent with previous studies [HFE mutations were associated with increased oxidative stress and Braak AD stage [Our results suggest that C282Y and H63D variants of  studies ,21 but c studies  and dataAD stage . The latAD stage ,23. SampAD stage . These sHFE and AD in the current study may be related to one or more of several factors: first, the present results may represent a false negative finding, driven in part by the low sample numbers; second, the role of HFE variants in risk for disease may vary between different populations (ie genetic background); third, as discussed below, the variants studied here may not be disease causing, but in linkage disequilibrium with disease causing mutations, thus discordant results will be seen in different populations.The lack of association between genetic variability in HFE variants and PD reported an opposite effect to the data presented here: the authors presented data suggesting that individuals with C282Y mutation have a decreased risk of developing PD [HFE variants in risk for PD [The data presented here show a significant increase of the prevalence of 282Y carriers in the PD cohort compared to controls. A previous study examining the relationship between oping PD , in contk for PD  and recek for PD .HFE and PD The small number of individuals in this study makes an ultimate assessment of the biological and genetic significance of these data clearly impossible. Thus we have analysed all previous studies published so far on this subject, in order to perform a meta-analysis of the data, and hopefully shed some light on these mechanisms.The discordant results may be explained by several factors: first, the results of the current study and those of Dekker and colleagues represent false positive findings; second, the results of Buchannan and colleagues represent false positive findings; third, 282Y is not a causal variant but is in linkage disequilibrium with another variant that underlies disease risk. The degree and direction of a disease association when genotyping what is in effect a tagging SNP, are both sensitive to the structure and content of a given block of linkage disequilibrium; these factors are both potentially different between populations. The observation that the 282Y allele is overrepresented in the PD cohort compared to the AD cohort demonstrates explicitly the main findings of this paper; that this variant may infer risk for PD, but not AD in the Portuguese population. While it is tempting to speculate that differences in iron handling may differentiate the molecular underpinnings of these two disorders, the current data is too far removed from this mechanistically and too preliminary to make this a convincing argument. The infrequency of C282Y mutations obviously limits the statistical power of this analysis, thus, studies in larger samples from diverse populations are needed to clarify the relationship between variability in Regarding AD, a total of five studies has been published aiming to find an association of these HFE mutations with the disease. No statistically significant differences were found in any of the studies. The combined data set can be observed in Tables As for PD, two studies have described a positive association between the C282Y variation and the disease ,11, as cWhen looking at all the results published so far, there is no statistically significant association between any of the variations and the development of any of the diseases Tables ,5,67.6HFE may be a risk factor for PD. The rarity of HFE 282Y limits the statistical power of this analysis, thus studies in larger samples and in diverse cohorts are needed to make clarify the relation between variability in HFE and PD.In conclusion we present data that suggests genetic variability in The author(s) declare that they have no competing interests.RJG and JMB performed the genotyping, statistical analysis, and drafted the manuscript. MHR, IS, BS, CJ and ASM contributed to collecting materials. JH, AS and CRO participated in the study design and coordination, together with drafting the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Public funding is aimed at facilitating the initiation, completion and publication of research study protocols. However, no evaluation is made to investigate the impact of grant success on the conduct of biomedical research. It is therefore of great interest to compare the fate of funded protocols versus not funded: Are they initiated? Are they completed? Did the results confirm the hypothesis? Were they published? The objective was to investigate the fate of protocols submitted for funding, whether they were funded or not.Retrospective cohort study of protocols submitted for funding to the Greater Lyon regional scientific committee in 1997. Initial characteristics of protocols  were abstracted from archives, and follow-up characteristics  from a mailed questionnaire to the principal investigators.Among the 142 submitted protocols, follow-up information was available for 114 (80%). As a whole, 38% of studies were funded by the Greater Lyon research committee. The rate of initiation varied from 62% for studies with no acknowledged funding to 100% for studies with both committee and other simultaneous funding. When initiated, the rate of completion was 62% for studies with at least one funding and 40% for studies without acknowledged funding. When completed, publication was reached for 77% of studies with either committee or external funding, for 58% of studies without acknowledged funding and for 37% of studies with both committee and external funding.Some protocols submitted for funding were initiated and completed without any funding declared. To our understanding this mean that not all protocols submitted really needed funding and also that health care facilities are unaware that they implicitly financially support and pay for biomedical research. Public total expenditures in biomedical research have grown in recent years.In the USA, the National Institutes for Health's (NIH) total appropriation rose to $28 billion in 2004 , 129 milDespite previous increases in public budget for clinical research, competition for obtaining funds is therefore rough since the success rates for grants remained fairly the same  and the It is expected that all research funded will lead to publication and this was explicitly mentioned in the law that settled the French National funding scheme. In thisThe French Ministry of Health has also planned regular assessment of its national funding scheme, and four evaluations have been conducted at the national level in France  of funded protocols versus not funded, as well as to compare the fate according to type of funding. Our objective was to follow all protocols submitted for funding to the regional scientific committee of Greater Lyon in 1997 and to investigate their fate according to the obtaining of funding: are these protocols funded elsewhere? Are they initiated? Are they completed? Did the results confirm the hypothesis? Do investigators consider the study results as important? Were the results published? Our hypothesis was that absence of acknowledged funding does not hamper implementation nor publication.All biomedical research protocols submitted for funding to a French regional scientific research committee  in 1997 were included.All protocols submitted are archived by the regional scientific committee. Data on initial characteristics , funding results  and amount (converted into 1997 euros) were collected by the same person (ED) to ensure homogeneity. To confirm data extraction, main data of interest were reviewed by FC. When variables were ambiguous or not clearly stated in protocols, data were revised by both FC and ED to reach an agreement. To obtain follow-up characteristics , a mailed questionnaire was sent to the principal investigator of each submitted protocol. The principal investigators were contacted up to three times to obtain the completed questionnaire in 2003.Committee funding: two kinds of regional scientific committee fundings were considered:- National funding scheme for clinical research: In 1992, the French Ministry of Health launched a national funding scheme aiming to support biomedical research in French hospitals .- Lyon hospital system's internal funding schemes include special funding for research assistants, young researchers, registered nurses, support for clinical research protected time and support for clinical research facilities.External funding: investigators had to declare if the protocol had been submitted to other funding schemes, and if so, if they succeeded.To avoid confusion between different sources of funding, protocols funded after evaluation by the regional scientific committee were labelled "committee-funded protocols" (and "not committee-funded protocols") and protocols funded after other applications were labelled "externally funded protocols" .Legal and methodological definitions used are provided in table Fate: specific endpoints of a research protocol .Rating of study results: Investigators were asked to self-rate the importance of their study , and results were considered as "unimportant" when this rating was lower or equal to 5.Scientific publication, oral presentation and grey literature  were basPublication bias is defined as the tendency on the parts of investigators, editors, and others to favour publication of research with positive results compared to research with null or negative results . As a paThis study was conducted according to the French law on epidemiologic and descriptive studies . Data we2 tests were used for qualitative variables.We obtained frequency distributions for all variables (means and percentages). When assessment of association was needed, χWe restricted analysis of publication to the cohort of completed studies. We excluded studies from the analysis when the rating of results was not provided by the investigator. To obtain an odds ratio for publication bias and oral presentation bias, we introduced these variables in a univariate logistic regression model .SAS software was used for all analyses. We considered associations to be statistically significant when p values were less than 0.05.During the year 1997, 144 protocols were submitted for funding to the Greater Lyon regional scientific committee: 63 (44%) to the national funding scheme and 81 (56%) to the Lyon Hospital system's internal funding schemes. Two