The association of genetic polymorphisms related to metabolism of homocysteine with inflammatory bowel disease has been evidenced in Crohn disease and remains an open question in ulcerative colitis. We evaluated the association of the polymorphisms of MTHFR, MTR, MTRR and TCN2 genes with ulcerative colitis in Central China.168 patients were genotyped for these polymorphisms and compared to 219 matched controls.Methionine synthase 2756G allele frequency was higher in ulcerative colitis than in controls 0.15 (95% C.I. 0.11–0.19) vs 0.09 (95% C.I. 0.07 – 0.12), (P = 0.0137) and predicted ulcerative colitis risk in logistic regression, with an Odds ratio at 1.8 (95% C.I. 1.15–2.84). Methylenetetrahydrofolate reductase 677TT genotype was 2.7-fold more prevalent in individuals with pancolitis than in those with left colitis or proctitis, with respective percentages of 27.3 (95% C.I.16.4–42.0) and 10.5 (95% C.I. 6.3–17.1) (P = 0.0123). The carriage of 677TT or 677CT/1298AC genotypes of methylenetetrahydrofolate reductase was more frequent in cases with pancolitis than in subjects with left colitis or proctitis (P = 0.0048), with an Odds ratio adjusted by age and sex at 3.3 (95% C.I. 1.4–7.9), P = 0.0084) in logistic regression.Methionine synthase and methylenetetrahydrofolate reductase are genes of vitamin B12 and folate cellular metabolism associated respectively with risk and extent of ulcerative colitis, at least in Central China. This finding may open new insights, particularly for the potential interest in treating patients carrying the 677TT MTHFR genetic trait and a deficit in folate. Plasma R C677T) ,3, methi A2756G)  and meth677T allele in IBD patients, compared to controls [MTHFR variants may explain, at least in part, discrepancies between case control studies on the association between MTHFR polymorphism and IBD [MTHFR, MTR, MTRR and TCN2 polymorphisms on the primary and secondary risk of ulcerative colitis have never been evaluated in a same sample population. Most of the case-control studies have evaluated Caucasian populations from North America and Europe, where the prevalence of Crohn disease is predominant, in contrast with populations from Asia, where UC is the most frequent form of IBD. In contrast to MTHFR 677T allele, the G allele frequency of TCN2 polymorphism is dramatically higher in Central China, compared with that reported in Caucasian populations [Hyperhomocysteinemia is recognized as a risk factor for both venous and arterial thrombosis in the general population ,8. In thcontrols . Neverthcontrols -17. Ethn and IBD ,19. Polyulations ,18,19.MTHFR, MTR, MTRR and TCN2 with the primary risk and the clinical manifestations of UC, in a case control study of a sample population from Central China.The aim of this study was therefore to evaluate the influence of genetic determinants of homocysteine metabolism, Eligible patients were males or females with established ulcerative colitis UC. 168 patients who were recruited from one single department  were prospectively enrolled in a case-control study, during a 2-years period. Information regarding concomitant medications was collected at study entry. Extent of UC was defined according to Montreal Classification . The terC677T and A1298C polymorphisms of MTHFR, as well as the A2756G MTR and the A66G MTRR polymorphisms, were based on polymerase chain reaction (PCR) amplification, restriction cleavage and separation of the DNA fragments by 15% non denaturant polyacrylamide gel electrophoresis (SDS-PAGE), as previously described [TCN2 C776G polymorphism was performed by the amplification-refractory mutation system, as described recently by us [MTHFR, MTR and MTRR genotypes were sequenced and subsequently used as controls in all series of genotype determination. Five % of the samples were re-genotyped to check for genotype calling consistency, with a genotyping success rate higher than 98%.Fasting venous blood was collected in EDTA-containing tubes, immediately centrifuged, and stored at -20°C until analysis. DNA was isolated from a lymphocyte-enriched fraction of whole blood with NUCLEON BACC3 for extraction of genomic DNA kit . The procedures for detecting the escribed ,19. Genoa priori hypotheses were the association of the genetic determinants of homocysteine metabolism with the primary risk and the clinical characteristics of the disease, respectively. Categorical variables were reported as counts, percentages and 95% confidence interval, and continuous variables as means ± SD. For categorical variables, continuity-corrected chi-square test or exact Fisher test  were used to assess differences. For continuous variables, a Mann-Whitney U-test was employed. The significance, odds ratios (OR) and 95% confidence interval of independent determinants regarding the risk of IBD were determined by logistic regression analysis using a model that included age, sex and the genetic and clinical variables. The residual model of logistic regression multivariate analysis considered only the variables with P-value < 0.10 in the univariate analysis. The minimal size of our sample was estimated at 150 patients, with a study power 1-β = 0.8 and α = 0.05, assuming a 1.5-fold difference in the less frequent alleles between controls and patients. P-value lower than 0.05 indicated statistical significance and Bonferroni correction for multiple testing was used as described previously, when several dependent or independent statistical tests were being performed simultaneously [The tested aneously . ; the age was respectively 41 ± 15 and 41 ± 12 years (P = 0.7428). The age onset was 38 ± 15, without difference among gender. The main clinical characteristic of the UC patients are summarized in table MTHFR C677T, A1298C, MTR, MTRR, and TCN could be determined in respectively 215, 213, 218, 219, 216 of the 219 DNA samples from controls and in respectively 168, 160, 156, 164, 146 of the 168 DNA samples from UC cases. The genotype distributions of MTHFR, MTR, MTRR, and TCN polymorphisms of patients were in Hardy-Weinberg equilibrium  and were not influenced by gender (data not shown). MTHFR 677T and 1298C alleles were in complete disequlibrium, as previously reported in other populations [There was no linkage desequilibrium among different genotypes. The genotype of MTHFR, MTRR and TCN polymorphisms did not differ between controls and UC patients  and 16.5 (12.2–22.0) (P = 0.0212). The same observation was made with the MTR 2756G allele frequency, with a difference even more significant  and 10.5 (95% C.I. 6.3–17.1) (P = 0.0123). The frequency of subjects who presented with either 677TT or the double heterozygous 677CT/1298AC genotype was also significantly