The associations of pesticide exposure with disease outcomes are estimated without the benefit of a randomized design. For this reason and others, these studies are susceptible to systematic errors. I analyzed studies of the associations between alachlor and glyphosate exposure and cancer incidence, both derived from the Agricultural Health Study cohort, to quantify the bias and uncertainty potentially attributable to systematic error.For each study, I identified the prominent result and important sources of systematic error that might affect it. I assigned probability distributions to the bias parameters that allow quantification of the bias, drew a value at random from each assigned distribution, and calculated the estimate of effect adjusted for the biases. By repeating the draw and adjustment process over multiple iterations, I generated a frequency distribution of adjusted results, from which I obtained a point estimate and simulation interval. These methods were applied without access to the primary record-level dataset.The conventional estimates of effect associating alachlor and glyphosate exposure with cancer incidence were likely biased away from the null and understated the uncertainty by quantifying only random error. For example, the conventional p-value for a test of trend in the alachlor study equaled 0.02, whereas fewer than 20% of the bias analysis iterations yielded a p-value of 0.02 or lower. Similarly, the conventional fully-adjusted result associating glyphosate exposure with multiple myleoma equaled 2.6 with 95% confidence interval of 0.7 to 9.4. The frequency distribution generated by the bias analysis yielded a median hazard ratio equal to 1.5 with 95% simulation interval of 0.4 to 8.9, which was 66% wider than the conventional interval.Bias analysis provides a more complete picture of true uncertainty than conventional frequentist statistical analysis accompanied by a qualitative description of study limitations. The latter approach is likely to lead to overconfidence regarding the potential for causal associations, whereas the former safeguards against such overinterpretations. Furthermore, such analyses, once programmed, allow rapid implementation of alternative assignments of probability distributions to the bias parameters, so elevate the plane of discussion regarding study bias from characterizing studies as "valid" or "invalid" to a critical and quantitative discussion of sources of uncertainty. The Agricultural Health Study (AHS) enrolled 57,311 applicators licensed to apply restricted use pesticides into a large prospective cohort study between 1993 and 1997. Among the initial objectives was to identify and quantify cancer risks among men, women, whites, and minorities associated with direct exposure to pesticides and other agricultural agents , (b) the odds ratio associating the disease with the confounders [ORDC], (c) the inverse of the prevalence of the confounder in the unexposed group [1/PC + E-], (d) ORDC/{(1-PC + E-) + ORDC*PC + E-}, and (e)OREC/{(1-PC + E-) + OREC* PC + E-} [The change in the rate ratio from 1.1 when adjusted only for age to 2.6 when adjusted for the additional factors suggests a substantial amount of confounding controlled by the adjustment or a substantial amount of bias introduced by the adjustment (or some combination of the two). The relative risk due to confounding is a measure of the direction and magnitude of confounding controlled by adjustment variables. In this case, the relative risk due to confounding by the additional adjustment variables would have to equal 2.36 to explain the change in the rate ratio from the age-adjusted estimate of 1.1 to the fully adjusted estimate of 2.6. The relative risk due to confounding is bounded by the following limits: (a) the odds ratio associating the exposure with the confounder [ORPC + E-} .I have assessed the potential for confounding by education to illustrate the value of bounding the relative risk due to confounding. The odds ratio associating glyphosate use with level of education (item a) equaled 1.94, the inverse of the prevalence of the confounder in the unexposed group equaled 1.46 (item c), and item (e) equaled 1.18. While I cannot estimate items b or d from above, I do know that the relative risk due to confounding cannot exceed the minimum of the three bounds that I can estimate (1.18). The observed relative risk due to confounding (2.36) is far in excess of that bound. None of the variables controlled after age, individually or jointly, would be expected to yield a relative risk due to confounding of the size observed (2.36), particularly conditional on the initial adjustment for age.Indeed, the authors acknowledged that the change in estimate from 1.1 to 2.6 derived more from restricting the sample size to those without missing data on the adjustment variables than from the adjustment itself. They wrote in the discussion section:Table 1 shows that the 54,315 subjects were included in the age-adjusted models, whereas because of missing data for covariates, only 40,719 subjects were included in fully adjusted analyses. The association of glyphosate with myeloma differed between the two groups, even without adjustment for any covariates, with no association among the full group and a positive association among the more restricted group.Unfortunately, the authors did not provide the age-adjusted rate ratio comparing ever-users with never-users restricted to subjects who had complete data, although they apparently calculated that rate ratio. Had they provided it, the reader would be able to judge the extent to which the fully adjusted rate ratio derived from restricting the sample to those without missing data. The authors went on to say in the discussion section:The increased risk associated with glyphosate in adjusted analyses may be due to selection bias or could be due to a confounder or effect modifer that is more prevalent among this restricted subgroup and is unaccounted for in our analyses.The prevalence of ever-use of glyphosate was equivalent in the full and restricted samples, so the strength of association between the confounder and the exposure would have to be larger in the restricted data set than in the full data set. That circumstance would describe selection bias, not confounding. The effect modification explanation is also implausible. If all applicators had the effect modifier in the restricted sample, then the rate ratio among the full sample (1.1) would have to equal an inverse variance weighted average of the 2.6 rate ratio in the 75% of applicators who had the effect modifier and were included in the restricted analysis and another rate ratio in the 25% of applicators who did not have the effect modifier and were excluded from the restricted analysis. That other rate ratio would have had to equal an extremely unlikely protective association between glyphosate use and rate of multiple myeloma among those with this hypothetical effect modifier for the weighted average effect to be null. Other scenarios would be even less plausible. The best explanation is that restricting the data set to those with complete data introduced an important selection bias.Misclassification of exposure to glyphosate was a second major source of systematic error, just as it was for the alachlor publication. As for the alachlor analysis, cohort members were originally split into three groups: (a) those with missing data on application of glyphosate, (b) those who reported any application of glyphosate, and (c) those who reported no application of glyphosate. The same concerns about the quality of classification of exposure apply to the glyphosate analysis as they did to the alachlor analysis.There were 32 cases of multiple myeloma, 75% of whom reported ever using glyphosate. Among all subjects, 79% reported ever using glyphosate. To calculate the expected number of exposed cases and persons, I used reliability data from the Agricultural Health Study reported by Blair and colleagues . AlthougEC/{(1-PC + E-) + OREC*PC + E- = 1.18). If the confounding impacts of all of the variables are completely independent, which would require them to be unassociated, then the relative risk due to confounding would equal the product of their individual bounds on the relative risks due to confounding, which equaled 1.56. State is unlikely to be related to risk of multiple myeloma, but disease risk is not available within levels of the confounders, so the association cannot be examined. Recalculating the independent relative risk due to confounding without including the contribution from enrollment state yields an expected relative risk due to confounding of 1.39. I parameterized the trapezoidal distribution of the relative risk due to confounding with a minimum of 1 (no confounding after adjustment for age), a lower mode of 1.18 (the bound on relative risk due to confounding by education), an upper mode of 1.39 , and a maximum of 1.56 .Since the relative risk due to confounding exceeded its plausible maximum, I adjusted for the potentially confounding variables in the bias analysis by drawing the relative risk due to confounding from a trapezoidal distribution. I parameterized the trapezoidal distribution using the data in the publication to calculate the bounds on the relative risk due to confounding for each individual confounder. For each potential confounder, the bound on the relative risk due to confounding was ultimately derived from item e. The greatest of these is item e from the education variable, so if adjustment for all variables were attained by adjusting for just this variable, then the relative risk due to confounding would equal its item e , not sensitivity and specificity. To calculate the predictive values, I used the following equations relating predictive values to sensitivity and specificity:PPV and NPV are the positive and negative predictive values, s is the sensitivity of exposure classification, t is the specificity of exposure classification, p is the prevalence of glyphosate exposure, and i is an index for the four combinations of exposure status (glyphosate exposed and unexposed) with disease status (cases and persons at risk).Where To conduct an iteration of the bias analysis, I drew a value of the sensitivity and a value of the specificity from the trapezoidal distributions, then calculated the positive and negative predictive values for cases and persons at risk, conditional on the observed prevalence of glyphosate exposure. I used a random binomial distribution to model the number of expected exposed cases and persons and the number of expected unexposed cases and persons. For example, I drew the number of expected exposed cases using the random binomial distribution with 24 trials (the number of observed exposed cases) and the positive predictive value for cases as the probability of a success. This procedure would yield between 0 and 24 exposed cases, and the difference between the number of modeled exposed cases and 24 would be reclassified as unexposed cases. Similarly, I drew the number of expected unexposed cases using the random binomial distribution with 8 trials (the number of observed unexposed cases) and the negative predictive value for cases as the probability of a success. This procedure would yield between 0 and 8 unexposed cases, and the difference between the number of modeled unexposed cases and 8 would be reclassified as exposed cases. The total number of exposed cases would equal the number modeled as correctly classified as exposed (out of 24) and the number modeled as incorrectly classified as unexposed (out of 8). I used a similar procedure to model the total number of persons at risk, but using that population size and the predictive values calculated using the prevalence of glyphosate exposure in the total population. I then calculated the modeled risk ratio and divided it by the crude risk ratio  to obtain an estimate of the bias due to misclassification.HRs) simultaneously accounting for both threats to validity. To account for random error, I calculated the standard error of the hazard ratio (SE) using the exposed and unexposed case counts generated by the misclassification procedure, drew a random standard normal deviate (z), and then calculatedTo combine the bias analysis applied to account for confounding and misclassification, I multiplied the conventional age-adjusted hazard ratio reported in the paper (1.1), by each iteration's relative risk due to confounding and its misclassification bias to obtain the hazard ratio (HRR).to yield the natural logarithm of the hazard ratio incorporating random error as well as systematic error (Figure Figure Figure When studies are designed without the benefit of randomized exposure assignment, conventional frequentist statistics underestimate the true uncertainty in an estimate of association. Despite qualitative descriptions of study limitations, the conventional frequentist statistics will often lead to overconfidence about the reported result and a tendency toward favoring the causal explanation for an association over bias explanations . Bias anI illustrate the value of bias analysis by applying it to two results reported from the AHS. Pesticide exposure is allocated by non-random influences, so studies of its association with health outcomes are candidates for bias analysis. Although both studies depicted the potential for systematic error in the description of limitations, neither applied a quantitative method to assess the additional uncertainty beyond random error contributed by these limitations. I implemented a bias analysis of the central findings from studies of the association of glyphosate exposure and alachlor exposure with cancer incidence. These bias analyses were conducted without access to record-level data, so relied entirely on published results, which illustrates that stakeholders can often address study limitations quantitatively even if original data are not available for analysis. Were record-level data available, additional bias analysis would have been possible, including further analysis of classification errors and an analysis of modeling error.An advantage of bias analysis is that alternative assumptions about the parameterizations and bias models can be readily examined, and the results compared with one another. These comparisons provide a quantitative basis for disagreements among stakeholders about the potential impact of systematic error, and thereby remove the debate from the realm of qualitative criticism heavily influenced by politics and polemics. Instead, the analysis places the debate into the realm of quantitative analysis, in which differences between stakeholders can be readily traced to differences in the parameterization of the bias analysis, and the consistency of the different parameterizations with external information can be examined. These differences – when they yield substantially different results – provide fodder for discussion and may inspire additional research to characterize better the values to assign to the parameters. This approach to examining differences in interpretation of study results is far more consistent with the scientific enterprise, an inherently quantitative undertaking, than simple categorization of study results as "valid" or "invalid," since all studies are susceptible to imperfections.Conditional on the validity of distributions assigned to the bias parameters, I showed that the central results from both studies were likely biased away from the null and that the conventional frequentist measures of error underestimated the true uncertainty. These methods of bias analysis are not difficult to implement and should be encouraged in future presentations of results.This project was supported, in part, by an unrestricted research grant from Crop Life America to the Boston University School of Public Health. The author has received $2000 in consulting fees from Monsanto Company, manufacturer of alachlor and glyphosate, in the past five years.TL conceived of the study, conducted the analyses, and wrote, read, and approved the final manuscript.