protocols were only "intention letter" with no complete protocol available and were therefore not included.A questionnaire was sent to the principal investigator of the 142 protocols included and an answer was received for 114 (80%): five investigators were not located, 18 did not answer and five others answered that they did not wish to complete the questionnaire. The association between funding status and response from the investigator was tested and did not reveal any response bias.Initial files were missing for five (4%) protocols. Half of the protocols involved human beings and had therefore to be evaluated by a research ethics committee as well.Table . The desn = 128; 90%) and one-half of them in a single centre.Most protocols were conducted in France only (The expected number of patients was provided for 100 (74%) protocols , not applicable for 21 (14%), no estimations were calculated for 16 (12%), and were missing for five (4%).An expected duration in months was provided for 96 (68%) protocols .Among the 142 protocols included, 54 were committee-funded (38%): 26 (42%) were submitted to the national funding scheme and 28 (35%) for HCL-internal applications. In 1997, the amount of funding after evaluation by the Lyon regional scientific committee was 3,139,000 euros . The funding granted ranged from 3,050 euros for a diagnostic strategy protocol to 274,400 euros for two regional studies, with a median of 34,200 euros.In the subset of protocols for which the investigator responded to the questionnaire (114/142), 46 (40%) were committee-funded.Simultaneous submissions for other grants or funding were declared by 38 investigators . At least two other submissions for funding were declared for 34% (13/38) and at least three for 13% (5/38). These submissions were addressed to private grant applications in 18% of cases (7/38), to other public schemes  including specific associations such as cancer associations. Another 11% (4/38) declared that they decided to use previous public funding already available from another study and 5% (2/38) did not provide any answer.Overall, simultaneous submissions for external funding were successful for 30 (79%), and for 6 out of 7 in case of submission to private companies. Only ten (9%) investigators declared that they obtained both committee-funding and external-funding Figure .n = 45), 26% that expenses were only partially covered (n = 30) and 10% declared that the funding was not sufficient at all (n = 11). Another 28 did not answer this question (25%).Among the 6 private fundings, 4 did not obtained committee-funding. Considering all funding sources globally, 39% of investigators considered that all their study expenses were covered  committee-funded and externally funded studies, and for 12/30 (40%) studies with no funding. When there was at least one private funding source involved, the completion rate was 67% and 54% otherwise (NS). Most studies were stopped due to subject recruitment difficulties (31%), technical  difficulties (31%) or funding difficulties (15%).p = 0.11).Publication rates varied from 37% for studies with both committee funding and external funding to 90% for studies with external funding only (When there was at least one private funding source involved the publication rate was 75% (3/4) and 66% (31/47) otherwise (NS).An answer from investigator was obtained for 75% (15/20) of randomised clinical trials. The initiation and the completion rates were respectively 93% and 100% but a publication was made only for 33% (n = 5).Investigators of initiated studies were asked to rate the importance of their study results. An answer was obtained for 74 of the 93 initiated studies. The study was still on-going for half of the non-responses. The overall mean ranking was 6.5/10 ,n = 11; 65%). Two investigators declared that they had no time to write (12%), one that the results were not interesting enough (6%) and three did not provide an explanation (17%).For publication bias analysis on 51 completed studies, we had to exclude four studies for which the rating of study results was not available Table . Among tThe odds ratio for publication of important results versus unimportant results was 1.58 (95% CI: 0.37–6.71).Oral presentations were made for 39 (83%) of the 47 documented and completed studies. When results were deemed unimportant, 2/10 (20%) resulted in neither publication nor oral presentation (Table n = 6), 15% internal reports only (n = 2), 15% an internal report and an abstract (n = 2), 15% an academic doctoral thesis only (n = 2) and 8% a doctoral thesis, an internal report and an abstract (n = 1).Fifty-one studies were completed (whether or not they were rated by the investigators) and 34 lead to scientific publication of which 50% resulted in multiple publications. Among the 17 studies not leading to scientific publication, there was no dissemination at all for four, and grey literature was produced for the remaining 13 studies. Grey literature comprised 46% abstracts only , confirming our main hypothesis. Moreover, we highlighted a tendency for publication bias as well as oral presentation bias, although this was not statistically significant for study power reason.Our study was the first to include protocols submitted on a comprehensive 1-year period to different types of public funding and was also the first to provide a follow-up of protocols that were not funded, whether they obtained other grants (e.g. private funding) or were conducted without any funding at all. It was based on only one regional scientific committee. However, this is the only regional committee not specialized and in charge of all health care facilities from general practice to highly specialised hospitals, i.e., cancer facilities, psychiatry and high-level tertiary teaching hospitals. The year 1997 was chosen to ensure enough time to conduct and complete a study and to publish the resultsp-value of the statistical test" was not used in the analysis since it was insufficiently documented by the investigators: in some cases, no statistical test could be performed, e.g. for descriptive non-comparative designs, but in other cases the data were truly missing. For these reasons, we relied on study results rated by the investigator over 5 . This threshold was chosen in order to ensure the minimal positive feeling of the investigator about his own study.Some variables definitions, such as study design, were a source of problems when we extracted data from the protocols. This was partly due to imprecise definitions generally used in the literature, but also to the sometimes poor quality of submitted protocols. The variable named "Self-rating by investigators is a subjective indicator, but it is very relevant since the choice to publish results is not always based on statistical significance but also on a subjective perception of the importance of the study results.The only article published on this topic provided information on protocols funded by the NIH . The resReports on the French national funding scheme have also been made public but were not published and restricted to funded protocols and to only one source of public funding -14.Our completion rate was 55% at 5 years, to be compared with 68% nine years later for the NIH funded protocols and at least 80% for the four French previously mentioned unpublished reports -14. HowePublication rates ,21-25, sIn our study, publication rate was higher for private fundings, which is not consistent with research on this subject ,21-26. TMost funded protocols were descriptive, reflecting the general situation in the 1990s when public funding was mostly aimed at the description of clinical or epidemiological situations. Today, one could find more funding for interventional/experimental protocols, and this could be checked in few years when the current ongoing protocols will have been completed and published.Since many protocols submitted for funding were initiated without any funding declared, does this mean that not all protocols submitted really needed funding? Or does this mean that health care facilities are unaware that they financially support and pay for biomedical research? To our understanding, both situations occur.More attention should be given to the follow-up of protocols submitted to grant application, this could be eased by the creation of local/national registries. Such registries could also help to describe the total number of applications and the total amount of money allocated per protocol, and should also be used to study the follow-up and the fate of submitted protocols, whether they are funded or not.The author(s) declare that they have no competing interest.ED coordinated the study, managed the data, did the statistical analysis, and drafted the manuscript.FC designed, submitted, and coordinated the study, interpreted data, and helped to draft the manuscript.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


I believe that their analysis is flawed, invalid, andnot appropriate as an input to policy decisions.If any MeHg exposure above the RfD reduced IQ, there would still be causefor concern. However, there is no evidence for IQ reductions even atexposures several times the RfD.Previous studies in the Seychelles Islands  and New a) the Seychellois are exposed to MeHg through ocean fish, similar to Americans, whereasthe Faroese are exposed through whale meat (b) the Seychellois are ethnically diverse, but the Faroese are homogeneouslyScandinavian (c) the Seychelles study used hair MeHg to measure exposure, and the Faroesstudy used cord blood. Hair MeHg has been calibrated with fetal brainlevels, but cord blood has not  the dinavian ; and c)  has not .Despite the advantages of the Seychelles study, They claimed that the New Zealand study reported IQ reductions, citing They claimed that the Seychelles study had only half the statistical powerof the Faroes study. The studies actually have similar power .They claimd the NRC concluded that MeHg reduces IQs even at exposures lowerthan the RfD. However, the NAS cautioned that the cohort studieswere incapable of assessing effects of exposures near the RfD, becausehardly any children had such low MeHg exposures .The weight of the evidence indicates that MeHg, even at exposures substantiallygreater than the highest U.S. levels, does not reduce children’sIQ. The evidence against IQ reductions is particularly strongfor MeHg exposures from fish.