different between subjects with pancolitis and those with left colitis or proctitis, with respective percentages of 43.2 (95% C.I. 29.6–43.2) and 20.2 (95% C.I. 14.1–28.1) (P = 0.0048).We next examined the association of polymorphisms of homocysteine metabolism-related enzymes with the clinical characteristics. None of the polymorphisms influenced the age onset of the disease. In univariate analysis, MTHFR. The sex and age adjusted odds ratio of the association between pancolitis and MTHFR 677 TT was estimated to 3.82 . When the age and sex adjusted analysis was performed by considering the carriers of 677TT or C677CT/1298AC genotypes of MTHFR instead of MTHFR 677TT only, the same conclusion was reached, with an Odds ratio at 3.3 .Finally, the association of gene polymorphisms of genes of the remethylation pathway with the risk of pancolitis was estimated by logistic regression analysis using a model that included age, sex, medications and genetic polymorphisms. We confirmed that the only significant independent gene predictor of extensive lesions was MTHFR, MTR, MTRR and TCN2, in a case population restricted to UC, in China. It evidenced a significant association of MTR and MTHFR variants with the primary risk and the extent of the disease, respectively; 677TT and C677CT/1298AC genotypes of MTHFR were predictors of extensive colitis, while no association was found with the primary risk of UC. In contrast, MTR 2756G allele was associated with an increased risk of UC and had no influence on the extent of the disease. MTR and MTFHR have a complementary role in this pathway. During the remethylation of homocysteine into methionine, a methyl group provided by 5-methyltetrahydrofolate (5-methylTHF) is transferred to homocysteine by MTR. In this remethylation pathway, cobalamin (vitamin B12) is involved as an intermediate carrier of the methyl group while the 5-methylTHF is synthesized by MTHFR. The phenotypic influence of the 2756G allele on the activity of MTR has not been clearly evidenced [677TT and 1298AC/677CT genotypes are known to produce a decreased catalytic enzyme activity of MTHFR [677TT genotype of MTHFR is encoding a thermolabile variant characterized by an alanine to valine substitution at position 222, and a 50% reduction in enzyme activity [MTHFR C677T polymorphism produces a decreased cellular level of methyl-THF and a cellular accumulation of homocysteine, particularly in patients with insufficient folate supply [Genetic factors possibly associated with ulcerative colitis remain poorly known. This is the first study that evaluated the association of genes of the remethylation pathway of homocysteine, videnced . In conte supply . InteresMTHFR C677T polymorphism include increased risk for several diseases, such as vascular, neurological and for birth defects [MTHFR with other diseases than IBD seems to be neutral in South European countries, where the status of populations in folate is better than in North Europe, while some studies have showed association with MTR and MTRR in these countries [MTHFR TT genotype [MTRR 66A allele in patients with Crohn's disease compared with controls, and we observed an influence of this gene variant on the extent of the disease [methionine synthase A2756G polymorphism, a vitamin B12-dependent enzyme, may predict ulcerative colitis. The data are also difficult to compare among contrasted populations since the frequency of these gene polymorphisms and their influence on homocysteine fluctuate greatly, worldwide [T allele seems to be influenced by folate status, the prevalence being the highest in Mexicans, Chinese and Italians and the lowest in West Africans [MTHFR that have been published in Europe by evaluating also the association with the other gene variants related with the remethylation pathway. Finally, the discrepancies observed in previous studies on association between MTHFR and IBD may also correspond in part to differences in the proportion of cases with extensive colitis, as suggested by our observation that MTFHR is a predictor of pancolitis, at least in UC. This observation was in agreement with our previous study of a smaller series of UC patients [MTHFR gene may result in reduced S-adenosylmethionine concentations, leading to enhanced TPMT enzyme degradation and possibly modulating azathioprine efficacy [Clinical implications of the  defects ,23. Its  defects . Similarountries . This cogenotype ,25, whergenotype ,14-17. I disease . We also disease . Consist disease . Taken torldwide ,19. The Africans ,19. It wpatients . In addipatients . Vitaminpatients . Homocyspatients . Unfortupatients , suggestefficacy .MTHFR C677T polymorphism have been regarded solely as potential contributors to the greater risk of thrombosis in IBD [Over the past decade, hyperhomocysteinemia and s in IBD ,15,30-32MTR and MTHFR are two genes of vitamin B12 and folate cellular metabolism associated respectively with the primary risk and the severity of UC, at least in central China. These findings might open new insights into the pathogenesis and assessment of UC, particularly for the potential interest in treating the patients presenting with the 677TT MTHFR genetic trait and a deficit in folate. However, our results await confirmation in a large cohort of patients with UC.The authors declare that they have no competing interests.J–LG was the guarantor and supervisor of the study. MC, LP–B, BX and J–LG contributed to conception and design, recruitment of patients, analysis and interpretation of data and drafting of the manuscript. R–MG–R carried out the biological and genetics analyses, interpretation of data, statistical analyses, revising of the manuscript. BX and MC recruited the patients and M–AB and J–PB contributed in interpretating the data. LP–B and J–LG revised the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Previously, we have shown a strong association between complement factor H (CFH) Y402H and AMD in the Finnish population. A carrier of at least one risk allele in each of the three susceptibility loci  had an 18-fold risk of AMD when compared to a non-carrier homozygote in all three loci. A tentative gene-gene interaction between the two major AMD-associated loci, LOC387715 and CFH, was found in this study using a multiplicative (logistic regression) model, a synergy index (departure-from-additivity model) and the mutual information method (MI), suggesting that a common causative pathway may exist for these genes. Smoking (ever vs. never) exerted an extra risk for AMD, but somewhat surprisingly, only in connection with other factors such as sex and the C3 genotype. Population attributable risks (PAR) for the CFH, LOC387715 and C3 variants