The frequency of homozygote for ACE D variant is significantly higher in the case subjects than that of the controls . The adjusted odds ratio of the D homozygous for the risk of OPL is 8.10 =2.04–32.19, P=0.003). In the allelic base analysis, the D allele is also significantly associated with higher risk of OPL. When grouping the study subjects by smoking status, the association between ACE I/D polymorphism and risk of OPL was only observed in nonsmokers. Our results support the theory that genetic factors may contribute to the susceptibility of OPL and suggest that smoking and genetic factors may be differently involved in the development of OPL.To investigate whether  Both oral submucous fibrosis (OSF) and oral leukoplakia (OL) are chronic potentially malignant lesions of the oral cavity, the former being characterised by a submucosal fibrosis, resulting in marked rigidity and progressive difficulty in opening the mouth, the latter by chronic white mucosal maculae . The mosACE insertion/deletion (I/D) polymorphism is associated with fibrotic and atherosclerotic cardiovascular disease  is expressed in a wide range of tissues  criteria  by the duration of betel quid chewing (in years). Valid questionnaires were examined further by a structured questionnaire. Blood pressure, body height and weight were measured and body mass index (BMI) was calculated. Blood was also collected; serum and white blood cells (WBCs) were separated on the day of blood collection and frozen at −70°C until subsequent analysis.μl reactions ; 50 mmol l−1 KCl; 0.001% gelatin; 10 mmol l−1 Tris-HCl, pH 8.3) with 4 min of denaturation at 94°C, followed by 35 cycles of 15 s at 94°C, 5 s at 67°C and 30 s at 74°C in a thermal cycler . Reaction was terminated at 72°C at 2 min. To avoid ID/DD mistyping of heterozygote as DD homozygote  amplification products were obtained using 25 mozygote . The PCRt-tests or χ2 tests were used to compare means and proportions between the groups of control and oral precancerous lesions (OPL) when appropriate. To determine whether an association existed between ACE genotype and allele frequency with OPL risk, the significance of the difference in the distribution of genotypes and alleles between OPL patients and control subjects was calculated by χ2 statistic or Fisher's exact test and shown by P-value. All P-values were two-sided. A P-value <0.05 was considered to be statistically significant. Association of ACE genotypes and OPL was analysed by conditional logistic regression of OPL patients vs controls matched by betel quid chewing duration. To control the potential confounding effects, sex, age, BMI, blood pressure, smoking and drinking status were used as independent variables for adjustment. Conditional logistic regression was carried out with the SAS procedure PROC PHREG. Genotypic data entered the regression model in the form of two dummy variables representing the effect of genotype ACE I/D and genotype DD vs the reference category II, respectively.Data are shown as the mean±s.d. All statistical analyses were performed using SAS statistical software . The two-sample There were 185 subjects from a total of 826 rural indigenous people who were found on screening to have oral mucosal lesions and signed consent for further pathologic diagnosis. Among these, 111 subjects were confirmed to have OSF or OL; none had oral cancer, while a total of 50 subjects were excluded, 21 because of missing blood collection and 29 with missing information. Finally, a total of 61 persons were included as case subjects, 10 with OSF, 26 with OL and 25 OL with both lesions related to betel quid chewing. In all, 61 betel quid chewers without any previous or present lesions in the oral cavity matched for betel quid chewing duration and dosage were used as control subjects. ACE gene I/D genotype distributions and allele frequencies of the study groups are presented in ACE genotypes in OPL patients was significantly different (P=0.0108) from that of controls. Patients with OPL had a higher distribution of DD genotype or D allele frequency, indicating the existence of a relationship between ACE gene polymorphism and OPL risk. The association between ACE DD and ID genotypes and risk of OPL still exist even after adjusting for age, gender, BMI, systolic blood pressure, smoking and drinking status by conditional logistic regression analysis (DD vs II: adjusted odds ratio (OR)=8.10, 95% confidence interval (CI)=2.04–32.19, P=0.0030; ID vs II: adjusted OR=3.69, 95% CI=0.98–13.81, P=0.0530). When analysis was performed comparing the risk of DD genotype vs II/ID genotype or ID/DD genotype vs II genotype and D allele vs I allele, DD genotype or D allele still had a significantly higher risk in the association with OPL . Further analysis comparing the risk of DD genotype vs II/ID genotype or ID/DD genotype vs II genotype and D allele vs I allele showed that DD genotype or D allele had a significantly higher risk in the association with OPL (As previous reports have indicated that smoking status acts as a contributing factor for OPL (hout OPL , while twith OPL .Although available epidemiological evidence indicates that the chewing of betel quid is an important risk for the development of OSF and OL (ACE gene I/D polymorphism is significantly associated with betel quid chewing-related OPL both in nonadjusted and adjusted models. To the best of our knowledge, this is the first report showing that ACE gene I/D polymorphism is associated with the risk of OPL in subjects who chew betel quid. Our results support the evidence that genetic factors may interact with environmental factors to contribute to the pathogenesis of OPL (Our previous studies confirmed that betel quid chewing acts as a major risk factor for OPL , while tStudies have suggested that molecular mimicry may explain the association among chronic inflammatory infiltrates, human leucocyte antigen and autoimmune disease with the pathogenesis of OSF and OL (ACE D allele in its homozygous form might confer susceptibility for autoimmune manifestations in sarcoidosis. ACE genotype could be associated with the disease activity of SLE. Thus, subjects with the ACE gene D allele may, through the increased effect of Ang II, on cytokine activation, chronic inflammation and immune modulation, contribute to the increased risk of OSF and OL.Various immunologic changes are believed to play a role in the pathogenesis of OSF and OL (ACE gene D allele. Recently, Wang and co-workers reported that GSTM1 deficiency is associated with the risk of vascular diseases, while they did not observe any interaction between the status of cigarette smoking and GSTM1 deficiency in relation to coronary arterial disease severity (GSTM1 non-null genotype of prostate cancer. Our results show that ACE gene I/D polymorphism and cigarette smoking status have no synergistic or additive effect in relation to the risk of OPL. This fact may suggest that smoking and genetic factors may be differently involved in the development of OPL disease.Cigarette smokers are more likely to develop many forms of disease than nonsmokers, including oral diseases. However, our study did not show an increased risk of precancerous lesions in smoking betel chewers with severity . SimilarACE gene I/D polymorphism with OSF and OL in a Taiwan aboriginal population. These results imply that susceptibility to OSF and OL could involve genetic mechanisms modified by betel quid exposure. The present study also suggests that the RAS may be involved in the pathophysiology of inflammatory reaction and immunologic derangement in OSF and OL. Further work is required to confirm these findings in populations of different races and these preliminary exploratory results should be confirmed in a larger study.In summary, our study shows an association of the


There were strong correlations between both systolic and diastolic blood pressure and all three urine albumin methods (p < 0.0001). There were also significant correlations between the different urine albumin measurements and serum amyloid A component, high-sensitivity C-reactive protein and interleukin-6. The three different urine albumin methods studied provided similar information in relation to cardiovascular disease. There was a strong correlation between systolic and diastolic blood pressure and microalbuminuria in both the whole study population and in nondiabetic males emphasizing the role of hypertension in glomerular damage. The good correlation between the studied urine albumin measurements show that all three methods can be used for monitoring urine albumin excretion.Microalbuminuria is associated with hypertension and is a strong risk factor for subsequent chronic disease, both renal and coronary heart disease (CHD), Presently there are several methods available for measurement of microalbuminuria. The aim of this study was to evaluate if the three different methods gave similar information or if one of the assays were superior to the others. Blood pressure, inflammatory markers and cardiovascular mortality and morbidity were correlated with urine albumin analysed with a point-of-care testing (POCT) instrument, nephelometric determination of albumin and albumin/creatinine ratio in elderly males. The study population consisted of 103 diabetic and 603 nondiabetic males (age 77 years) in a cross-sectional study. We analyzed urine albumin with a HemoCue Hypertension is a highly prevalent disorder that affects the majority of adults . It is a2 . POCT offers rapid test results, which facilitates the use of the results to motivate the patient to lifestyle changes and increased compliance . To achi® urine albumin (POCT instrument), a central laboratory instrument (ProSpec®) and albumin/creatinine ratio (ACR).The aim of the present study was to correlate hypertension with different markers for microalbuminuria in a group of elderly males. The methods used in the study were HemoCueThis study is a cross-sectional investigation of Swedish men, 77 years of age, which were participants in Uppsala Longitudinal Study of Adult Men (ULSAM) . This heTwenty-four hour urine was collected without additives. Blood samples were drawn from an antecubital vein in the morning after a 12-hour (overnight) fast. The samples were immediately frozen at −70°C until analysis. The same urine samples were used for all three assays.Medical history and information on medication were obtained by a self-administered questionnaire. A total of 706 urine samples were analysed. The patients completed a questionnaire and had a fasting plasma glucose value. Type 2 diabetes was diagnosed according to the WHO definition: fasting plasma glucose ≥7.0 mmol/L, or intake of oral anti-diabetic drugs alone, or in combination with insulin treatment (n = 103). Control men had plasma glucose <7.0 mmol/L and no anti-diabetic drug (n = 603). Information of a history of CVD  was obtained from the Swedish Hospital Discharge Registry. In the study population, 44.3% of the participants had hypertension, 24% had ischemic heart disease , 16% had a history of myocardial infarction, 18% had cerebrovascular diseases (including stroke) and 16% had a history of stroke.