Excessive consumption of carbonated soft drinks has been putatively linked to health effects including dental caries, obesity, and osteoporosis. An August 2006 study by the Centre for Science and Environment (CSE), a private research group in New Delhi, now reiterates another concern first raised three years ago: pesticide contamination.Analysis of Pesticide Residues in Soft Drinks, the authors report finding an average pesticide content of 11.85 ppb in 11 soft drink brands sold in India by The Coca-Cola Company and PepsiCo. An average of 5.37 ppb lindane, a possible human carcinogen, was detected in the 57 samples analyzed, as was an average of 4.71 ppb chlorpyrifos, a neurotoxicant. Heptachlor was found in 71% of the samples at an average concentration of 0.41 ppb. All these values surpass safe limits of 0.1 ppb for individual pesticides and 0.5 ppb for total pesticides in soft drinks that were proposed by the Bureau of Indian Standards in October 2005. The CSE had released similar findings in 2003.In Industry has contested the findings. Coca-Cola commissioned the analysis of more than 180 samples at Central Science Laboratory (CSL)—a British executive agency—and Vimta Labs in India, and the results showed less than 0.1 ppb of any pesticide. John Gilbert, research director for food at the CSL, says the CSE did not achieve sufficiently good matches between compounds in the samples and known pesticides to confirm identification. “If an identification is not confirmed unequivocally,” he says, “then going on and measuring something of uncertain identity makes no scientific sense.”In October a panel of India’s health ministry examined the report and rejected the findings, noting that confirmation of pesticide residues in soft drinks by gas chromatography–mass spectrometry (GC-MS) analysis was done for only three samples and did not represent the pesticide residue status of all the 57 test samples. In addition, the CSE improperly used U.S. EPA methodology meant to estimate pesticide concentrations in solids and liquids without validating it for soft drinks. The panel said data received from the CSE relating to “percentage of recovery of different analytes” differed from those in the August 2006 report, raising doubts about the reliability of the results.Sapna Johnson, lead author of the CSE report, defends the findings, saying they were quantified by two separate detectors. She thinks one reason for the difference in findings of the two studies may be that the CSE used GC-MS only for confirmation of pesticides, whereas the CSL used GC-MS for quantification as well. A CSE statement on the methodology reads, “Test products are fortified with target compounds and are analyzed to demonstrate precision and accuracy of the analyses. Replicate analyses of samples are conducted to confirm that the original results are reproducible. This is how the world tests products. And this is a scientifically valid and socially prudent method.”“Although the levels of pesticide residues reported [in the CSE] study are unlikely to cause immediate harm to consumers, chronic effects following long-term exposure may occur, particularly in susceptible populations such as children,” says Rolf U. Halden, cofounder of the Johns Hopkins Center for Water and Health. He says the CSE’s data may hint at a larger problem—pesticide-impacted drinking water in the region—and that confirmatory analyses using the best available analytical techniques should be the next step.


Polyvinyl chloride is used in production and manufacturing of many essential tools . Its production is impossible without the use of vinyl chloride monomer (VCM), which can cause liver damage in long-term. In this study we intend to assess the effects of mild to moderate long term exposure to VCM on liver and to assess the importance of liver enzyme measurements as a screening tool.In this study, liver enzyme levels of 52 workers were compared to 48 control workers using the T-test. The cases all worked in a PVC production unit in a petrochemical complex and the controls were randomly selected from office personnel of the same complex. A questionnaire was also filled in about information such as age, weight, work history, etc. in both groups.Mean comparisons for ALP and GGT using T-test showed statistically significant differences between the two groups. For AST, ALT and bilirubin  the mean was higher in the case group but this difference was not statistically significant.This study showed that mild exposure to VCM can cause mild liver cholestasis. So, using cholestasis assessment tests such as ALP and GGT should be considered in periodic assessment of liver function in PVC producing units. PVC application is increasing day by day. It is used in the production of plastic pipes, cable and wire coatings, floorings, photography films and also electronic industry -3. VCM iVCM is hepatotoxic and carcinogenic and can cause liver damages such as hepatic fibrosis, hepatic angiosarcoma, hepatocellular carcinoma, portal hypertension, etc.The mechanism of toxicity and carcinogenicity of VCM is hypertrophy and hyperplasia of hepatocytes and sinusuidal cells, sinusuidal dilatation and destruction, hepatocyte destruction, portal tract fibrosis and binding of chloroethylene oxide (VCM metabolite) to DNA and RNA -10.An important subject in health preservation of workers exposed to VCM is the early detection of is effects. Unfortunately minor liver damages can not be detected through routine screening tests such as aminotransferase measurement and needs more specific tests such as the measurement of serum bile acid levels, liver and spleen sonography and indocyanin clearance test .Several studies have been conducted on detection of early effects of VCM on workers with contradictory results. In addition, the role of confounding factors such as BMI, cigarette smoking and alcohol consumption were not taken into account in lots of these studies. Most of the studies were conducted on above the threshold levels of VCM, while regulatory organizations such as ACGIH reduce the threshold day by day . This shows that VCM can have detrimental effects on human health even below the threshold level.In many petrochemical complexes workers are exposed to below the threshold levels of VCM. Regarding the increase in the number of such complexes in our country and the few studies conducted about the problem, we designed this study.This is a cross-sectional analytic study. The study was conducted in a PVC production unit in a petrochemical complex in 2005. In this unit, PVC is produced from VCM by a rather long process.The case group was 52 male workers who worked in the PVC production unit and the control group was 48 male office workers from the same complex. Having at least 2 years of working experience was the inclusion criteria.Environmental monitoring showed a concentration of 0.7 ppm for VCM. Sampling tube method was used and its analysis was performed by Gas chromatography.Environmental sampling was done in several consecutive days and during the whole working days, i.e. 8 hours. Maximum exposure level was 0.85 ppm and the mean exposure level was 0.8 ppm in 8 working hours. Previous years' samplings also showed a maximum environmental exposure level of 0.8 ppm.So the cases were workers who had long time exposure to less than allowable levels (1 ppm) of VCM.Demographic data including age, weight height, work experience, alcohol consumption, tobacco smoking, past medical history, drug history, performing heavy exercises, work history including any changes of the job and second job, history of surgery and history of blood transfusion were all gathered through a questionnaire.Workers with systemic diseases  or active hepatic diseases , those consuming drugs that could affect liver enzyme levels , workers who had undergone coronary bypass surgery or had received blood in the past 3 months and workers who performed heavy exercises were excluded. HBs-Ag, HBc-Ab and HCV-Ab tests were used to rule out active infectious diseases. Blood samples were also used to assess the following 6 biochemical markers:1 – Liver transaminases  which show hepatic necrosis2 – Liver cholestasis tests 3 – Liver clearance tests Lab tests along with the questionnaire data were analyzed with SPSS-10 using the T-test and linear regression analysis.All samples were Iranian males so there was no difference between the two groups. In addition, no statistically significant difference was found between the groups for age, job experience and BMI. Table Viral hepatic disease tests were performed for all cases and controls. 11 workers (6 cases and 5 controls) were positive and thus excluded. Alcohol consumption was considered positive even if consumed occasionally. 7% of cases and 8% of controls consumed alcohol. 