were 58.2%, 51.4% and 5.8%, respectively, the summary PAR for the three variants being 65.4%.We investigated the association between the CFH and LOC387715 was obtained using three methods, logistic regression, a synergy index and the mutual information (MI) index.Evidence for gene-gene interaction between two major AMD associated loci  It is characterized by drusen deposits in its early forms. The late forms are geographic atrophy and exudative AMD, which especially affects central vision complement component 3 (C3 [MIM 120700]) gene of complement cascade was associated with AMD in two case-control sets CFH this is an interesting observation because CFH is known to inhibit activation of C3 in the complement system Most recently, a common polymorphism (rs2230199) in the CFH Y402H polymorphism is associated with AMD in Finnish patients LOC387715 A69S (rs10490924), HTRA1 promoter (rs11200638), or C3 R102G (rs2230199) variants are associated with AMD in the Finnish population. In addition, we compared three different statistical methods to estimate the combined risks and gene-gene and gene-environment interaction of CFH Y402H, LOC387715 A69S, C3 R102G, and smoking to the development of AMD.We have previously shown that the The patient material comprised 332 Finnish patients with AMD attending the Departments of Ophthalmology of Helsinki (n = 201), Oulu (n = 10) and Kuopio (n = 39) University Hospitals, or private offices and outpatient clinics (n = 82) In the family members of the index patients, the AMD stage was verified from fundus photographs or angiograms in 45 (60.0%), from the medical records in 28 (37.3%) and from an examination by a retinal specialist belonging to the study group in two of the patients (2.7%). In the rest of the subjects  the AMD stage was verified from fundus photographs or angiograms in 338 (93.4%), from an examination by a retinal specialist belonging to the study group in four (1.1%) and from medical records in 20 (5.5%) of the subjects. The verification of the AMD status of patients was carried out as described in Information about the smoking history of the study participants was obtained by telephone. The following data were recorded: Had a participant ever smoked, the ages at which he/she started and quit (if he/she had quit) smoking, as well as the numbers of cigarettes smoked per day during that period. Then, the numbers of pack-years were calculated (cigarettes smoked per day×years smoked/20 [cigarettes per pack]). A binary variable, never/ever, was based on the information obtained on whether a study participant had smoked <one pack-year (never-smoker), or >one pack-year (ever-smoker) in his/her lifetime.CFH was described in the previous article LOC387715 rs10490924 forward 5-GGTGGTTCCTGTGTCCTTCA-3 and reverse 5-GGGGTAAGGCCTGATCATCT-3HTRA1 rs11200638 forward 5-ATGCCACCCACAACAACTTT-3 and reverse 5-CGCGTCCTTCAAACTAATGG-3C3 rs2230199 forward 5-GGAACAGACCCCTGACAATG-3 and reverse 5-CTTGTGGTTGACGGTGAAGA-3. Amplification was performed in a DNA 2720 Thermal Cycler . The polymerase chain reaction conditions were as follows: 5 min at 94°C followed by 35 cycles of the denaturation step: 30 s at 94°C; the annealing step: 30 s at 59°C (rs10490924), 52°C (rs 11200638) and 60°C (rs2230199); the elongation step: 45 s at 72°C; and final extension for 7 min at 72°C terminated the reaction after final annealing. Sequencing was performed using cycle sequencing with the Big Dye Terminator kit (version 3.1) supplied by Applied Biosystems , and reactions were run on an ABI 3730 capillary sequencer according to the manufacturer's instructions.The genotyping procedure for LOC387715 rs10490924 and HTRA1 rs11200638 were found to be in almost perfect LD with each other (D′ = 0.99).Allele and genotype frequencies were estimated by direct counting. Deviations from Hardy-Weinberg Equilibrium (HWE) were tested with the standard Chi square test separately in cases and controls, to identify possible genotyping problems. No deviations were found . The overall success rate in genotyping was 99.5%. The LOC387715 rs10490924, HTRA1 rs11200638, C3 rs2230199) were measured by the standard Pearson's Chi square test with one degree of freedom . Marginal odds ratios (OR) and their confidence intervals were estimated for all significantly associated loci to assess the strength of the association. This was carried out with R scripts freely available on the Internet . Furthermore, population attributable risks (PAR) were estimated with Levin's formula; (100%×proportion of exposed×(OR−1))/(proportion of exposed×(OR−1)+1)), where the proportion of exposed is the frequency the allele or genotype in the blood donor controls).The allelic and genotypic associations of the individual loci  were calculated for each 2-locus genotype separately, using the non-risk double homozygote genotype  as a reference  and environmental effects  and various G×G, G×E and E×E interactions were fitted to the data. The non-nested models were compared by the means of Akaike's information criterion (AIC), where there is a difference of 2 or more between the AICs of the models including and excluding the term in question, respectively, is taken as evidence of a significantly better fit to the data  Multivariable logistic regression was used for assessing the relationship between the independent variables and the outcome, estimating the effects of the individual SNPs and covariates , and for dissecting potential gene-gene (G×G) and gene-environment (G×E) interactions in our data. This was carried out with the SPSS Binary logistic regression modeling procedure, in which stepwise backward variable selection procedure was used to screen out the informative covariates from the uninformative. We coded the loci genotypes following the notation presented by Cordell (2002) and North et al (2005) www.epinet.se) 1 and G2, with each locus having two alleles.In addition to the logistic regression approach, which is known to have only modest powers for distinguishing interactions LOC387715 A69S risk allele T was over-represented in our patient material with the T allele frequency reaching 48% in AMD cases compared to 19.5% in non-AMD controls (p = 3.75×10−9) and 24.7% in blood donor controls (p = 2.78×10−19) . The difference between the genotype frequencies in the familial and sporadic cases (p = 0.09).The AMD-associated 8×10−19)  and S4. ic cases  was not HTRA1 rs 11200638 polymorphism , was associated with AMD in familial cases when compared to non-AMD controls (p = 0.008) or to blood donor controls (p = 0.039)   and S4. CFHY402H, LOC387715 A69S and C3 R102G (using blood donor controls as a reference group) were 58.2%, 51.4%, and 5.8%, respectively. The