® urine albumin . CV calculated on duplicate samples was 3.9%. Urine albumin was also measured by nephelometry  using a Behring BN ProSpec® analyzer (Dade Behring). The total analytical imprecision of the method was 3.7% at 9.5 mg/L and 5.0% at 85 mg/L. Urine creatinine were analyzed with a modified kinetic Jaffe reaction on an Architect Ci8200® analyzer  and reported as S.I. units (μmol/L) and creatinine related urine albumin was calculated from the Prospec® results. The total analytical imprecision of the creatinine method was 4.8% at both 94 and 337 μmol/L. All assays were performed independently without prior knowledge of other patient data.Urine albumin was analyzed with HemoCueHigh sensitivity IL-6 was analyzed by an ELISA kit . The total CV of the method was 7% and interassay coefficient of variation (CV) was 5%. High sensitivity CRP and SAA measurements were performed by latex enhanced reagent  using a Behring BN ProSpec analyzer (Dade Behring). The intraassay CV of the CRP method was 1.4% at both 1.23 mg/L and 5.49 mg/L and the intraassay CV of the SAA method was 5.9% at 12.8 mg/L and 3.2% at 81.7 mg/L.® and 3 mg/L for ProSpec®) was assigned the value of the respective detection limit in the statistical analysis. When the ProSpec® urine albumin values were below 3 mg/L, ACR could not be calculated by the laboratory information system. In the statistical analysis these cases was assigned an ACR of 0.3 g/mol creatinine.All urine albumin values below the detection limits . Differences between diabetic patients and controls were tested with Mann-Whitney test, Kruskal Wallis test or partial correlation in multivariate analyses. Associations between continuous variables were tested with Spearman’s rank correlation analysis. P-values <0.05 were regarded as statistically significant throughout the study.® (R2 = 0.999). There were also good agreements between the two urine albumin methods (R2 = 0.956) (® and ACR (R2 = 0.937) (results not shown). The Hemocue® method gave slightly lower values than ProSpec®.There was a strong correlation between duplicate analysis of 100 samples on Hemocue= 0.956)  and betw® instrument. The corresponding figures for ProSpec® were 124 (20.6%) and 106 (17.6%), respectively. 111 (18.5%) of the nondiabetic males had creatinine related urine albumin >2.5 g/mol creatinine and 102 (17%) individuals had values >3 g/mol creatinine.114 (18.9%) of the nondiabetic males had urine albumin >20 mg/L and 101 (16.7%) individuals had urine albumin >25 mg/L with the HemoCueNo significant correlations were found between any of the microalbuminuria measurements and total cholesterol, HDL-cholesterol or LDL-cholesterol in either the whole study group or the nondiabetic group. In contrast, most of the correlations were significant with triglycerides and the inflammatory markers hsCRP, IL-6 and SAA. The most pronounced correlations were found with SAA. Significant correlations are presented as Spearman rank correlations in There were significant correlations between urine albumin values and cardiovascular disease and angina but not with ischemic stroke, myocardial infarction and cardiac failure . The onl®, DCA 2000® and HemoCue®  and a variety of immunochemical methods, such as radioimmunoassays, enzyme-linked immunoassays, nephelometric and turbidimetric assays . The insHemoCue® .Screening for microalbuminuria are usually performed by one of these methods: measurement of total urine albumin in 12 or 24 h collection, measurement of the albumin-to-creatinine ratio in morning urine or random sample or measurement of urine albumin in morning urine . The 24 ® and ProSpec® are sufficient to detect even low levels of microalbuminuria.The lower cut-off values for microalbuminuria varies between studies, but it is often in the range of 15–25 mg/L for albuminuria and 1.8–3 g albumin/mol creatinine for ACR . The Eur® gave slightly higher values than HemoCue®  and ACR in a group of elderly males showing that all three methods can be used for monitoring urine albumin excretion. Age, sex and ethnicity composition were homogenous in this study, but the results may therefore have limited generalizability to other age and ethnic groups and women. The strong correlation between systolic and diastolic blood pressure and microalbuminuria in both the whole population and in nondiabetic males shows that the blood pressure has an important role for the development of albuminuria.In conclusion, this study shows a good correlation between urine albumin analysed with HemoCue


The etiology of esophageal squamous cell cancer (ESCC) in high prevalence regions of China remains unclear.Endoscopic biopsies were conducted among 7381 inhabitants aged from 25 to 65 of Anyang, China.in situ and invasive ESCC. Using deep well (depth >100 meters) as water source  was negatively associated with ESCC and its precursors, whereas tobacco and alcohol use were not significantly associated with ESCC.In this study, 2.57, 0.20 and 0.16% of the participants had mild, moderate and severe squamous dysplasia, respectively; 0.19 and 0.08% showed squamous carcinoma Water source and other factors in this region need further evaluation by longitudinal studies. Esophageal cancer is the sixth most common cancer among men and ninth most common among women . TobaccoIn Anyang, a region in north central China, mortality from esophageal squamous cell cancer (ESCC) is among the highest in the world  being 10In 2007, we initiated a population-based esophageal cancer cohort study in Anyang, China using endoscopic biopsy and a questionnaire investigation to determine its age specific prevalence and that of its precursor lesions in this region, and investigate the possible risk factors for ESCC. The results would also serve as baseline data for our subsequent longitudinal investigation.Anyang, an agricultural region in the northern part of Henan Province, PR China, consisting of five counties, Linzhou, Hua County, Anyang County, Tangyin County and Neihuang County, with a population of 5.37 million; its gross domestic product (GDP) per capita is about US $1760. In 2007–09, we conducted an endoscopic survey for esophageal cancer and precursor lesions in nine villages of rural Anyang, which were selected on the basis of the population size and location. Five were in Hua County, two in Linzhou, one in Anyang County and one in Tangyin County. Criteria for participant eligibility were as follows: (1) permanent residency in one of these villages; (2) age 25 to 65; (3) no previous cardiocerebral vascular diagnoses or psychological disorders; (4) voluntary participation in this study and agreement to complete all phases of the examination, including endoscopy.in situ (CIS), invasive squamous cell carcinoma and adenocarcinoma based on commonly accepted criteria  was sampled if no abnormalities were found. Biopsies were fixed in 10% formaldehyde, embedded in paraffin, sectioned at 5 mm, and stained with hematoxylin and eosin. Biopsy slides were read independently by two pathologists at Anyang Cancer Hospital, without knowledge of the subjects’ history or endoscopic findings. Diagnoses included normal, esophagitis, basal cell hyperplasia (BCH), squamous dysplasia , squamous carcinoma A questionnaire was completed covering socio-demographic characteristics, pesticide exposure, living environment, alcohol and tobacco use and dietary habits. Interviews were carried out one-on-one in a room situated so as to ensure privacy.χ2-test and Fisher’s exact test. Univariate and multivariate unconditional logistic regression was employed to identify risk factors for ESCC and its precursor lesions. All subjects were categorized either as ‘case group’  or ‘control group’  for logistic regression analysis. Two cases of adenocarcinoma were excluded from this analysis. All statistical analyses were conducted using SPSS for Windows version 11.5. All P-values were two-sided and P-values less than 0.05 were considered statistically significant.Crude detection rates of all histological diagnoses were standardized by the age structure of the World Health Organization world standard population of 1985  and there were no other significant gender differences in detection rates of esophageal cancer or its precursor lesions  completed all required blood testing, endoscopy and biopsy and the questionnaire. Of these individuals, 7381 (96.57%) had at least one biopsy technically adequate for pathology diagnosis. As a recent study in this region demonstrated that esophagitis was not associated with risk of esophageal cancer . Figure  lesions .Ptrend=0.017) was associated with increased risk of ESCC and its precursors, whereas use of water pumped from a deep well (over 100 m depth)  showed a negative association with ESCC and its precursor lesions. After adjusting for age and sex, use of deep well water remained as having a negative association with ESCC and precursor lesions . Cigarette smoking, alcohol intake, pesticide exposure and hot food intake were not found to correlate with ESCC and precursor lesions.Potential ESCC risk factors, including tobacco and alcohol use were evaluated using unconditional logistic regression analysis . UnivariThe Anyang region of China is a high-risk region for ESCC, and a series of studies on ESCC have been carried out there. To obtain the greatest number of patients with ESCC or precursor lesions as possible, participants chosen for almost all these studies were of generally greater age (Water source is not a common risk factor for ESCC, especially in developed countries. Until about 20 years ago, the main sources of drinking water in rural area of Anyang were rivers or shallow wells. However, since the 1980s, the local government has committed significant resources to an extensive water improvement project, and now-a-days deep groundwater has become the main water source for most of the inhabitants of rural Anyang. A previous ecologic study reported that the incidence and mortality from esophageal cancer in Linzhou showed a decreasing trend in villages included in this water improvement project as compared with those without water improvement (Although tobacco and alcohol are well-established risk factors for ESCC in western countries  Changes In summary, 7381 individuals of rural Anyang participated in this cross-sectional endoscopic survey. Detection rates of mild, moderate and severe squamous dysplasia, CIS and ESCC were 2.57, 0.20, 0.16, 0.19 and 0.08%. Use of water pumped from deep wells was negatively associated with ESCC and precursor lesions, whereas tobacco and alcohol use had no significant relationship with ESCC. Longitudinal studies are needed of other risk factors that may contribute to ESCC in this high-risk region.