19 cases (36.5%) and 18 controls (37.5%) smoked tobacco. No statistically significant difference was found between the two groups for alcohol consumption and tobacco smoking and so these two can not be confounding.Mean comparison between two groups and the related P-values are shown in Table For further assessment of the relationship between liver enzyme levels and VCM exposure and for proper control of all confounders we used the linear regression analysis. The results are shown in Tables Cases were divided to 3 subgroups regarding their job experience. There was no statistically significant difference for BMI, age and the number of alcoholics and smokers among these 3 subgroups; but for GGT and ALP the differences of means were significant.Liver enzyme levels were compared in each group of cases and controls separately regarding the consumption of alcohol and smoking. Liver enzyme levels were not significantly different between smokers and nonsmokers, and alcoholics and non-alcoholics, in each group.In fact, the liver enzyme levels were compared between 4 cases who consumed alcohol and 48 cases that didn't. There was no statistically significant difference in any of the liver enzyme levels between these 2 subgroups. The liver enzyme levels were also compared between 4 controls who consumed alcohol and 44 controls that didn't. There was no statistically significant difference in any of the liver enzyme levels between these 2 subgroups. The same applies for smoking and no statistically significant difference was found in any of the 2 groups of cases or controls. The only statistically significant difference was between GGT and ALP P < 0.05) .We omitted the effect of confounding factors such as age, sex, weight, alcohol consumption, tobacco smoking, job experience and infectious hepatic diseases (hepatitis B & C) which was not done systematically in previous studies.In workers with positive viral hepatic disease tests, mean comparison showed higher ALT in the case group (P < 0.05). This shows that VCM exposure can aggravate viral hepatic disorders and further increase liver enzymes. Between these two groups there was no statistically significant difference for factors such as age, tobacco smoking, alcohol consumption and job experience, which is in accordance with previous studies.Maybe the increase of sample size would lead to significant difference in the level of other liver enzymes between the 2 groups. But we must take into consideration that in chronic liver diseases not led to end stages, such as fibrosis, ALT is higher than AST . The meaA statistically significant relation was seen between ALP and GGT increase and exposure to VCM which can show mild liver injury (cholestasis) due to VCM. Although such relation was not seen for other liver enzymes but their absolute values were higher in case group.Previous studies have shown increase in liver transaminases in workers exposed to high levels of VCM ,14-16. OThere was a statistically significant relation between job experience in PVC production unit and increase in GGT and ALP which draws our attention to the importance of periodic exams and screening tests especially among those with higher job experiences.Previous studies have shown contradictory results in the increase of liver enzyme levels in workers exposed to VCM ,17,18.In this study a statistically significant relation was found between ALP and GGT and VCM exposure which was in accordance with some previous studies . Some stFor example it has been suggested that in the screening of VCM exposed workers at first ALP and indocyanine clearance test should be done to reduce the number of false positives and then GGT must be used to reduce the number of false negatives .Assessment and discussion about biologic monitoring and early detection of VCM effects on liver is complicated. The most important urine metabolite of VCM is thiodiglycolic acid but it is of little value in biologic monitoring due to its non-specificity and that its concentration is not directly related to the concentration of VCM .Different organizations have suggested different tests and biologic monitoring to detect the early effects of VCM on body (such as PFT and liver enzymes). In several studies the measurement of serum bile acids in workers exposed to VCM has been proposed as the best and most sensitive test for screening of VCM liver effects -23.In some other studies hepatic and splenic sonography or platelet level was used to assess this effect and the VWF was used for the early detection of angiosarcoma ,11,24-26On the whole, most studies have approved the suitability of measuring serum bile acids in the screening of VCM exposed workers. Unfortunately due to financial problems we didn't measure bile acid levels and didn't perform hepatic and splenic sonography.It is important to say that workers at this PVC production unit have simultaneous exposure to other materials such as ethylene gas, but the measured levels of these materials were zero or negligible and they can not affect liver enzyme levels.Liver tests performed in this study for cases were usually in the normal range and our findings in fact show the changes of these tests in working population. The clinical importance of these subtle changes of liver function in working populations is not clear and further longitudinal studies are necessary. Considering the results of this study, performing biochemical liver tests is recommended for screening and surveillance of workers exposed to low level VCM.Naturally, liver transaminases and especially ALP and GGT are available, noninvasive and affordable tests for screening of workers exposed to VCM to detect early liver dysfunction.


Differences on whether results were nominally statistically significant (p < 0.05) or not with the two designs were common (opposite classification rates 14% and 17%); this reflected largely differences in power. Conclusions were largely similar in diverse subgroup analyses. Unrelated case-control and family-based designs give overall similar estimates of association. We cannot rule out rare large biases or common small biases.The most simple and commonly used approach for genetic associations is the case-control study design of unrelated people. This design is susceptible to population stratification. This problem is obviated in family-based studies, but it is usually difficult to accumulate large enough samples of well-characterized families. We addressed empirically whether the two designs give similar estimates of association in 93 investigations where both unrelated case-control and family-based designs had been employed. Estimated odds ratios differed beyond chance between the two designs in only four instances (4%). The summary relative odds ratio (ROR) (the ratio of odds ratios obtained from unrelated case-control and family-based studies) was close to unity . There was no heterogeneity in the ROR across studies (amount of heterogeneity beyond chance I Different types of designs are used for the assessment of genetic associations for complex diseases. Case-control studies of unrelated people and family-based designs are the most widely used. Each has its advantages and disadvantages. This paper compares the estimates of the two types of design using a meta-analytic approach, i.e. a systematic selection of data and quantitative synthesis of results across many studies. The authors examined 93 associations where both unrelated case-control and family-based designs had been employed. Both designs gave overall similar estimates of association and the conclusions were very similar in subgroup analyses that considered various design features that might affect in theory the degree of agreement between the two designs. No heterogeneity between studies was observed. Hence, there was no consistent pattern of over-estimation or under-estimation of the probed association with one or the other design. However, one cannot exclude the possibility that rare large differences or common small differences may occur between the two designs. Genetic associations for complex diseases may be probed either with case-control studies of unrelated people or with family-based designs. Both designs have advantages and disadvantages. Studies of cases and unrelated controls are the most commonly used approach; sufficiently large study populations can be readily assembled without the need to enroll also family members of the recruited participants. However, a disadvantage of this approach is that confounding due to unaccounted population admixture remains a possible threat to the validity of the obtained results –3. On thThis problem of most appropriate versus most feasible study design may become even more pressing in the era of whole-genome strategies. Modest confounding due to population stratification may create unacceptable noise in the search for significant associations across the genome. Conversely, sample sizes need to be large enough to avoid type I error both in the screening process, as well as in the validation of what are likely to be modest genetic effects . Of courStrong views have been expressed on the relative merits of and preference for family-based versus unrelated controls designs ,5. A numWe analyzed a total of 93 eligible comparisons between family-based and unrelated case-control designs where both designs had been used by the same investigators to address the same postulated gene-disease association . The medn = 10, unrelated case-control n = 15) and in 15 comparisons the results for one design had been selected for presentation based on their own statistical significance  or the statistical significance of the results obtained with the other design . Finally, three studies violated Hardy-Weinberg equilibrium (HWE) assumption (exact test p < 0.05 for the distribution of genotypes in the unrelated controls), and another two studies had absolute fixation coefficients