joint population attributable risk for the three loci was 65.4%. Since carrying a risk allele in one locus does not exclude also carrying a risk allele in the other locus the summary PAR is less than the sum of the three single PARs. The PAR for smoking was 47.7%, note, however that it had to be estimated using non-AMD controls as a reference group; thus it cannot be interpreted as a true population-wise figure. Also, it is not directly comparable to the gene-wise PAR:s given above due to different reference group used.The estimated population attributable risks (PAR) for a carrier of the risk allele of p = 4.69×10−5, non-AMD controls), and the joint OR for the three loci and smoking was 74.3 .Odds ratio for smoking was 3.22  polymorphism, previously shown to be associated with AMD in the Finnish population LOC387715 A69S polymorphism. Joint analysis of ORs for the two variants showed that the risk of AMD was 27-fold  if an individual had both homozygous risk genotypes, CC (CFH) and TT (LOC387715), compared to the non-risk genotype TTGG, respectively, with all the other joint OR:s ranging from 2 to 21  joint OR:s are given in We also assessed the joint OR:s of  2 to 21 . Three-lp<0.001) additive gene effects for the LOC387715 and CFH loci, both having approximately the same effect size (C3 an additive effect was seen (p = 0.007), which was slightly weaker than the individual effects of CFH and LOC387715, but still made the overall fit of the three-locus model statistically significantly better than any two-locus models. No dominance effect could be demonstrated for either CFH Y402H, LOC387715 A69S, or C3 R102G.In the logistic regression modelling, we found highly significant , with the estimated effect being only a bit smaller than the additive effect of C3 locus alone. With the departure-from-additivity model the attributable proportion due to interaction of the loci was 70% (95%CI 51–89%) and S, the synergy index 3.79 (95%CI 1.82–7.89)  whereas the G×E interaction with sex (p = 1.04×10−6) was shown using MI statistics. Here the higher number of female patients in our study material might affect the result.Interestingly, an interaction between 82–7.89) . Also, mty model . No evidp = 0.795). Instead, E×E and G×E interactions were suggested: sex×smoking (p = 0.043) and C3×smoking (p = 0.086). When combined into a 3-way interaction C3×sex×smoking (p = 0.016) the 2-way interaction C3×smoking disappeared. The sex×smoking interaction term preserved borderline significance   ever-smoker women had 4.68-fold  risk of AMD when compared to never-smoker women, whereas in men the odd's ratio was smaller (OR = 2.57) and of borderline significance . Thus, the effect of smoking was more pronounced in women, which also explains the significance of the sex×smoking interaction. At the same time it explains why no independent main effect for smoking alone was obtained. ii) C3×smoking: smoking seemed to have a significant risk-conferring effect on the non-risk genotype CC, but the effect is not that strong (and non-significant) in G-carrying genotypes . In the never-smokers, the C3 G allele predisposed to AMD as expected, whereas, interestingly, in ever-smokers the effect of G allele was virtually indistinguishable. iii) C3×sex×smoking: here we saw the highest OR for ever-smoker women with the non-risk genotype CC , and non-significant effects in G-carrying genotypes . Overall, both smoking and carriership of the G allele confers risk for AMD, but the effect could be mediated via a different pathway.No evidence for an independent effect of smoking could be detected in the logistic regression modelling in our data (= 0.065) . Based o= 0.065)  failed tCFH, LOC387715 and C3) loci, and smoking in AMD. We found tentative evidence for a gene-gene interaction between CFH and LOC387715, the two major susceptibility genes of AMD using three different statistical approaches. The first evidence for interaction was obtained using logistic regression; however, because the power of logistic regression to detect interactions is low de facto necessitated the use of other, more sensitive methods. Thus we applied the departure-from-additivity model that is based on additive disease rates CFH risk genotype carriers with high C-reactive protein serum levels, elevated sedimentation rate (ESR), leucocyte count and smoking and more recently by Baird et al., CFH Y402H in the aetiology of AMD. The third method, mutual information statistics, has not been previously utilized in analyzing risk factors for AMD also suggested strong evidence for interaction of CFH and LOC387715.Here we report joint risks of three gene  and Fritsche et al. (2008) showing that LOC387715 is localized to mitochondria CFH and the LOC387715 genes indeed belong to a common causative pathway.The strong association of the CFH Y402H and smoking could be detected in our material using MI statistics  and leucocyte count were reported using the departure from additivity model (synergy index of 2.38 [95%CI 1.06–5.35]) CFH would be plausible, since cigarette smoke has been shown to activate the complement system CC or CT variants of CFH (Y402H) has been detected CFH Y402H have consistently been found to be statistically independent risk factors for AMD A G×E interaction between We did not find an interaction between LOC387715 and smoking with any of the three statistical methods used that is in agreement with several previous findings C3 seems to have a milder, though a statistically significant effect on AMD, as noted earlier CFH Y402H and LOC387715 A69S loci, while adding the risk allele of the C3 R102G increased the OR to only 18 using logistic regression analysis  and LOC387715 A69S (51.4%) are in line with other reports where PARs for CFH Y402H range from 43 to 68 % LOC387715 A69S from 36 to 57% C3 R102G in Yates et al. was higher (22%) C3 G allele effect is diluted in our data by the stronger effect of CC genotype in ever-smoking women (C3×sex×smoking). Schmidt et al. CC of the C3 gene whereas in the never-smokers the risk effect comes from the reported risk allele warrants further studies.The population attributable risks (PAR) for the In conclusion, analysis of gene-gene and gene-environment interaction of risk factors in AMD benefits from combination of several statistical methods and the available biochemical data.Methods S1(1.23 MB DOC)Click here for additional data file.Table S1(0.04 MB DOC)Click here for additional data file.Table S2(0.08 MB DOC)Click here for additional data file.Table S3(0.05 MB DOC)Click here for additional data file.Table S4(0.05 MB DOC)Click here for additional data file.Table S5(0.04 MB DOC)Click here for additional data file.