Exertional rhabdomyolysis syndrome is recognised in many athletic horse breeds and in recent years specific forms of the syndrome have been identified. However, although Standardbred horses are used worldwide for racing, there is a paucity of information about the epidemiological and performance-related aspects of the syndrome in this breed. The objectives of this study therefore were to determine the incidence, risk factors and performance effects of exertional rhabdomyolysis syndrome in Standardbred trotters and to compare the epidemiology and genetics of the syndrome with that in other breeds.GYS1) mutation responsible for type 1 polysaccharide storage myopathy. A total of 44 susceptible horses was reported, resulting in an annual incidence of 6.4 (95% CI 4.6–8.2%) per 100 horses. Female horses were at significantly greater risk than males  and nervous horses were at a greater risk than horses with calm or average temperaments . Rhabdomyolysis-susceptible cases performed better from standstill starts (p = 0.04) than controls and had a higher percentage of wins (p = 0.006). All exertional rhabdomyolysis-susceptible horses tested were negative for the R309H GYS1 mutation.A questionnaire-based case-control study  was conducted following identification of horses that were determined susceptible to exertional rhabdomyolysis (based on serum biochemistry) from a total of 683 horses in 22 yards. Thirty six exertional rhabdomyolysis-susceptible horses were subsequently genotyped for the skeletal muscle glycogen synthase (Exertional rhabdomyolysis syndrome in Standardbred horses has a similar incidence and risk factors to the syndrome in Thoroughbred horses. If the disorder has a genetic basis in Standardbreds, improved performance in susceptible animals may be responsible for maintenance of the disorder in the population. Equine exertional rhabdomyolysis syndrome (ERS) commonly affects the athletic breeds, including Thoroughbred, Quarter Horse, Arabian, Warmblood, and Standardbred GYS1)) Epidemiological studies in specific breeds have defined the risk factors and welfare and economic implications of ERS. For example, between 5 and 7% of Thoroughbreds are affected worldwide To the authors' knowledge, there have been no epidemiological studies of ERS in Standardbred horses even though this is a popular racing breed in many countries. Despite this, an early genetic association study suggested that the disorder in Standardbred horses may have an underlying genetic basis in vitro experimental evidence GYS1 mutation.Although isolated reports exist of Standardbred horses with polysaccharide storage myopathy Of 66 Standardbred trotting trainers licensed by the Swedish Trotting Association and contacted either via letter, email or telephone in March 2007, 57 were willing to participate in the study and were contacted again via telephone . Nine weOut of the 35 trainers reporting at least 1 case of ERS in the previous year, 13 (37.1%) relied on clinical signs only while 22 (62.9%) routinely utilised veterinary assessment of measured serum CK and AST activities to confirm the diagnosis. All of these 22 trainers were willing to participate in the case-control study . They trForty four of the 683 horses from the 22 yards were affected by ERS over the 12-month period; therefore the overall trainer-reported annual incidence in this study population was 6.4 cases (95% CI 4.6–8.2) per 100 horses whilst the annual incidence in individual yards ranged from 1.7 (95% CI 0–6.0) to 20 (95% CI 0–44.7) cases per 100 horses. Eleven of the 22 yards reported more than 1 case of ERS in the previous year and 28 of the 44 (63%) affected horses had had more than 1 episode. Multiple episodes ranged from 2 to 20 (median of 2) per horse.Clinical signs included stiffness in all 44 cases, sweating in 38 (86.4%), pain or distress in 19 (43.2%), swollen or hard gluteal muscles in 17 (22.7%) and recumbency in 2 (4%). The 44 cases had had a total of 139 disease episodes (median of 2) in the last year with 135 episodes (97.5%) observed within an hour after training exercise and 2 (1.4%) episodes noticed during training. One episode was reported just prior to a race and another was observed just after finishing a race.Fifteen of 44 affected horses (34.1%) had a history of lameness. In 26 cases (59.1%) the episodes had occurred after a period of rest, ranging from 1 to 14 days, with a median of 2; in 4 of these 26 cases (15.4%) the rest was due to another disease. Thirty three of the 44 cases (75%) were given a day or more to recover from ERS ranging from 1 to 28 days with a median of 7 training days lost per episode. Twenty two of 44 cases had been prevented from racing for between 1 and 10 weeks as a result of ERS.Most  ERS cases were considered to be in full fitness when the episode occurred, 4 cases (9%) were in medium condition while 2 (4.5%) were reported as unfit. Sixteen of the 44 affected horses (36.4%) had episodes with no associated seasonality while 9 occurred in the spring (20.5%), 6 during the summer (13.6%), 4 in the autumn (9.1%) and 9 (20.5%) during winter months. In 1 mare, the trainer believed that ERS was associated with persistent oestrus in the spring, in another case it was believed associated with a virus, while 2 horses, both stallions, were reported to be “burnt out” when the episodes occurred. In other cases no clear predisposing factor was reported.Although it was not possible to apply statistical analysis to training and feeding practices between groups, management was broadly similar for all horses in each yard and between yards. Trainers fed a mean of 4.5 kg (range 3–6 kg) of concentrate and 9 kg (range 6–11 kg) roughage feed per horse.The 22 trainers reported various methods of preventing and managing ERS and most yards used a combination of methods . Most coAlterations in feeding regimes were commonly employed, with 9 trainers reducing the amount of concentrates fed when rested. Six trainers ensured that affected horses had set daily routines while 5 trainers tried to minimize stress as much as possible. One trainer reported using low dose oral acepromazine when returning horses to work however no trainers used dantrolene sodium or electrolyte supplementation for prevention.Descriptive statistics of continuous variables for cases and controls are shown in Results of multivariable conditional logistic regression are shown in GYS1 mutation.DNA samples were available from 36 of the ERS-susceptible Standardbred horses and all were negative for the R309H This study is the first that has examined epidemiological aspects of ERS in Standardbred horses. Results indicate that approximately 6% of horses were affected by ERS per year and that being female and having a nervous temperament were significant risk factors. Common regimes used to manage ERS included alterations to diet and exercise. For certain variables, ERS-susceptible horses performed better than matched controls. Additional risk factors or associations were not found, perhaps because of the relatively low number of animals studied.www.travsport.se), hence at approximately 6% incidence, ERS in this breed likely has substantial financial and welfare implications.The annual incidence of ERS in the telephone study (4.1%) was lower than that reported for the case control study (6.4%) perhaps because recognition of low grade ERS was more readily achieved when trainers routinely measured CK and AST for diagnosis (i.e. the case control group). This incidence of ERS in Standardbreds is similar to that reported in other breeds: 6.7% and 4.9% in UK and USA flat racing Thoroughbreds respectively Female horses were more likely to have ERS than male horses; this finding supports those in several other studies Unlike some studies in Thoroughbreds where age was a significant risk factor Almost 90% of Standardbreds were considered at full fitness at the time of their ERS episode. This contrasts findings in polo ponies where the majority of episodes occurred after a chukka in which the horse was perceived to be not fit enough for the level of play demanded in vitro studies report that muscle from Thoroughbred horses affected by recurrent exertional rhabdomyolysis contracts and relaxes more quickly than muscle from unaffected horses Performance is a multifactorial variable likely affected by training, level of fitness, genetics, behavior, temperament, experience, track position, surface conditions, coach driver and trainer. Standardbreds with ERS had a significantly faster best lap time from a standstill start than controls, and significantly more wins and/or placings. No significant difference was found between groups in fastest lap time from a running start or in any other performance indicators. Surprisingly therefore, improved performance occurs despite an underlying susceptibility to ERS: perhaps environmental or management effects on occasion create the conditions necessary for an episode of rhabdomyolysis, whereas the rest of the time, the horse is not subject to any detrimental effects; indeed there is statistically significant enhanced performance. Further studies are however required to determine the reason for the faster (standing start) lap times in the affected horses, though one conceivable explanation may be better acceleration: Questionnaire-based studies have certain limitations. For example, even though trainers were encouraged to elaborate regarding disease episodes, their answers may be restricted by, and dependent on, ability to recall information and by the questions asked. It is conceivable that answers given in response to the questionnaire may have been biased by prior knowledge of the risk factors and management of the disorder in Thoroughbreds; however, in the current study we believe that this is unlikely to be a major factor since trainers were (for example) unaware of recommendations to manage ERS in Thoroughbreds with prophylactic administration of oral dantrolene GYS1 mutation The epidemiological profile and risk factors identified in this study in Standardbreds are more suggestive of recurrent exertional rhabdomyolysis in Thoroughbreds than of polysaccharide storage myopathy in Quarter Horses, Warmblood or draught breeds It is currently unknown whether all Thoroughbreds with ERS have an identical disease, however a genetic predisposition with autosomal dominant inheritance has been proposed in Thoroughbreds in the USA Animal procedures in this study were conducted by licensed veterinary surgeons with informed consent in accordance with the Royal Veterinary College's ethics and welfare committee regulations.Data were gathered using a purpose-designed form and comprised general information about horses in training  and the procedure used to confirm the diagnosis .Horses with trainers who reported using serum or plasma muscle enzyme activities (CK and AST) routinely to diagnose cases of ERS and who had 1 or more ERS cases in the last year were selected for inclusion in the case-control study. Of the 22 trainers who satisfied these criteria, 9 were based in southern Sweden and these were interviewed in person while the remaining 13 were spread throughout Sweden: for these trainers a telephone interview was conducted. All interviews were conducted by the same person and a purpose-designed standardised questionnaire was used for each interview (information requested is summarised in www.travsport.se); performance indicators that were assessed are listed in Trainers listed all horses considered free from ERS within their yards and subsequently, for each ERS case, 2 ERS-free horses were randomly selected  from the list as matched controls. For each case and control horse the age, sex and trainer-perceived temperament  was recorded. Information about management in each yard was collected and for each case, further information regarding the episode(s) of ERS was also recorded. Performance data for case and control horses were obtained from online records  and/or improved model fit . All analyses were conducted in Stata SE 9.2  and the level of statistical significance was set at p<0.05.R309H mutation using the restriction fragment polymorphism assay as reported previously Blood samples derived from jugular venipuncture from 36 Standardbred horses identified in the study as being susceptible to ERS on the basis of routine measurement of CK and AST activity were stored in EDTA-tubes (Vacutainer) at −20C and shipped on dry ice. DNA was extracted from whole blood using a specific kit  and the DNA was tested for the presence of the GYS1