exceeding 0.03 in the unrelated controls, even though this deviation from HWE was not formally significant.In these 93 comparisons, the populations analyzed in the family-based and unrelated case-control studies overlapped  in 47 and in 16 comparisons it was clearly stated that this was the first published investigation addressing the specific gene-disease association. Moreover, in 25 comparisons, it was clearly stated that one design was applied first (family-based F) and the unrelated case-control design (ORU) for the minor versus major allele in order to obtain a summary OR for the strength of each probed association. When this summary OR was <1, we inversed the allele contrast, so that all summary ORs would be ≥1. We then estimated the ratio of the ORU over ORF. This relative odds ratio (ROR) reflects the difference in the effect size between the two designs. It is expected to be 1 when the two estimates agree, >1 when the family-based design gives a smaller estimate of association than the unrelated case-control design, and <1 when the opposite occurs. When the 95% confidence interval (CI) for ROR does not contain 1, then the difference between ORF and ORU is beyond chance at the 0.05 level of significance.We combined the odds ratios (OR) in the family-based design (ORThe difference between these two estimates was significant only in four (4%) probed associations . An eval2 = 0). Several studies had wide 95% CIs. Such studies would tend to increase the degrees of freedom and thus may hide some between-study heterogeneity. Analyses strictly limited to the exact same “ethnic” groups in both types of design yielded an identical summary ROR estimate of 0.96 , and again there was no heterogeneity between the 93 ROR estimates.Despite these isolated significant discrepancies, the summary ROR estimate across all 93 associations was 0.96  showing high overall agreement. There was no heterogeneity across the ROR estimates across the 93 probed associations beyond what would be expected by chance alone , the two designs estimated effects in the opposite direction . In 64 comparisons (69%), the relative risk increase (OR−1) of the unrelated case-control design was less than half or more than double compared to the family-based design.Although the differences in the results of the two designs were rarely nominally significant, the exact point estimates of the ORp = 0.05 level of significance. We estimated the probability that the unrelated case-control design gives a significant result and family-based gives a non-significant result, and the probability of the inverse scenario. These probabilities of opposite classification were 14%  and 17% , respectively.We further examined the concordance in the level of nominal statistical significance, i.e. whether both designs found significant or non-significant results at the In theory, the design, conduct, and reporting of the two types of studies may influence the degree to which they agree. Therefore, we examined whether the results obtained with the two types of designs were more or less likely to differ systematically when there were overlapping populations in the two designs; when one design had clearly been applied first; when studies claimed to be the first article on the probed association; when results were presented based on the statistical significance of their findings or the findings of the other design; when there was violation of or major deviation from HWE in the unrelated controls; or when we had selected only one association among many presented in an article; when the sample size of unrelated case-controls contained more that 1,000 alleles; and when TDT studies used families with multiple affected sibs or not. There was no suggestion that these characteristics influenced systematically the overall summary ROR in the respective subgroup analyses .The ROR estimate is expected to fluctuate more around 1, when the data obtained by either design are more limited. Unrelated case-control and family based-designs gave overall similar estimates of association. This means that there was no consistent pattern of over-estimation or under-estimation of the probed association with one or the other design. One might wonder whether family-based studies give much larger estimates than unrelated case-control studies in some occasions, and much smaller estimates in other occasion, but on average these differences cancel out. However, the absence of heterogeneity that we observed does not support this claim. Our analyses were more consistent with the interpretation that typically there is agreement in the estimates obtained by the two designs. Our findings should be interpreted cautiously given the small sample sizes in several of these studies.Considerable differences in the OR point estimates between the two types of design are common and they reflect mostly the uncertainty that accompanies the estimates of small studies. If inferences are made categorically for the presence or not of formal statistical significance, discrepancies between the two study designs are common and the same applies, when inferences are made based on the magnitude of the point estimates of the genetic effects, if the uncertainty thereof, is ignored. Power deficit is a major concern for small studies and this is a greater concern for family-based designs , where gWe should acknowledge that with large studies of many thousands of cases and controls, even modest stratification problems may yield spuriously formally statistically significant results. Since most genome-wide approaches use formal significance rather than effect sizes to select genes for further testing, modest errors could create considerable problems. Therefore our findings should not be interpreted as evidence that unrelated case-control studies may be designed without careful standards and proper attention regarding the recruitment of the study population. Furthermore, although not common, confounding of substantial magnitude may occur sometimes even within so-called “racial” or even “ethnic” descent groups. This would not be captured by our analyses. With such confounding, even careful matching at the ethnicity level would not suffice. Our analysis suggests that large confounding effects are not common, but we cannot rule out rare large confounding effects or common small confounding effects. The latter could still bias well-powered studies using small alpha levels, a situation that is increasingly demanded in current genome wide association studies.We should also caution that some researchers may preferentially report associations that show similar and confirmatory results with the two designs. However, our protocol excluded upfront studies where such selection was stated to have been applied. We cannot exclude the possibility that an investigator may be less likely to publish studies that found very different results in the two types of design, whereas two independent investigators may not have the same problem. Unfortunately, by default, unpublished data cannot be retrieved to see how and to what extent they might influence our conclusions. Selection choices may not be stated at all in the published reports. This publication bias would tend to increase concordance in the examined sample of investigations. However, publication bias may also decrease the overall observed concordance, if some studies with highly concordant, but “negative” results with both designs are not published .We also performed subgroup analyses considering a wide range of other more subtle selection features that may in theory affect the extent of agreement between the two designs. Reassuringly, the two designs gave consistent results in all subgroup analyses. Finally, it is possible that discrepancies may be greater when the two designs have been applied by different teams of investigators. However, this concern would apply also to studies with the same design performed by different investigators.Overall, our analysis suggests that despite the dangers of population stratification ,30, on aAllowing for these caveats, both types of design can yield useful and complementary information. Methods to improve efficiency of design and combination of data from both designs are welcome –16 and sWe considered published studies that examined genetic associations for the same polymorphisms using both family-based and unrelated case-control designs in the same article. Eligible studies were those where we could extract or compute the allele-based log-odds ratio and its variance for at least one association with a phenotype that had been probed with both designs. For consistency, we focused on biallelic markers and binary phenotypes. We excluded studies where data had been obtained for many genetic markers and/or phenotypes, but the results had been selected for presentation based on the concordance of the two designs. We excluded studies considering microsatellites, non-biallelic markers and continuous traits; and non-English language articles.We searched PubMed using the terms “transmission disequilibrium test,” “TDT,” “STDT,” “PDT,” “Sib-TDT,” “ETDT,” “RC-TDT,” or “family based,” combined with the terms “unrelated” or “case-control.” The search was last updated on 31 July 2005, and 2,151 items were retrieved and screened for eligibility: 1,993 could be excluded from inspecting the