Arsenic from drinking water has been associated with malignant and nonmalignant respiratory illnesses. The association with nonmalignant respiratory illnesses has not been well established because the assessments of respiratory symptoms may be influenced by recall bias or interviewer bias because participants had visible skin lesions.We examined the relationship of the serum level of Clara cell protein CC16—a novel biomarker for respiratory illnesses—with well As, total urinary As, and urinary As methylation indices.n = 241) selected from a large cohort with a wide range of As exposure (0.1–761 μg/L) from drinking water in Bangladesh. Total urinary As, urinary As metabolites, and serum CC16 were measured in urine and serum samples collected at baseline of the parent cohort study.We conducted a cross-sectional study in nonsmoking individuals (p = 0.01). We also observed a positive association between secondary methylation index in urinary As and CC16 levels  in the overall study population; the association was stronger among people without skin lesions , indicating that increased methylation capability may be protective against As-induced respiratory damage. In a subsample of study participants undergoing spirometric measures (n = 31), we observed inverse associations between urinary As and predictive FEV1 (forced expiratory volume measured in 1 sec) .We observed an inverse association between urinary As and serum CC16 among persons with skin lesions (β = −0.13, The findings suggest that serum CC16 may be a useful biomarker of epithelial lung damage in individuals with arsenical skin lesions. Also, we observed the deleterious respiratory effects of As exposure at concentrations lower than reported in earlier studies. The second study (n = 287), reported a higher risk  of common respiratory symptoms and lower FEV1 (forced expiratory volume measured in 1 sec) and FVC  among people with arsenical lesions. In their most recent study (n = 258), Although several international studies have shown the effects of arsenic on elevated lung cancer risk, there has been relatively little research on its role in nonmalignant respiratory illnesses , Bangladnd study , with a In a study in an As-endemic area of Chile, , most of these studies were conducted among people with visible arsenical lesions that may have biased the assessment of respiratory symptoms in the study participants  with a Hitachi Z-8200 system  in the Geochemistry Laboratory at Lamont Doherty Earth Observatory of Columbia University. A detailed description of the water-collection procedure has been reported elsewhere .Spot urine samples were collected in 50-mL acid-washed tubes and kept in portable coolers with ice packs (carried by the research team) until storage at −20°C at the end of the day. All samples were frozen until shipment on dry ice to Columbia University. Urinary As concentration assays were performed with GFAA using a PerkinElmer Analyst 600 graphite furnace system  in the Department of Environmental Health Sciences of Columbia University, as described by Urinary As metabolites were assayed by inductively coupled plasma-mass spectrometry with dynamic reaction cell (ICP-MS-DRC) coupled to high-performance liquid chromatography (HPLC). ICP-MS-DRC was used as a detector for As metabolites chromatographically separated on an Anion Exchange Column  with 10 mM ammonium nitrate/ammonium phosphate, pH 9.1, as mobile phase . The excBlood samples were collected in 10-mL vacutainers with silica clot activator and polymer gel separator. Blood samples were stored in a cold box at 4°C immediately after collection. To separate serum from the whole blood, the blood samples were centrifuged 10 min at 4,000 rpm. Separated samples were then stored at −20°C until they were shipped on dry ice to Columbia University for analysis. The serum samples were aliquotted into small plastic tubes and shipped on dry ice to the laboratory at the Catholic University of Louvain for analysis of CC16.oC for 30 min and by adding polyethylene glycol  and trichloroacetic acid . After overnight precipitation at 4°C, the serum samples were centrifuged  and CC16 was determined in the supernatants. This assay has a detection limit of 0.5 μg/L and an average analytical recovery of 95%. All laboratory assays for serum CC16 were performed in duplicate at two different and independent dilutions of the samples to achieve maximum accuracy. The within- and between-run coefficient of variation (CV) was between 5 and 10%, and the values were in the normal range  (The concentration of CC16 in serum was determined using the assay method described by  of age) , 1999b.  of age) . The CC1At the baseline interview for each participant, information on daily water consumption and duration of well use was collected for wells used on a regular basis. In addition, similar information was also collected on any other well that a participant used currently (or had used in the past). When the previously used well was within the study area and the As concentration was known, As concentration of the previous well was incorporated in the calculation of the cumulative As exposure index (CAI). Based on well utilization history, we calculated the CAI, which is the sum of the products of the amount of water consumed per day (liters per day) × As concentration in the well(s) (milligrams per liter) × the duration of well use (days) for each well . The CAIThe percentages of InAs, MMA, and DMA were calculated after subtracting AsC and AsB  from the total. An alternative method for describing As metabolite data employs primary (PMI) and secondary (SMI) methylation indices. PMI is the ratio of MMA to InAs, and SMI is the ratio of DMA to MMA.n = 4) or who produced readings of poor quality (n = 5), a total of 31 spirometry test results were available for analysis. We used a portable, battery-operated ultrasound transit-time based spirometer  for pulmonary function tests. The device is standardized to meet American Thoracic Society guidelines for lung function tests. The EasyOne has been used in a number of research studies in different countries and at a number of teaching hospitals in the United States . The distributions of the outcome and As variables were skewed; therefore, they were log-transformed. Pearson correlation coefficients were used to evaluate the relationship between CC16 and As exposure variables, as well as the relationships of lung function indices with urinary As and As methylation indices. Multivariate linear regression was used to evaluate the associations of As exposure variables and urinary As methylation indices with log-transformed