Normal aging involves a decline in cognitive function that has been shown to correlate with volumetric change in the hippocampus, and with genetic variability in the APOE-gene. In the present study we utilize 3D MR imaging, genetic analysis and assessment of verbal memory function to investigate relationships between these factors in a sample of 170 healthy volunteers (age range 46–77 years).Brain morphometric analysis was performed with the automated segmentation work-flow implemented in FreeSurfer. Genetic analysis of the APOE genotype was determined with polymerase chain reaction (PCR) on DNA from whole-blood. All individuals were subjected to extensive neuropsychological testing, including the California Verbal Learning Test-II (CVLT). To obtain robust and easily interpretable relationships between explanatory variables and verbal memory function we applied the recent method of conditional inference trees in addition to scatterplot matrices and simple pairwise linear least-squares regression analysis.APOE genotype had no significant impact on the CVLT results  or the ICV-normalized hippocampal volumes. Hippocampal volumes were found to decrease with age and a right-larger-than-left hippocampal asymmetry was also found. These findings are in accordance with previous studies. CVLT-LD score was shown to correlate with hippocampal volume. Multivariate conditional inference analysis showed that gender and left hippocampal volume largely dominated predictive values for CVLT-LD scores in our sample. Left hippocampal volume dominated predictive values for females but not for males. APOE genotype did not alter the model significantly, and age was only partly influencing the results.Gender and left hippocampal volumes are main predictors for verbal memory function in normal aging. APOE genotype did not affect the results in any part of our analysis. MR imaging and segmentation of brain volumes have been increasingly applied in studies of the human brain and its functions. Several studies on aging and age-related cognitive decline have combined neuropsychological tests with MRI findings. These studies have revealed a relationship between regional volumetric atrophy as measured with 3D MRI, decline in memory functions as measured with neuropsychological tests, and the presence of early signs of dementia -3.Although environmental factors contribute to the variation in cognitive function during aging, recent studies have identified genetic markers as significant factors, not only for cognitive function, but also for volumetric variability in aging ,5. StudiCombining genetics with volumetric imaging has indicated that regional hippocampal volumes correlate negatively with the zygosity of ApoEϵ4 -16. TherFurthermore, Tupler et al.  combinedIn the present study of 170 normal elderly subjects, we set out to assess the relationship between (i) volumetry of brain structures involved in memory, (ii) genotype of the polymorphic ApoE gene and (iii) scores on the long delay free recall subtest from CVLT (CVLT-LD). Few studies other than Tupler et al. , Lind etAlthough we have performed only a cross-sectional study, in contrast to the repeated and predictive design by Tupler et al. , a majorThe subject material consisted of 170 individuals . There were 86  in the Bergen sample and 84  in the Oslo sample. The participants were interviewed, and those with previous or present neurological or psychiatric disorders, head trauma, a history of substance abuse, or other significant medical conditions were excluded. Based on information from interviews and evaluation of neuropsychological test results, none of the subjects were defined as demented. All subjects signed an informed written consent to participate in the study, and the study was approved by the Regional Committee for Medical Research Ethics of Southern Norway. The age, education and IQ-range  at voxel-size 0.94 × 0.94 × 1.4 mmAt both sites, the DICOM images were then transferred from the scanner to a Linux workstation for further processing as described below.The volumetric analysis, using the FreeSurfer software package  -39 was bThickness measurements were obtained by reconstructing the gray/white matter boundary surface and the gray/pial boundary surface Figures  and calcSubcortical segmentation uses somewhat different methods than in the surface processing step, as described in  and 41]41]. ThisDue to biological variability and differences in image quality, it was necessary to control the results after both the surface reconstruction and the subcortical segmentation processes. This had to be done for each subject. Errors in the surface reconstruction or subcortical labeling were corrected using the methods described in , and theBy including 170 individuals of both genders and with different body size, one can expect a wide range of values in morphology and measures of body structures. In the brain, this can be appreciated by the fact that large human beings have larger somatosensory areas and more muscle to control and thus, larger brains. This is evident in the different brain sizes in men and women ,45,46. WμL whole blood using MagNA PureLC DNA Isolation Kit-Large Volume on the MagNA Pure LC (Roche), eluted and diluted to 1 mL, of which 5 μL was applied in each assay. The typing of ApoE-zygosity was performed using the LightCycler APOE Mutation Detection Kit (Roche). The assay was performed as specified by the supplier, except for scaling down the total assay volume from 20 to 10 μL. The laboratory participates in an external quality assurance program  that includes APOE-genotyping. Individuals were characterized by their APOE allele combinations ϵi/j , and classified as ApoE-ϵ4-positive (ϵ4-carriers) or ApoE-ϵ4-negative (non-carriers) based on the presence or absence of at least one ϵ4 allele.The APOE genotype was determined with polymerase chain reaction (PCR) with allele-specific fluorescence energy transfer probes and melting curve analyses on the LightCycler system . As also explained in the study by Espeseth et al. , DNA wasA Norwegian translation of the California Verbal Learning Test-II (CVLT) ,49 was p® scientific programming package was used for this analysis and graphics. To assess possible complex relationships between morphometric, behavioural, and genetic variables we used the novel technique of conditional inference trees as implemented in R (version 2.7.0) software environment for statistical computations and graphics [For descriptive analysis we used matrix scatterplots and simple linear regression models with estimation and plotting of the probability distribution of each variable Figures , 4 and 5graphics . This pan = 7), ϵ3/4 (n = 48) and ϵ4/4 (n = 8). Thus, 4.7% of the sample was ϵ4/4 homozygote. In the non-carrier group of 107 subjects the allele combinations were ϵ2/3 (n = 21) or ϵ3/3 (n = 86). No subject had the allele combination ϵ2/2. Wilcoxon rank sum test for equal medians revealed no statistically significant difference for CVLT-LD scores between subjects with two, one, or zero ApoEϵ4 alleles at 5% significance level. Using the same test to compare homozygote ϵ4ϵ4 ApoE-carriers (n = 8) to the rest of the sample did not reveal any significant difference, neither in normalized hippocampus volume, nor in CVLT-LD. Median values for normalized hippocampal volumes were 0.0049 (range 0.0031–0.0061) and 0.005 (range 0.0036–0.0064), and CVLT-LD scores were 12 (range 7–16) and 13 (range 3–16) for ApoEϵ4ϵ4 versus others, respectively.The ApoEϵ4 carrier group comprised 63 subjects (37% of the study group), with allele combinations ϵ2/4 .CVLT-scores by sample and gender are presented in Table When including age, gender and normalized hippocampal volume as explanatory variables and CVLT-LD as response variable, we calculated the conditional inference tree as depicted in Figure For women under 70 years, which constituted the majority of our sample, the bilateral hippocampal volumes created a split at normalized volumes below and above 0.005. Women less than 70 years who had normalized volume below 0.005 (n = 17) performed significantly poorer on the verbal memory score than those with a higher volume (n = 82).left normalized hippocampus volume as the more powerful predictor, creating a split at normalized volume of 0.008 (Bonferroni adjusted p = 0.011). Moreover, ApoEϵ4 status did not provide any useful predictive value in this sample, as was expected due to the findings mentioned above. The other hippocampus-related parameters were also shown to be little influential in prediction of verbal memory scores.When we included additional morphological variables related to left and right hippocampus, as well as ApoEϵ4 status to the set of explanatory variables, we obtained an even simpler regression tree result. As expected, gender was still the most important variable, and males were separated from the sample and no other variables could reject the null-hypothesis for men, i.e. explain the variability in CVLT-LD score. However, age for women now vanished as a predictor, leaving only the The present study is one of few studies of normal aging where image-derived hippocampal volumes, APOE genotype, and verbal memory performance have been jointly investigated. In our investigation we also included other variables  that could be important a priori for prediction of verbal memory performance (i.e. CVLT-LD score). Applying the conditional inference model to our multivariate sample, we expected to reveal the sequential importance for each variable in predicting CVLT-LD.Investigating our results Figure  we see tWe also found that APOE genotype did not have any important predictive value regarding CVLT-LD score. This was also confirmed by the more simple Wilcoxon rank sum test. This result is in accordance with Smith et al. , who repThe role of left hippocampal volume in verbal memory function has been reported in several previous studies -61, but One other finding was that the hippocampus volume declines relatively more than the cortical volume with age Figure . This isA possible confounder in our study is the different scanners used to acquire the brain volumes. This is however, a substantial difficulty in multi-center studies, as various scanner vendors provide a wide range of models with different specifications such as field strength, gradient system, coils, and pulse sequence principles and parameters. This might not present as a problem in clinical practice, or when manual deliniations and segmentations are performed, whereas automated methods can be more sensitive to subtle differences in image properties . HoweverThe automated image segmentation method used is well proven, and correlates well with more conventional, but time-demanding and subjectivity-prone methods ,38. ThisA follow up study will be