title and abstract, and 158 were examined in full-text for eligibility. A total of 84 articles were eligible; one gene-phenotype association was systematically selected for each of them, except for nine articles where two very different phenotypes were addressed.For eligible reports, when data were available for the comparison of the two designs for two or more polymorphisms, we selected the one that was first mentioned in the text. We set this rule to avoid subjectivity in the selection of the polymorphism to be analyzed and to avoid having many correlated data stemming from the same compared study groups. When two or more entirely different phenotypes were examined in the same article, we considered each one of them separately.U was estimated as the ratio of the products of the two diagonals and the variance of its natural logarithm was estimated by the sum of the inverse of the four cells of the 2 × 2 table. We also recorded the number of transmitted (T) and non-transmitted (NT) alleles in the respective family-based design. The ORF was estimated as the ratio of T over NT and the variance of its natural logarithm was estimated by the sum 1/T + 1/NT. For studies where this information was not directly available, we extracted information in order to calculate ORF and ORU from other presented data . OR estimates were first derived for the minor versus major allele.We recorded the numbers in the 2 × 2 table of each analyzable unrelated case-control design for an allele-based analysis (only this would be feasible for the family-based designs). The ORIn 18 studies that had used data from people of different “racial/ethnic” descent, the OR was estimated first in each “racial/ethnic” subgroup and data were then combined for each design across the available “racial/ethnic” subgroups using stratified analyses (Mantel-Haenszel method) to obtain a single OR estimate per design.a as the risk allele, the odds ratio of disease risk can be estimated by USE =T is the number of transmitted high risk alleles and NT the umber of non-trasmitted alleles the odds ratio of disease risk can be estimated by FSE =F and ORU for the minor allele for each association in order to obtain a summary OR. This summary OR was estimated as the weighted sum of the ORF and ORU using the natural logarithms of the two ORs and their inverse variances as weights. For consistency, all summary ORs were then coined to be ≥1.00. This means that when the summary OR was <1 using the minor versus major allele contrast, then we used the major versus minor allele contrast instead for both the unrelated case-control and family-based data. The purpose of coining the summary OR to be ≥1.00 was to make this metric show consistently the strength of the association, regardless of whether the minor allele might be protective or conferring susceptibility. This allowed to evaluate whether unrelated case-controls consistently suggests a less strong gene-outcome association compared to family-based designs or vice versa.Suppose the frequency of alleles in cases and controls in the unrelated case-control study are U and ORF was calculated to obtain the ROR [F and ORU.The ratio of the OR the ROR  for each2 statistic which is calculated by I2 = 100%(Q−df)/Q, where Q is the Cochran's heterogeneity statistic and df the degrees of freedom [2 ranges between 0% and 100% and estimates the amount of heterogeneity that is beyond chance. Heterogeneity is considered low, moderate, large and very large for I2 values of 1%–24%, 25%–49%, 50%–74%, and 75% or higher, respectively. In the absence of any heterogeneity, fixed and random effects estimates coincide.The natural logarithms of the ROR estimates were combined to obtain the summary ROR ,41 using freedom . I2 rangp = 0.05 level of significance.The main analysis examined whether the summary ROR is different from 1. The summary ROR provides an estimate of the average deviation between the odds ratios in the two study designs, i.e. whether studies with unrelated case-controls provide consistently stronger (ROR >1) or consistently weaker (ROR <1) associations than family-based studies. In addition, we evaluated whether, for specific studies, the 95% CIs of the ROR excluded 1, meaning that the results of the two types of design differed beyond chance at F and ORU were in the same direction (both above 1 or both below 1) and whether the magnitude of the relative risk increase (OR−1) of the unrelated case-control design was less than half or more than double compared to the family-based design. Differences in direction are important for inferences, but estimates in the opposite direction may still be very close and not incompatible with each other. The OR−1 comparison focuses on the magnitude of the difference, but does not address its statistical significance. Finally, we evaluated differences in the level of formal statistical significance, i.e. what is the probability that one design might give non-significant results, when the other design gives significant results at the 0.05 level of significance. These complementary approaches have been previously used in the comparison of effect sizes from different studies in the medical sciences [Identifying a statistically significant difference does not depend only on the magnitude of the difference, but also on the power of the compared study designs, since very small studies may give very different point estimates, but with very large uncertainty. Therefore, we also evaluated whether the ORsciences ,45,46.AAP and aaP are the proportions of the homozygotes and Ap and ap are the proportions of the corresponding alleles. The coefficient takes values from 1 to −1 depending on the extent of excess or deficit of homozygotes compared with the proportions expected under the Hardy-Weinberg law [We recorded whether one design had clearly been applied first and the other was performed at a second stage. We also recorded various characteristics that may influence the observed concordance between the two types of designs. We noted whether there was any stated overlap between populations for each design and whether this was claimed to be the first article on this association. In theory, concordance may be larger when there is overlap in populations and first studies may also be biased towards presenting concordant results. Moreover, we recorded whether results were generated or selected for presentation based on their statistical significance, or the significance of the results of the other design; in theory, concordance may then be smaller due to regression to the mean and the winner's curse phenomenon . We recoberg law . We finaWe examined with a linear regression, the dependence of the absolute value of the natural logarithm of the ROR on the standard error of the summary ROR (square root of the variance). The regression shows the magnitude of the absolute deviation in the OR estimates obtained with the two designs as a function of the amount of data. Although there is a certain amount of structural correlation between ROR and its standard error, this would not affect considerably the slope and 95% CIs for the regression.Dataset S1(44 KB XLS)Click here for additional data file.Text S1(47 KB DOC)Click here for additional data file.


Pre-eclampsia is thought to have an important genetic component. Recently, pre-eclampsia has been associated in some studies with carriage of a common eNOS gene Glu298Asp polymorphism, a variant that leads to the replacement of glutamic acid by aspartic acid at codon 298.Healthy women with singleton pregnancies were recruited from 7 district general hospitals in London, UK. Women at high risk of pre-eclampsia were screened by uterine artery Doppler velocimetry at 22–24 weeks of gestation and maternal blood was obtained to genotype the eNOS Glu298Asp polymorphism. Odds ratios (OR) and 95%CI, using logistic regression methods, were obtained to evaluate the association between the Glu298Asp polymorphism and pre-eclampsia. A meta-analysis was then undertaken of all published studies up to November 2005 examining the association of eNOS Glu298Asp genotype and pre-eclampsia.89 women with pre-eclampsia and 349 controls were included in the new study. The Glu298Asp polymorphism in a recessive model was not significantly associated with pre-eclampsia . In the meta-analysis, under a recessive genetic model (1129 cases & 2384 controls) women homozygous for the Asp298 allele were not at significantly increased risk of pre-eclampsia . A dominant model (1334 cases & 2894 controls) was associated with no increase of risk of pre-eclampsia for women carriers of the Asp298 allele .From the data currently available, the eNOS Glu298Asp polymorphism is not associated with a significant increased risk of pre-eclampsia. However, published studies have been underpowered, much larger studies are needed to confirm or refute a realistic genotypic risk of disease, but which might contribute to many cases of pre-eclampsia in the population. Normal pregnancy is associated with a substantial change in maternal cardiovascular haemodynamics. Despite an increase in blood volume and cardiac output, blood pressure falls in the first half of pregnancy, as a result of systemic arteriolar vasodilatation, before returning to pre-pregnancy levels towards term . EnhancePre-eclampsia is thought to have an important genetic component ,7. SeverWe have undertaken a new, case-control study of the eNOS Glu298Asp polymorphism and pre-eclampsia and combined our results with all available studies evaluating such an association in a meta-analysis.Maternal genotyping was performed in women who underwent screening by uterine artery Doppler velocimetry at 22–24 weeks of gestation. This sub-study was part of a multi-centre randomised controlled trial that evaluated aspirin vs placebo as an intervention for the prevention of pre-eclampsia and the rationale and design of the study has been described in detail elsewhere . Women wth centile (1.6) and 650 women with a mean PI <1.6, that consented to give a blood sample were followed up to delivery. Of the 1273 women, DNA was available for analysis in 89 women of the 121 who developed pre-eclampsia. Baseline characteristics of the 32 women that were not included in the DNA analysis for logistical reasons were not significantly different from the 89 cases included in the present report (Data not shown). Of the 1152 women who did not develop pre-eclampsia, 349 women (ratio 1:4) were selected as controls using frequency matching by maternal age and ethnic background.For the present study 623 women who had a mean pulsatility index (PI) of the uterine arteries above the 95Pre-eclampsia was defined according to the International Society for the Study of Hypertension in Pregnancy . It requDNA was extracted by means of the QIAamp blood minikit, the 894 G/T polymorphism in exon 7, which predicts a Glu298Asp amino acid substitution in the mature protein, was genotyped by polymerase chain reaction (PCR) with primer pairs 5'-CCCCTCCATCCCACCCAGTCAATCC-3' and 5'-AGGAAACGGTCGCTTCGACGTGCTG-3' and allele-specific restriction enzyme digestion. PCR was performed for 35 cycles in a volume of 30 μL. Denaturation was at 95°C, annealing at 63°C and a final extension at 72°C, all for 45 seconds. Ten microliters of each PCR product (151bp) was then subjected to restriction digestion with 2U Dpn II, which cuts only in the presence of T-allele at position 894 (corresponding to Asp298). Digested samples were resolved by electrophoresis. Genotyping was conducted in a blinded manner to the clinical status of the subjects included in the study.t, χ2, or Mann-Whitney tests were used as appropriate. Tests for Hardy-Weinberg equilibrium were performed by χ2 analysis.For the case-control study medians, proportions and their corresponding interquartile ranges (IQR) were used for descriptive purposes. To evaluate the presence of differences between groups unpaired Student's As an a priori hypothesis, the association between the eNOS Glu298Asp polymorphism and pre-eclampsia was evaluated under a recessive model of inheritance, based on previous results from the eNOS genotype on ischaemic heart disease risk . HoweverQ test : 1.61), Asians 1.41% (SE: 0.95), Hispanics 2.55% (SE: 1.53) and Afro-Caribbean 1.11% (SE: 0.71). All control samples from the studies included on which genotype frequency was available were in Hardy-Weinberg equilibrium (P>0.05) ,22,23,312 = 27.2%, P value for Heterogeneity [PHet] = 0.20). The distribution of the ORs from individual studies in relation to their respective standard deviations (the funnel plot) was symmetric and the Egger test suggested a low probability of small-study bias (p = 0.78).After combining our results in a meta-analysis, the summary OR was 1.28  for women homozygous for the Asp298 allele compared with carriers of Glu298 allele  with this model, partially explained by ethnicity , while disease severity  and use of blinding of genotyping staff to the case-control status  did not account for much of the heterogeneity  . The funnel plot was symmetric and the Egger test suggested a low probability of small-study bias (p = 0.20).The main finding of the case-control study and the meta-analysis was that on evidence obtained to date, women homozygous for the Asp298 allele were not associated with a significantly increased risk of pre-eclampsia when compared with carriers of the Glu298 allele . Similar results were obtained using other genetic models of inheritance. However, an exploratory analysis that must be treated with appropriate caution suggested that the effect of this candidate-gene might be different according to ethnic background.The aetiology of pre-eclampsia remains complex. Identification of candidate-genes for pre-eclampsia could aid substantially in the understanding of this important public health problem and provide clues for the prevention and treatment. It might also have wide relevance since women with a history of pre-eclampsia are at increased risk of cardiovascular disease in later life . EndothePre-eclampsia has an inherited component and it is likely that many genes are involved . PreviouThe lack of evidence of increased pre-eclampsia risk for the eNOS Glu298Asp variant may be explained by: (i) the absence of a biological effect, (ii) failure to detect a small but important increase in susceptibility to pre-eclampsia because of inadequate statistical power or (iii) the presence of real genetic heterogeneity according to ethnic background . It has been recently suggested, that for complex diseases with a low sibling recurrence risk such as pre-eclampsia, several candidate-genes with a small to moderate effect, rather than a few candidate-genes with a high effect on incidence of the disease, is the more likely model to explain such genetic susceptibility . This sude novo" for a single centre, an alternative approach, which has been suggested in the cardiovascular arena, involves the recruitment and genotyping of smaller numbers of patients and controls from many centres according to uniform selection criteria and outcome definitions, and submission of the data to a common web-based repository for on line continuously up-dated meta-analysis. Such an approach would also help to minimise publication bias, which is a potential concern with meta-analysis of published genetic association studies. However, an alternative approach would be to conduct a very large multi-centre case-control study of several thousand of cases, which should be less prone to bias, in order to confirm or refute the role of candidate gene-variants with small to moderate effects in pre-eclampsia [In order to increase the statistical power, we therefore combined all available studies in a meta-analysis. In addition to our study, eleven other studies were identified, but only nine studies providing 3513 women (1129 cases & 2384 controls) under a genetic recessive model were included in our main comparison. A very large case-control study or a meta-analysis of smaller studies, including about 5600 Caucasian women would be needed to have an 80% of power at a significance level of 5% to detect an OR of 1.3 under a recessive model of inheritance. A sample size approximately three times higher would be needed to detect the same OR among non-Caucasian populations because of the lower allele frequency of Asp298. Thus, a small to moderate effect on incidence of pre-eclampsia for the Glu298Asp cannot be totally excluded on current evidence. Since recruitment of data sets of the required size may be difficult "In summary, currently available data from genetic association studies do not provide positive evidence for a role of the eNOS Glu298Asp polymorphism in pre-eclampsia. Very large studies, in both Caucasians and non-Caucasian subjects, are required before a role for this gene can be excluded, and the development of very large datasets is an important research priority for genetic studies in this area .The author(s) declare they have no competing interest.CY recruited study subjects, abstracted medical records, entered data and drafted the manuscript.JP contributed to the study design, performed statistical analysis, interpretation of the results and preparation of the manuscript.MS contributed to the study design and interpretation of the results.MKS performed all the PCR tests.KN provided clinical authorisation and support for the study.AH conceived methodology and supervision of PCR tests, conceived and co-ordinated the study, reviewed the statistical analyses and preparation of the manuscript.All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The independent prognostic impact, as well as the possible causal role, of hyperhomocysteinemia (HHcy) in coronary artery disease (CAD) is controversial. No previous study specifically has addressed the relationship between HHcy and mortality after coronary artery bypass grafting (CABG) surgery. The aim of this study is to evaluate the prognostic impact of HHcy after CABG surgery.th percentile (25.2 µmol/L) of the population's distribution, was significantly associated to total and cardiovascular mortality . The PML test had no prognostic value. After multiple adjustment for other univariate predictors by Cox regression, including statin therapy , high-sensitivity C Reactive Protein (hs-CRP) levels, and all known major genetic (MTHFR 677C→T polymorphism) and non-genetic  tHcy determinants, HHcy remained an independent prognostic factor for mortality .We prospectively followed 350 patients who underwent elective CABG between May 1996 and May 1999. At baseline, fasting total homocysteine (tHcy) levels were measured in all participants, and a post-methionine loading (PML) test was performed in 77.7% of