CC16 in all the participants, and was performed separately by skin lesion status. Potential confounders including age, sex, and body mass index (BMI) were included in the multiple linear regression models. A conventional cutpoint  . All statistical analyses were conducted using the SAS 8.2 statistical package for Windows .Preliminary analysis involved calculations of frequency distributions, means, and tabular statistics. We performed Student’s e value)  was seleThe average age of the study participants was 37 years . Cases wp < 0.01). Conversely, the secondary methylation index (DMA/MMA) was lower among people with skin lesions (5.6) than among those without (7.8).The mean As concentration in drinking water was 134 μg/L . Individr = −0.11, p = 0.07), although the association did not reach statistical significance at p < 0.05 among all individuals. However, among individuals with arsenical lesions, we found significant associations of urinary As  and CAI  with CC16 levels. The association between water As and CC16 was not statistically significant .Serum CC16 levels were inversely related to urinary As concentrations (p = 0.10) adjusting for age, sex, and BMI in the overall study population (p = 0.01) and a marginally significant association of CC16 with CAI .Multiple linear regression analyses also revealed no significant association between urinary As and log-transformed CC16 values  but a marginally significant positive association with the DMA/MMA ratio  (p = 0.04), suggesting that As metabolism may be protective of respiratory injury. In addition, our overall data showed a significant association of serum CC16 with MMA%  but not with DMA%  in linear regression models after adjusting for age, sex, and BMI.This analysis revealed a weak negative association between CC16 and the MMA/InAs ratio  . Among in = 31) to ascertain the association between CC16 and clinical lung function. We observed strong inverse associations between urinary As and predictive FEV1 , FVC  and FEV1/ FVC ratio  among this subset of the study population. Our data show a strong inverse association between FEV1/FVC and PMI  , confirming that individuals with chronic respiratory illness have low levels of serum CC16  . Furtherrum CC16 .1/FVC reinforce the fact that serum CC16 is a biomarker of lung function, and it may be useful in assessing early respiratory damage induced by As, especially among individuals with skin lesions.To our knowledge, this is the first study to systematically assess the effects of As from drinking water on respiratory illness using biomarkers of As exposure and lung injury. We found an inverse association between urinary As and serum CC16 level among cases with skin lesions. This could be because individuals with skin lesions are either exposed to a higher levels of As or they are more susceptible to other health effects  of As exposure. The significant associations between serum CC16 and FEVOur analyses also showed positive associations of CC16 levels with secondary As methylation index (MMA/DMA), particularly among individuals without skin lesions, suggesting that those with better methylation capacity are less prone to respiratory effects of As. The inverse association of CC16 with MMA% also suggests that individuals with incomplete methylation are more susceptible to the respiratory effect of As. In a large case–control study of As-related skin lesions within the parent cohort study, we also found a dose–response relationship between risk of skin lesions and %MMA .Although the mechanism of As-induced nonmalignant respiratory illness is not known, several studies have shown that a large amount of As is deposited and stored in the lung, especially in the epithelium . It is pCC16 is one of the 20 proteins secreted by Clara cells in the lung’s alveolar epithelium, and it plays a major role in protecting the alveolar epithelium from pollutants . CC16 re1, FVC, and FEV1/FVC ratio in the 31 persons with no skin lesions confirms the positive associations between respiratory symptoms and As exposure observed in populations with very high concentrations and individuals with skin lesions . In contrast, urinary As reflects the aggregate exposure from all sources. Thus urinary As is considered a better measure of recent total As exposure.A limitation of our study is that we did not collect detailed clinical information on respiratory illness and that lung function data were only available among a subset of study participants. However, given that the subset was randomly selected, results on lung function observed in the subset of the study population may also apply to all of the study participants. It is also unlikely that the observed association is due to other factors that may influence serum CC16 concentrations. There has been no evidence that As exposure from drinking water is related to factors such as exposure to ozone, occupational exposure to nitric oxides, or asbestos that may influence serum concentrations of CC16. In fact, > 95% of the population in our study area use biofuels for cooking. Several studies have shown that smokers with As exposure are at a higher risk of developing respiratory illness . Our finPresently, invasive procedures for detecting respiratory illnesses, such as bronchoscopy or bronchoalveolar lavage techniques, are not suitable for large-scale population studies in rural Bangladesh. Also, self-reported symptoms or lung function tests usually detect disease with relatively late-stage lung damage. In contrast, an appropriate biological marker such as CC16 can be used to detect respiratory illnesses at an early stage and it is easy to use, making it especially attractive for tracking respiratory damage from As or other exposure in populations such as in Bangladesh.1 and FVC. These novel findings need to be confirmed in future larger studies.In conclusion, we observed an inverse association of serum CC16 with As exposure and a positive association with As methylation capacity in this Bangladeshi population. These associations differed by participants’ skin lesion status. We infer that the methylation of InAs to DMA is protective against respiratory effects of As, because those with a higher ratio of DMA/MMA appeared to be at decreased risk for lung dysfunction. We also observed a deleterious effect of urinary As on lung function, as assessed by FEV