required to further investigate our findings. Such a study will provide an opportunity to do follow-up analysis much like Tupler et al. , and theUsing automated brain morphometric analysis, APOE genotyping, assessment of verbal memory function, and multivariate statistical methods based on conditional inference trees in a sample of 170 subjects (age range 46–77 years), we found that gender and left hippocampal volumes are main predictors for verbal memory function in normal aging. APOE genotype seems not to have any significant effect on verbal memory function. Reduced left side hippocampus volume seems more important than right side reduction for verbal memory decline in women, especially in women 70 years of age or less. Moreover, men seem to have more affected verbal memory function in this age group than women, and this memory decline in men seems not to be related to hippocampal volume. To further investigate these findings, we are about to conduct a follow-up study, using the same data collection protocols in both Bergen and Oslo.The authors declare that they have no competing interests.MY was involved in data preparation, morphometric analysis, statistical analysis, main author. AJL was involved in study design, supervisor, internal review. EW was involved in data collection, neuropsychological assessment, internal review. TE was involved in sata collection, neuropsychological assessment, internal review. HR was involved in APOE-genotyping, internal review. LTW was involved in data preparation, morphometric analysis, internal review. MA was involved in data collection, neuropsychological assessment, internal review. SA was involved in data processing, internal review. JTG was involved in data acquisition, internal review. AMF was involved in data processing, internal review. IR was the principal investigator of the main study in Oslo and Bergen, and participated in study design, internal review. AL was the project supervisor, and participated in MRI acquisition protocol design, statistical analysis, internal review.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2342/9/17/prepub


The objective was primarily to describe short term intra-individual variation in serum levels of soluble adhesion molecules  and vascular cellular adhesion molecule-1(sVCAM-1)) in healthy subjects. Secondly, sCAMs were correlated to brachial artery flow mediated vasodilation (FMD).Forty healthy subjects aged 24–66 years had sCAMs measured twice with 4 week intervals and short-term intra-individual variation was estimated as variation in the paired measurements after correcting for the analytical precision of the used method. At baseline, brachial FMD was measured.No difference was observed in mean sCAMs in the whole study group. Estimated intra-subject variations in sCAMs were 7.6–11.3%. In a regression analysis, significant negative association was found between sE-selectin and FMD after controlling for possible confounders (p < 0.04) while no significant correlation could be demonstrated between the other sCAMs and FMD.In conclusion, short term intra-individual variations in sCAMs were 7.6–11.3% in healthy subjects. We also found a significant negative association between sE-selectin and FMD, indicating an possible association between inflammation and dysfunction of the vascular endothelium; however further studies are required to confirm this preliminary finding. The recruitment, adhesion and subsequent transendothelial migration of circulating leucocytes is important in the initiation and progression of atherosclerosis . These pThe biological variability of sCAMs needs to be known and be low enough to enable reliable risk stratification. The intra-individual variability in serum levels of sCAMs has not previously to our knowledge been investigated in detail.Flow mediated vasodilation (FMD) in the brachial artery is a non-invasive method to access endothelial function. FMD correlate negatively with known risk factors of CVD, such as dyslipidemia, diabetes, hypertension, smoking , adiposiIn the present study, we have investigated the short time variation in sCAMs in healthy volunteers and (as a pilot study) correlated FMD to plasma levels of sCAMs.Forty healthy subjects were recruited from the medical staff, bank employees and students from Aalborg, Denmark. None of the volunteers took any medication or had any known disease. The procedures followed were in accordance with the ethical standards of the responsible Regional Committee on Human Experiments and in accordance with the Helsinki Declaration of 1975 as revised in 1983. All subjects gave written informed consent.Blood samples were taken from the left cubital vein twice with an interval of 4 weeks . Venous blood was collected between 08:00 h and 09:30 h after fasting overnight for at least 10 hours. Blood samples were allowed to clot for 1 hour at room temperature, and serum collected after centrifugation at 2500 × g for 20 minutes and stored at −80 °C until analysis. Determination of serum levels of VCAM-1, ICAM-1, E-selectin and P-selectin were performed using commercially available test-kits , BBE 1B (sICAM-1), BBE 6(sP-selectin), BBE 2B (sE-selectin)). Analysis was performed as batch-operation using the same analysis-kit. The samples were analyzed twice and the average of the two measurements is reported.Subjects were fasting overnight and instructed to abstain from tobacco smoking and coffee. After blood samples had been drawn from the left arm, subjects were placed supine at rest for 15 minutes in a quiet room at normal room temperature. A continuous ECG was recorded for timing of measurements. The right radial artery was imaged in a longitudinal section 3 cm below the antecubital fossa. The probe was held in a constant position by a stereotactic clamp. Baseline dimensions were recorded at the center of the vessel with optimal contrast between the anterior and posterior vessel wall and the lumen. Doppler blood flow images were recorded from the center of the vessel. Then an arterial occlusion cuff, placed above the antecubital fossa was inflated to 300 mmHg for five minutes. Immediately after deflation of the cuff, a continuous 90 seconds recording of blood flow and vessel dimensions was obtained. The peak diameter of the artery was recorded 60 seconds after deflation of the cuff, and the percentage change from the baseline diameter was calculated.Analyses were conducted using STATA statistical software, version 9.1. Short term variation in sCAMs was estimated by a paired t-test and coefficient of variance was calculated as standard deviation of differences between paired measurements divided mean difference in sCAMs within-subjects (in sCAMs between baseline and after 4 weeks). Analytical precision was calculated as a coefficient of variation based on double measurements of all blood samples taken at baseline and after 4 weeks (80 double measurements).Pearson’s correlation coefficient was used as a measure of linear association. Linear regression analysis was conducted to correct for possible confounding effects. Age was included as a numerical variable and age-groups as an indicator variable, allowing for non-linear relation between age and sCAMs. Body Mass Index, serum total cholesterol, systolic blood pressure were included as numerical variables and (current) smoking status and gender as indicator variables. A p-value <0.05 (two tailed) was considered statistically significant.Characteristics of the forty participants are given in study% of the measurements is given. Also the table provides an estimate of the intra-individual variation, CVintra-subject% , based on the assumption that the measured CVstudy% consists of two components: variation  of the assay  and variation in the difference between the paired measurements . Then intra-individual variation were calculated using CVstudy2 = CVanalytical2 + CVintra-subject2. Overall, the CVintra-subject% for each sCAM was between 7.6–11.3%.The results of measurements of sCAMs with four weeks interval are given in No significant correlations were found between FMD and each of sCAM, systolic blood pressure, BMI, smoking habits, lipids and lipoproteins or age. By gender, scatter plots of the data revealed that the variability were higher in the females (data not shown). Variation also increased with age. Therefore, a regression analysis was performed with FMD as independent variable and sCAMs, gender and age. Age was included both as a numerical variable and with two age groups according to median age (51 years) as an indicator variable, allowing for non-linear relation between age and sCAMs. This effect remained significant after including following dependent variables (possible confounders): serum total cholesterol, BMI, systolic blood pressure and smoking habits. The results of the regression analysis are given in sCAMs may become a routinely used risk marker of preclinical atherosclerosis and therefore information about the within-subject biological variation is essential. In the present study, our primary objective was to estimate within-subject short term biological variation in serum level of sCAMs in healthy volunteers. The estimated within-subject variation coefficients were between 7.6–11.3% for all measured sCAMs. The difference in the study population mean serum sCAMs was negligible (less than 1%) in samples taken 4 weeks apart. Also analytical precision of commercially available analysis assays is excellent (CV% of approximately 2%). We have no knowledge of other studies investigating the short term variation in sCAMs under standardized conditions. However, variations in sCAMs have been investigated both as diurnal variations and variation due to physical or psychological stress. The diurnal intra-subject variation in sCAMs in healthy subjects and in patients with CHD have been reported for P-selectin, ICAM-1 and E-selectin ; only P-In the second part of our study we aimed to investigate an association between two important aspects in the progression of atherosclerosis, that is the lowgrade endothelial/systemic inflammation and the endothelial dysfunction. There is little knowledge of interrelations between FMD and sCAMs in healthy individual. In contrast, several reports have related either FMD or sCAMs to the presence of traditional risk factors for CVD, such as dyslipidemia, diabetes , hyperteLimitations of our study are primarily the small number of subjects under investigation. However, the differences in sCAMs between within subject paired measurements showed good approximation to a normal distribution. Population size was of greater importance in the investigation of a possible association between sCAMs and FMD. Because data analysis revealed a marked age-dependency and marked gender dependency on the association between sCAMs and FMD, a regression analysis including possible confounders were performed. However, care should be taken to make firm conclusions based on this part of the study. This part of our study should only be viewed as a pilot study. A large scale study is required to establish an association between sCAMs and FMD.In conclusion, short term intra-individual coefficients of variation in sCAMs in healthy subjects are estimated to be 7.6–11.3 percent under standardized condition. This intra-subject variation need consideration in evaluation of sCAMs in experimental trials and in clinical use. We also found a weak significant negative correlation between sE-selectin and FMD, indicating an association of dysfunction and inflammation in the vascular endothelium. However, due to the small population size, a larger study is required to establish possible association between FMD and sCAMs.