them (n = 272). After a median follow-up of 58 months, 33 patients (9.4%) had died, 25 because of cardiovascular events. HHcy, defined by levels higher than the 90HHcy is an important prognostic marker after CABG, independent of modern drug therapy and biomarkers. Details about the enrolment criteria have been described elsewhere Subjects were followed until death or May 2004. Study subjects status was determined by search of the national population register, and by an ambulatory or telephone survey. Ascertainment of mortality status was complete (100 percent). The date of death was obtained from the National Population Register. The causes of death were obtained from death certificates kept at the Italian Institute of Statistics (ISTAT). Death from cardiovascular causes was defined as death caused by CAD, heart failure, peripheral vascular disease, or cerebrovascular disease. Three subjects died peri-operatively , so that they were excluded from the survival analysis.MTHFR 677C→T polymorphism were determined as previously described Samples of venous blood were drawn from each subject after an overnight fast. Serum lipids, other routine biochemical parameters including creatinine, as well as plasma folate, vitamin B12, and the 2 test. Correlations between quantitative variables were assessed using Pearson's correlation test. Significant associations between tHcy levels and other continuous or categorical variables, were assessed by multiple regression models estimating R2 and standardized β-coefficients. Categorical variables were used after transformation into binary variables . Hyperhomocysteinemia (HHcy) was defined by tHcy levels higher than the 90th percentile of population's distribution, i.e. ≥25.2 µmol/l. Survival was assessed by the Kaplan-Meier method (log-rank statistic) and Cox regression. Kaplan-Meier methods were used for survival plots. Multivariate Cox proportional hazards analyses were performed considering sex, age, other tHcy determinants , and all predictors of mortality at univariate analyses, including drug therapy at discharge. Final models were obtained by a backward stepwise logistic regression approach, with P = 0.10 as the critical value for entering/excluding variables in the model. Hazard ratios (HRs) and 95% CIs are reported with two-tailed probability values. A P value less than 0.05 was used to indicate statistical significance.All the calculations were performed with SSPS 13.0 statistical package . Distributions of continuous variables in groups were expressed as means±standard deviation. Logarithmic transformation was performed on all skewed variables, including tHcy, folate, vitamin B12, vitamin B6 and hs-CRP. Hence, geometric means with 95% confidence intervals (CIs) are given for these variables. Quantitative data were assessed using the Student's t-test, or by ANOVA with Tukey's post-hoc test when appropriate. Associations between qualitative variables were analyzed with the χT allele of MTHFR were increased. The PML increase of tHcy over basal levels (Δ PML tHcy) was similar in the two groups.r = 0.143, P = 0.008), and negatively with levels of folate , vitamin B12 , and estimated GFR . The relations with vitamin B6 levels  and with age  were not significant. As expected, MTHFR 677TT subjects had increased mean levels of tHcy  as compared to either 677CT  or 677CC  subjects (P<0.001 by ANOVA with Tukey post-hoc test). A multiple regression model including all the above mentioned variables showed folate, vitamin B12, GFR, and MTHFR TT homozygosity, as independent predictors of tHcy levels (Fasting tHcy levels correlated positively with hs-CRP (y levels .P<0.05 by χ2-test in both cases). In HHcy patients, mortality was nearly twice (20%) as much than in the others, and all deaths in HHcy group were from cardiovascular causes (P = 0.023), and 3.67, 95% CIs 1.53 to 8.76 (P = 0.004), respectively. P = 0.018; and 9.76, P = 0.002, respectively). Since literature often consider a tHcy level of 15 µmol/l as the upper limit beyond which the cardiovascular risk may start to increase n = 139) or without (n = 176) baseline tHcy levels≥15 µmol/l. However, with respect to either total or cardiovascular (After a median follow-up of 58 months (range 3 to 87), 33 patients (9.4%) had died. Twenty-five deaths  were because of cardiovascular events. The 8 deaths due to noncardiovascular causes were mainly due to cancer (6), one patient died from pneumonia, and one from undetermined cause . The prevalence of HHcy was significantly higher in total and cardiovascular mortality groups  as compared to survivors . Other predictive factors in univariate analyses were hs-CRP levels, age, LVEF, and β-blocker therapy at discharge. Details about the relative HRs with 95% CIs are shown in MTHFR 677TT genotype was not significantly associated to an increased mortality, while we observed a non significant trend toward the contrary: for example, the prevalence of TT genotype was only 6.3% in the total mortality group, 8.3% in the group died for cardiovascular causes, as compared to 17.5% in survivors (P = NS). Moreover, none of the other determinants of tHcy in the multiple regression model  showed a predictive value at a P level<0.10).Statin therapy at discharge was the most powerful prognostic indicator . All univariate predictors of total or cardiovascular mortality with P<0.10  were initially entered. Statin therapy was confirmed as the most significant protective predictor. Noteworthy, in multivariate models hs-CRP levels were no longer independent predictors of either total or cardiovascular mortality. Additional stepwise models revealed that this loss of statistical significance was due to adjustment for statin therapy (data not shown). On the contrary, HHcy remained a significant independent predictor even after adjustment for all potential confounders, including major tHcy determinants such as GFR, folate, vitamin B12, and MTHFR 677 TT genotype  and non-genetic  determinants of tHcy, as well as modern drug therapy. To put our results into perspective from a clinical standpoint, we propose the following considerations.In this prospective study, hyperhomocysteinemia (>25.2 µmol/l) was an important and independent prognostic factor of mortality after CABG. A particular effort was made to take into account all possible confounders, including the known major genetic , and details on current drug therapy after CABG. The predictive value of tHcy remained statistically significant even after correction for a major determinant of mortality such as statin therapy.Most of the prospective studies published so far on tHcy focused on subjects without evident cardiovascular disease at entry, yielding variable results The PML test has been suggested as an useful tool to uncover subtle abnormalities of the homocysteine metabolic pathway MTHFR 677TT genotype was not at all a predictor of mortality, even in univariate analysis. Meta-analyses have quantified into ∼2.5 µmol/l the usual mean increase in tHcy levels of TT versus CC subjects at a population level MTHFR 677C→T polymorphism increased tHcy levels only in individuals with low folate status, while it was neutral in individuals with adequate folate levels MTHFR genotypes and CAD, if any exists, is a complex phenomenon whit marked inter-individual variations. Thus, our null finding for MTHFR genotypes and mortality is not at all surprising, and in line with the reports from Anderson  MTHFR 677C→T polymorphism Despite being confirmed as a major independent predictor of hyperhomocysteinemia, the In this study, statin therapy was the most powerful independent predictor of mortality (Relative Risk Reduction in fully adjusted models: 76–85%). About 60% of our patients, recruited between 1996 and 1999, assumed statin therapy, which was formally introduced as a recommendation into guidelines for CABG surgery only in 1999 The association between hyperhomocysteinemia and mortality after CABG represents a clue, but not a proof, of causality. Recently, large randomized trials showed no clinical benefit of tHcy-lowering by means of B-group vitamins in secondary prevention A limitation of this study lies on the relatively low number of events. It also implies the limitations of nonrandomized observational studies , and possible inaccuracy in assigning causes of death by certificates. Nevertheless, it has some advantages. First, the clinical homogeneity of the sample (post-CABG patients with advanced atherosclerosis), which, on the other hand, may well represent the real world. Indeed, in terms of age, and burden of classical risk factors see , our patIrrespective of causality, hyperhomocysteinemia appears as a prognostic marker of mortality after CABG. Its effect was independent of the most significant predictor, which was represented by the lipid-lowering, and probably anti-inflammatory, therapy with statins. Measuring tHcy before CABG may help to identify a high-risk subgroup of patients for whom it may be especially important to follow a healthy lifestyle and diet, as well as to receive optimal treatments for known causal risk factors. Further studies with adequate design are needed to clarify whether or not patients with HHcy should take advantage from tHcy-lowering therapy, as well as to identify the tHcy threshold level that would be worth to treat, to reduce mortality after CABG.