Essential hypertension is a complex genetic disorder influenced by diverse environmental factors. Of the various physiological pathways affecting the homeostasis of blood pressure, the renin-angiotensin system (RAS) is known to play a critical role. Angiotensin-I converting enzyme (ACE) is a significant component of RAS and an insertion/deletion (I/D) polymorphism in its gene has been implicated in predisposition to hypertension.The present study is aimed to determine the association, if any, of ACE I/D polymorphism with essential hypertension in a rural population of Haryana, India.The blood samples were collected from the patients visiting M. M. Institute of Medical Sciences, Mullana, Haryana. DNA from the patients (106) and control (110) specimens were isolated, amplified by PCR and analyzed employing agarose gel electrophoresis.There was no significant difference in the distribution of DD, II and I/D genotypes of ACE polymorphism in essential hypertensive patients  and their ethnically matched normal control , respectively. The two groups also presented with very similar allelic frequencies and were also found to be in Hardy-Weinberg equilibrium.The present study demonstrates that ACE I/D polymorphism is not a risk factor for essential hypertension in the hitherto unstudied rural population of Haryana. Cardiovascular diseases are becoming a major health burden in developing countries. About 2.6 million Indian people are estimated to die due to coronary heart disease (CHD) alone by the year 2020. Hyperten2Angiotensin-I-converting enzyme (ACE) is a zinc metallopeptidase widely distributed on the surface of endothelial and epithelial cells and participates in producing arteriolar constriction and a rise in systolic and diastolic blood pressure. The ACE is encoded by a 21 kb gene that consists of 26 exons and is located on chromosome 17 and contains a polymorphism in the form of either insertion (I) or deletion (D) of a 287 base pair Alu repetitive sequence in intron 16. This pol7In the present investigation, the blood samples of 106 essential hypertensive patients and 110 samples of age and sex matched normal, healthy individuals as control group were collected with informed consent from M. M. Institute of Medical, Sciences, Mullana, Haryana. It is prudent to mention that it is the only mutlifacility hospital in Mullana and caters to a rural region within a radius of around 30 km. Patients were initially not on any medication and subsequently, consented for regular check up and treatment. Their follow up is up to date. The blood samples were collected in tubes containing EDTA as an anticoagulant. The samples were transported on ice to the laboratory and were processed on the same day. The isolated DNA samples were stored at −20°C till further analysis.Various parameters like age, sex, BMI, blood pressure, and dietary patterns were recorded in a questionnaire. Blood pressure (supine) was measured after the subject had rested at least 15 minutes with the help of mercury sphygmomanometer and stethoscope by ausculatory method. The recoet al.[2, 50 mM KCl, 0.01% gelatin, and 0.3 U of Taq DNA polymerase . Amplification was carried out for 35 cycles, each cycle consisting of denaturation at 94°C for 30 s, annealing at 58°C for 20 s, extension at 72°C for 20 s and finally a 3 m extension at 72°C. The PCR products were resolved in 2% agarose gel and visualized following ethidium bromide staining. All samples, identified as DD after initial amplification, were reconfirmed with an insertion-specific primer pair:DNA samples were isolated from peripheral blood lymphocytes by the standard modified inorganic method as described by Miller et al. and quanet al. The optiForward primer: 5’-GCCACTACGCCCGGCTAAT-3’;Reverse primer: 5’-GATGTGGCCATCACATTCGTCAGAT-3’).The reaction conditions and amplification parameters for this confirmatory reaction were the same as stated above. Known controls of each genotype were amplified with each set of samples for the ACE I/D polymorphism.† test. Allele frequencies were calculated from genotype frequencies and were compared using chi-squared (χ2) statistics. P value < 0.05 was considered statistically significant.Data analysis was done with the help of an SPSS version 7.5. Continous variables are expressed as means ± SD. Intergroup comparisons are made using students P >0.05).The clinical details of the hypertensive and control subjects are presented in Incidence of hypertension is increasing alarmingly in various populations of India and other developing nations.–18 It isPooling of epidemiology studies show that hypertension is present in 25% urban and 10% rural subjects in India. Clearly It is important to ascertain gene(s) that are involved in hypertension. This would help in identifying individuals at an increased risk of developing this disease and to initiate appropriate actions in them to avoid development or delay the onset of disease. Genome wide scan and candidate gene approach are two strategies used in dissecting complex genetic diseases. The formThe gene product must be functionally relevant to hypertensionPolymorphism within the gene must alter its functionHypertension needs to link to the chromosomal region harboring the candidate gene.Available studies demonstrate that the ACE I/D polymorphism fulfills above mentioned criterions in the context of hypertension22–24 andThe frequencies of different genotypes were found to be similar in patient and the control population . The fre28298The frequency of D allele of ACE I/D polymorphism in different hypertensive populations of India varied within 0.522 to 0.409 . The higIdentifying association between a gene and a complex genetic disease is difficult. One possible reason for this is the involvement of a large number of genes in the etiology of essential hypertension. Furthermore, these genes may interact with each other in different combinations to give rise to a similar disease phenotype. The magnitude of this problem makes the frequency of any polymorphism contributing to a disease phenotype marginally higher in disease group compared with unaffected controls. Linkage The results of the present study has triggered two very valid questions, i) what is the effect of different ACE I/D genotypes on the progression of the disease? and ii) are different drug regimens required for individuals with different ACE I/D polymorphism? Interestingly, our preliminary observations do suggest that hypertensive patients with DD phenotypes require higher doses of ACE inhibitor in their drug regimen as compared to their II counterparts (data not shown). The information will be of immense use in tailoring individualized therapy to hypertensive patients based on the ACE I/D genotypes.Our study suggests that the ACE I/D polymorphism is not a risk factor for the development of essential hypertension in the studied rural population from Haryana.