Mycobacterium tuberculosis infection (LTBI). Their performance may depend on assay format and may vary across populations and settings. We compared the diagnostic performance of an in-house T -cell and commercial whole blood-based IGRAs for the diagnosis of LTBI and TB disease in The Gambia.IFN-γ Release Assays (IGRAs) have been licensed for the diagnosis of latent M. tuberculosis exposure according to where s/he slept relative to a case: the same room, same house or a different house. We assessed the relationship between exposure and test results by multiple logistic regression.Newly diagnosed sputum smear positive cases and their household contacts were recruited. Cases and contacts were bled for IGRA and contacts had a Mantoux skin test. We assessed agreement and discordance between the tests and categorized a contact's level of M. tuberculosis exposure gradient and were positively associated with increasing TST induration (p = 0.003 for ELISPOT and p = 0.001 for QFT-GIT).In 80 newly diagnosed TB cases, the sensitivity of ELISPOT was 78.7% and for QFT-GIT was 64.0% (p = 0.047). Of 194 household contacts 57.1% and 58.8% were positive for ELISPOT and QFT-GIT respectively. The overall agreement between both IGRAs for LTBI in contacts was 71.4% and there was no significant discordance (p = 0.29). There was significant discordance between the IGRAs and TST. Neither IGRA nor TST had evidence of false positive results because of Bacille Calmette Guérin (BCG) vaccination. However, agreement between QFT-GIT and TST as well as discordance between both IGRAs and TST were associated with BCG vaccination. Both IGRAs responded to the The ELISPOT test is more sensitive than the QFT-GIT for diagnosing TB disease. The two tests perform similarly in the diagnosis of LTBI in TB contacts. Significant discordance between the two IGRAs and between each and the TST remain largely unexplained. Mycobacterium tuberculosis  and 0.92  respectively, both remaining non-significant after adjustment for possible confounding variables. The agreement between ELISPOT and TST did not differ significantly according to BCG scar status being 69.6%  in those contacts with a BCG scar and 62.4%  in those without a BCG scar. In contrast, agreement between QFT-GIT and TST was significantly different by BCG scar status (p = 0.03), being 76.5%,  in those with a scar and 68.2%  in those without a scar.2 = 21.6, p < 0.001 and χ2 = 50.6, p < 0.001 respectively). With increasing TST induration there was a trend towards increase in the proportions of contacts with positive ELISPOT and QFT-GIT results and this was statistically significant (p = 0.003 and p = 0.0001 respectively).In contacts, when a positive TST was defined as ≥5 mm, 86  of 106 TST positives were also QFT-GIT positive compared to 73  of 102 for the ELISPOT but this difference in proportions was not significant (p = 0.10). With a positive TST defined as ≥10 mm, 73  of 90 were QFT-GIT positive and 72  of 88 had positive ELISPOTs. When a definition of ≥15 mm was used, 36  of 46 had positive QFT-GIT, similar to the ELISPOT with 37  of 47. These differences in proportions were also not significant (p = 0.18 and p = 0.96 respectively). Figure M. tuberculosis exposure categories (represented as sleeping proximity to a case) are shown in Table The odds of test positivity were also not related to sputum smear grade , or duration of cough . The univariate odds of a positive result across the M. tuberculosis infection and disease in a resource poor TB-endemic tropical setting. Our results suggest both IGRAs, the QFT-GIT and our in house ELISPOT perform similarly in the diagnosis LTBI in contacts after recent exposure to a known TB case. However, the ELISPOT is more sensitive in diagnosing active PTB in cases.This study now provides a comparison of an ELISPOT test with QFT-GIT for the diagnosis of The sensitivity of 78.7% for the ELISPOT is within our previously published range 72.6–89.8%  and 77–9M. tuberculosis in The Gambia. Both tests responded appropriately to a gradient of recent exposure to M. tuberculosis and there was no significant divergence in results according to level of exposure. While we do not find strong evidence for sensitivity difference in case contacts, the tentative consensus from published literature is that ELISPOT does have better sensitivity with respect to LTBI [This study has taken advantage of a reproducible gradient of exposure to  to LTBI .Consistent with other studies, we show considerable discordance between the TST and the IGRAs and between the IGRAs themselves. Our results show significant discordance between the IGRAs in cases. Although about a quarter of ELISPOT or QFT-GIT positive contacts were negative with the other test, the discordance was not statistically significant. Discordance between IGRAs in cases and contacts has been described in several reports ,21,26. SThe absence of any significant effect of BCG vaccine on the TST, ELISPOT and QFT-GIT has remained a constant feature in Gambian studies, ,32 and hThat the TST was not done at the same time as the IGRAs may also be a considered a weakness of this study, as it makes any comparison of IGRA with the TST, subject to mis-classification bias. However, our primary goal was not to compare the IGRAs with the TST but to evaluate the IGRAs in relation to each other. The TST is subject to boosting. Although, there are some reports of possible boosting, by a previous TST, of the QFT-GIT and not the ELISPOT, large conclusive studies are lacking -39. The The higher test failure rate of the ELISPOT, compared to the QFT-GIT, in our study is inconsistent with other studies, where the opposite has been reported ,21. We nM. tuberculosis. This will require large, possibly multi-site, follow-up studies of TB case contacts and molecular subtyping of case and secondary case isolates. Such follow-up studies are underway in The Gambia.A limitation of IGRAs is their relatively high cost and need for laboratory infrastructure. The greater through-put of samples for QFT-GIT gives it some advantage in this light. However, one would need to demonstrate significant further advantage over the TST than is seen here, to justify the investment for developing countries in particular. The outstanding question to resolve now is whether IGRAs show improved predictive value for progression to active disease, once an individual is infected with M. tuberculosis infection in The Gambia.This study has shown that an in-house ELISPOT has greater sensitivity than the QFT-GIT for diagnosing active TB disease, and perform similarly with respect to The author(s) declare that they have no competing interests.IMO contributed to study design, conducted field and clinical activities, and was involved interpretation of laboratory data and data analysis.PC conceived of the study, and participated in its design and coordination, and was involved in data analysis.DJ contributed to design of the study and data analysis.SD contributed to study design and managed the data generated.ML carried out ELISPOTs and was involved in data interpretation.AH carried out QFT-GIT assays and was involved in data interpretation.RA provided overall supervision and was responsible for TB diagnosticsAll authors read and approved the final manuscript.This study was funded by the Medical Research Council (United Kingdom), QFT-GIT kits were provided free by Cellestis Limited, Carnegie, Australia. IMOA receives financial support from the European and Developing Countries' Clinical Trials Partnership (EDCTP).The pre-publication history for this paper can be accessed here:


While structural and functional alterations in mitochondria (mt) and their metabolites have been implicated in the pathogenesis of chronic neurodegenerative and vascular diseases, the relationship of inherited variants in the mitochondrial genome and mt haplogroup MT-ND4 and A14233G of MT-ND6). We genotyped T haplogroups in an independent sample of 490 cases and 61 controls from the same study (Stage II) and in 56 cases and 246 controls from the 10-year study (Stage III). People in the T2 haplogroup were approximately 2.5 times more likely to have advanced AMD than their peers  after considering the totality of evidence. Findings persisted after considering the impact of AMD-associated variants A69S and Y402H .We examined the relationship of inherited mtDNA variants with advanced AMD in 1168 people using a three-stage design on samples from 12-year and 10-year prospective studies on the natural history of age-related eye disease. In Stage I we resequenced the entire genome in 99 elderly AMD-free controls and 215 people with advanced AMD from the 12-year study. A consistent association with AMD in 14 of 17 SNPs characterizing the mtDNA T haplogroup emerged. Further analysis revealed these associations were driven entirely by the T2 haplogroup, and characterized by two variants in Complex I genes (A11812G of Loci defining the mtDNA T2 haplogroup and Complex I are reasonable targets for novel functional analyses and therapeutic research in AMD. We applied a rapid high-throughput method for this purpose on the basis of six lines of evidence: 1) cells most affected in AMD  and photoreceptors) are densely packed with mitochondria; 2) ultrastructure of RPE and photoreceptor mitochondria is altered in people with AMD MT-ND4  and MT-ND6 (subunit 6).Age-related macular degeneration (AMD) is the leading cause of blindness in elderly people of western European ancestry P≤0.05 . The term ‘AREDS A’ is used to designate this cohort. We identified 998 unique variants and analyzed all 315 SNPs that were both called reliably at ≥90% of samples and present in at least 1% of cases or controls. Seventeen SNPs yielded associations with advanced AMD at P≤0.05 Table 1.P≤0.68). However, there was a 10.5-fold increased likelihood having advanced AMD if they carried the T2 variants .P≤0.27). Results for the T2 association were also concordant: OR for advanced AMD in the sex-, age-, and smoking-adjusted model for T2 in AREDS B was 6.12 . To obtain a more accurate assessment of AMD risk associated with the T2 haplogroup, we pooled samples from both AREDS A and AREDS B. As ORs were not different .In Stage II we validated our findings by genotyping A4917G and A11812G in an independent AREDS sample of 490 cases and 61 controls. The term ‘AREDS B’ is used to designate this cohort. We used exact methods to confirm that allele frequencies for A4917G and A11812G were not significantly different across AREDS A and AREDS B. T1-associated SNPs were balanced across cases and controls in both samples. T2 distributions show that the advanced AMD group contained a higher proportion of people with 11812G and 4917G in each sample: 4917G/11812G existed in 2.02% of 99 controls vs. 14.42% of 215 cases in AREDS A and in 1.64% of 61 controls vs. 7.96% of 490 cases in AREDS B. Change in magnitude and precision of ORs was negligible after concurrent adjustment for age, sex, and smoking history. As in the AREDS A cohort, people in the T1 haplogroup were equally likely to have advanced AMD as their peers. The OR for advanced AMD in the sex-, age-, and smoking-adjusted model for T1 in AREDS B was 0.64 . T2 frequency was 7.72% (95%CI 5.0%–11.6%) for the AMD-free group and 10.71% (95%CI 5.1%–21.5%) for the advanced AMD group. T2-advanced AMD measures of association did not attain statistical significance within the BMES cohort . Combining our independent replication samples (AREDS B and BMES) yielded an OR = 1.87 .In Stage III we genotyped A4917G and A11812G in Blue Mountains Eye Study (BMES) participants with advanced AMD (n = 56) or who were ≥74 years-of-age and AMD-free (n = 246). T1 frequency was 1.23% (95%CI 0.45%–3.6%) in the AMD-free group and 3.57% (95%CI 1.10%–12.11%) in the advanced AMD group. Sex-, age-, and smoking-adjusted multivariable models yielded a non-significant increased odds of having advanced AMD among people carrying the T1-defining variants . We also combined raw data from AREDS A, AREDS B, and BMES in our final multivariable logistic regression models with a term for sample source (AREDS vs BMES), as differences in ORs for T2 between AREDS and BMES cohorts were not statistically different (P = 0.08). T2-AMD results persisted and were of the same magnitude of those from the DerSimonian & Laird estimates . Considering the totality of evidence from these 2 large longitudinal studies, people carrying the T2 variant  were more likely than their AMD-free peers to have advanced AMD.T2 haplogroup frequency for AREDS  and BMES  was similar and within limits reported from U.S. cohorts of European ancestry P≤0.028)). Data on Y402H existed for 52 BMES participants with advanced AMD and 238 of their AMD-free peers. The T2-advanced AMD relationship persisted after adding these data to the pooled AREDS A and AREDS B cohort . We conclude that presence of the Y402H or A69S risk allele did not alter the T2 haplogroup-advanced AMD association.We tested the independence of the T2-advanced AMD relationship by simultaneously entering A11812G with AMD susceptibility loci Y402H and A69S in age-, sex-, and smoking-adjusted logistic regression models. The distribution of A11812G did not vary by Y402H or A69S Table S2we consider our findings most valuable for exploratory purposes. BMES was a population-based cohort and although T2 frequency was similar to AREDS in the overall sample and AMD groups, this study contributed only ∼10% of our advanced AMD cases. While members of AREDS and BMES cohorts were all of European origin we did not have specific information on ethnic background. This may partially explain our findings, as clear differences in allele frequencies exist between European populations by geographic origin of ancestry MT-ND4. MT-ND4 codes for NADH dehydrogenase subunit 4 – a homologue of the E. coli NDH-1 proton translocation module polypeptide nuoM. NuoM shares strong sequence similarity with the antiporter multiple resistance and pH locus (Mrp) D of numerous Bacillus strains and for this reason is thought to have been recruited as a functional unit (instead of evolving from gene duplications). The nuoM link with this class of antiporter suggests a H+ channel composition and activity within proton translocation systems. MT-ND4 has also been implicated in ubiquinone biosynthesis and is essential for complex I assembly MT-ND4 variant characterizing the T2 haplogroup existed in a small case study of people with Leber hereditary optic neuropathy (LHON), a blinding disease MT-ND6 (NADH dehydrogenase subunit 6). MT-ND6 products compose elements of the proton translocation module. Like MT-ND4, MT-ND6 is essential for complex I assembly MT-ND6 is thought to be incorporated into complex I early in initial phases of the assembly process, while MT-ND4 follows after assembly of a number of intermediate subunits Our aim was to elucidate novel mtDNA-AMD relationships and mut mouse cause premature aging phenotypes without altering reactive oxygen species production, suggesting that respiratory chain dysfunction itself is the etiologic factor in many age-related diseases Neither A11812G nor A14233G are non-synonymous substitutions. While there is currently no evidence to conclusively demonstrate that these variants may impact translation dynamics for complex I , our results provide a reasonable basis for examining this possibility. We also note that 1–2% of the mt genome does not yield high-quality sequence data, and our resequencing algorithm is not designed to detect insertions/deletions. Thus, it is possible that the T2-associated functional variant is actually in an unsequenced region or a copy number variant, so that evaluating this possibility is a high priority. Somatic mtDNA mutations induced in the PolgACFH gene as a strong risk factor for advanced AMD P-value≤0.0001) were similar to those from extant reports. Results for T2  were similar to those we report for data combined from AREDS A+AREDS B. In summary, modeling Y402H and T2 simultaneously did not alter the standard error of either variable and yielded a negligible change in the magnitude of ORs. A strong AMD-LOC387715 (A69S) association also exists in the AREDS cohort P-value≤0.0001 in the model described above was similar to those reported in independent samples; in our analyses T2 retained significance . Modeling Y402H, A69S and T2 simultaneously did not impact the variance in either variable and resulted in no appreciable change to the magnitude of ORs , 2-sided P≤0.029). We conclude that presence of the Y402H or A69S risk allele does not alter the effect of the T2 haplogroup on advanced AMD . AREDS data were used in the first genome-wide association study to identify the Y402H variant of the In conclusion, we used a rapid high-throughput method to determine that prevalence of two variants defining the mtDNA T2 haplogroup was higher in people with sight-threatening AMD than in their healthy peers. Both variants existed in highly conserved genes of NADH ubiquinone oxidoreductase (Complex I). If these results are substantiated by functional studies demonstrating OXPHOS deficits in people with AMD, they may provide a basis for planning clinical trials with compounds exhibiting antioxidant and mitotropic properties.Our inferences are based on a 3-stage research design in 406 AMD-free people and 762 people with advanced AMD. We applied a mitochondrial genome-sequence scan, evaluation of single marker- and mtDNA haplogroup-AMD associations, and validation with representative SNPs in independent cohorts examined in large prospective studies designed to systematically measure AMD endpoints. All data are from elderly self-identified white, non-Hispanic U.S. and Australian residents participating in The Age-Related Eye Disease Study (AREDS) Details of the Age-Related Eye Disease Study (AREDS) study design and methods exist elsewhere Details of the Blue Mountains Eye Study (BMES) exist elsewhere AREDS Report 1 contains information on the process of outcome ascertainment Our central aim was identification of AMD susceptibility loci and haplogroups. Our AMD-free group had good vision in both eyes and no existing eye disease. Controls were selected to be the oldest participants with DNA available who had no or less than 5 small drusen (<63 µm diameter) and no pigmentary abnormalities in both eyes at all visits for which fundus photographs were gradable. Both eyes had visual acuity of 20/32 or better measured by a standard protocol, ocular media was clear enough for good quality fundus photographs, and there was absence of any ocular disorder that might obscure assessment of either AMD or lens opacities.Our controls have three distinguishing characteristics that make them the best existing comparison group for this purpose:Phenotype was determined annually over a 12-year period (AREDS) or across a 10-year period with a standardized protocol by multiple professional graders who were masked to phenotypic information from previous years. Adjudication with a standardized protocol occurred when discrepancies emerged.The criteria for AMD-free classification (<5 drusen of ≤63 µm in both eyes for the entire follow-up period) is stringent relative to those applied in previous association studies for AMD.The age of the AREDS AMD-free group is in the range that AMD prevalence increases ∼3 times (from ∼4% in those aged 74-to-79 to ∼12% in those aged ≥80-years) in population-based studies.The likelihood of having AMD increases 2-to-6 fold after age 75 and it was therefore essential to select our oldest AMD-free participants to reduce the false negative rate that would otherwise result from non-random misclassification in the youngest members of the control group. The mean age (±standard error) of controls in our combined cohorts was 77.7 (±0.29) years. R. Klein and M. Knudtson at the Beaver Dam Eye Study (BDES) applied our definition of AMD-free people in their population-based sample of U.S. residents of European ancestry. Only 3.3% of the 4,628 participants were both aged 75-to-86 years and AMD-free P-values are computed accordingly. Results from these analyses provided an empirical basis to pool samples.We examined the disparity between ORs in AREDS A, AREDS B, and BMES cohorts by calculating the difference between their corresponding logarithms (log odds) and dividing this value by the square root of the standard errors of the log odds. This ratio has an approximate standard normal distribution under the hypothesis of no difference between samples, and Haplotype frequency for T2 in our combined AREDS and BMES sample was 8.21% (95%CI 6.78%–9.94%); this value is within expected limits of those reported for U.S.-based cohorts of European ancestry et al.We applied the method of Samuels 2 index to quantify the degree of heterogeneity attributed to between-studies variation. For T2, 50% of the total variability in the magnitude of OR can be explained by actual heterogeneity between studies. On this basis we selected the point estimate derived from the random-effects model.We used the method of DerSimonian and Laird http://www.dpgp.org/RA/ra.htm) based on the ABACUS algorithm under the assumption of a haploid genome Resequencing of the entire mitochondrial genome was performed using the Affymetrix GeneChip Human Mitochondrial Resequencing Array 2.0 according to manufacturer's protocols. PCR and hybridization conditions are as previously described In the AREDS cohort the 11812G variant always existed with the 14233G variant. We believe that the likelihood that an individual would carry only one of these variants is extremely low, given the absence of this event in any of the 400 independent mitochondrial samples we have analyzed for work outside of the current study.Genotyping for mtSNPs 4917 and 11812 were performed using TaqMan Assays by Design (Applied Biosystems). Primers and probes are listed below:SNP Forward Primer Sequence Reverse Primer SequenceGACATCCGGCCTGCTTCT TGGGTTTGGTTTAATCCACCTCAMITO4917 ACAGTCGCATCATAATCCTCTCTCA GAGGTTAGCGAGGCTTGCTMITO11812 SNP Reporter 1 Sequence Reporter 2 SequenceCTCCCTCACTAAACGTAAG TCCCTCACTAGACGTAAGMITO4917 ACTTCAAACTCTACTCCCAC TTCAAACTCTGCTCCCACMITO11812 Table S1Demographic and life-style characteristics of AREDS and BMES samples. All participants are self-identified as being of being of non-Hispanic white origin. AMD = age-related macular degenration; Adv. AMD = advanced AMD (geographic atrophy and or neovascular) AREDS = Age-Related Eye Disease Study; BMES = Blue Mountains Eye Study; GA = geographic atrophy; NV = neovascular; stderr = standard error. AREDS A and AREDS B cohorts are independent. Demographic data are for all participants with T1 or T2 base calls.(0.02 MB XLS)Click here for additional data file.Table S2Cross-tabulations of percent allelic frequency in T2-associated variants for Y402H and A69S(0.02 MB XLS)Click here for additional data file.