CYP2E1 encodes a member of the cytochrome P450 superfamily of enzymes which play a central role in activating and detoxifying many carcinogens and endogenous compounds thought to be involved in the development of cancer. The PstI/RsaI and DraI polymorphism are two of the most commonly studied polymorphisms of the gene for their association with risk of head and neck cancer, but the results are conflicting.CYP2E1 PstI/RsaI and DraI polymorphism and head and neck cancer risk, especially by interacting with smoking or alcohol.We performed a meta-analysis using 21 eligible case-control studies with a total of 4,951 patients and 6,071 controls to summarize the data on the association between the Compared with the wild genotype, the OR was 1.96 (95% CI: 1.33-2.90) for PstI/RsaI and 1.56 (95% CI: 1.06-2.27) for DraI polymorphism respectively. When stratified according to ethnicity, the OR increased in the Asians for both polymorphisms , suggesting that the risk is more pronounced in Asians.Our meta-analysis suggests that individuals with the homozygote genotypes of PstI/RsaI or DraI polymorphism might be associated with an increased risk of head and neck cancer, especially in Asians. CYP2E1, which is a major component of the microsomal system involved in the metabolism of ethanol and acetone among phase I enzymes, has been widely studied as a cause of susceptibility to head and neck cancer [Squamous cell carcinoma of head and neck (HNSCC), including the oral cavity, pharynx, and larynx, is the sixth most common cancer in the world . Epidemik cancer .CYP2E1 gene, located on chromosome 10q26.3, is constitutively expressed in human liver and is responsible for the catalysis of xenobiotic. The protein encoded by this gene specifically activates N-nitrosamines and benzene, contained in cigarette smoke [CYP2E1 gene. Two point mutations in the 5'-flanking region , which are in complete linkage disequilibrium, was found to be associated with higher-transcription and increased enzyme activity [CYP2E1 wild (c1) allele and the less common (c2) allele and have been reported as conferring higher risk for developing oral, pharyngeal [CYP2E1 gene [CYP2E1 are therefore believed to be risk factors for HNSCC.The te smoke , and catte smoke . Severalaryngeal  and lungaryngeal . Another2E1 gene . PolymorCYP2E1 polymorphisms and HNSCC susceptibility. However, these studies have yielded contradictory results, with some studies showing a significant association, while others showing no such association. Such inconsistency could be due to the small effect of the polymorphism on HNSCC risk and the relatively small sample-size in each of the published studies. We therefore performed a meta-analysis of the published studies to clarify this inconsistency and to establish a comprehensive picture of the relationship between CYP2E1 and HNSCC.A number of studies have investigated the associations between The literature included in the current analysis was selected using PubMed with keywords "head and neck" or "oral-neoplasms" or "larynx" or "pharynx" and "cancer" or "carcinoma", "HNSCC" or "SCCHN", "cytochrome p450 2E1/IIE1", "polymorphism" or "variant" and abbreviations of the gene "Cyp2e1". No other limits were used during electronic search. All references cited in these studies and previously published review articles were reviewed to identify additional work not indexed by MEDLINE. The analyzed data is drawn from all English language publications up to August 2009.CYP2E1 gene polymorphisms were included. Eligible studies had to meet the criteria: (1) They were original papers containing independent data, (2) identification of HNSCC was confirmed histologically or pathologically, (3) they provided sufficient data to calculate the odds ratio (OR) or P-value, (4) they used a case-control design, (5) they described genotyping method, equipment and protocols or made reference to them. The major reasons for exclusion of studies were (1) they were family studies, (2) they contained overlapping data, (3) they were review papers.Only those studies assessing the association between head and neck cancer and the Each article was read and assessed according to the score scale for randomized controlled association study proposed by Dawei et al . In brieFor each study, the following information was extracted independently by two investigators: first author's surname, publication date, diagnosis criterion, gender, ethnicity, genotyping method, cigarette smoking status, alcohol consumption, total number of cases and controls and the numbers of cases and controls with the three genotypes. The results were compared and disagreements were discussed and resolved with consensus. Where essential information was not presented in articles, every effort was made to contact the authors.CYP2E1 polymorphism and head and neck cancer risk was measured by odds ratios (ORs) with 95% confidence intervals (CIs).For the control group of each study, the distributions of genotypes were tested for Hardy-Weinberg equilibrium (HWE) using the Chi-square test. If controls of studies were found not to be in HWE, sensitivity analyses were performed with and without these studies to test the robustness of the findings. For the RsaI/PstI polymorphism, we first estimated the risks of the c1c2 and c2c2 genotypes on HNSCC, compared with the wild-type c1c1 homozygote. The risk of c1c2+c2c2 versus c1c1 on cancers was then evaluated in dominant model. The same evaluation was carried out for the DraI polymorphism. The strength of the association between the 2 based Q-statistic test [I2 -test [Cochran's χtic test ,12 and II2 -test  were perI2 -test , which yI2 -test . In addiI2 -test . The sigI2 -test . In ordeCYP2E1-PstI/RsaI polymorphism, including a total of 4951 cases and 5995 controls, and for the DraI polymorphism 9 studies were identified covering a total of 2510 cases and 2162 controls. The detailed characteristics of these studies are summarized in table Figure Q = 0.87) for head and neck cancer risk among c2 homozygotes of CYP2E1 RsaI/PstI polymorphism compared with the wild type. However, no other genetic model gave statistically significant results.Our meta-analysis gave an overall OR of 1.96  among homozygotes (c2c2 versus c1c1). On the other hand, no such association was detected in Caucasian populations . When studies were stratified for study quality, an overall OR of 1.81  for c2 homozygotes emerged for the high-quality scored studies. These positive associations were also present among low-quality scored studies. Data on genotypes of the PstI/RsaI polymorphism among cases and controls stratified by smoking were available in five studies and by alcohol consumption in four studies. The results of the stratified meta-analyses according to smoking status and alcohol consumption are shown in Table When stratifying for ethnicity, c2 homozygotes were significantly associated with HNSCC in Asian populations with an OR of 2.08 for homozygotes compared with wild genotype, but no association was observed between T7632A and head and neck cancer using other genetic models.We also carried out a comprehensive meta-analysis of ls Table . The oveQ = 0.79) for the DraI polymorphism in Asian populations among homozygotes (AA versus TT), whereas no such association was observed in Caucasian populations. After stratification for study quality, an overall OR of 2.36  for homozygotes emerged for the low-quality scored studies but no such association was observed among high-quality studies , whereas deviations from HWE were observed in three studies of the PstI/RsaI polymorphism ,28,30. HBegger's funnel plot and Egger's test were used to assess publication bias. The shape of the funnel plots was symmetrical  and head and neck cancer risk. We found that the variant homozygote of the CYP2E1 PstI/RsaI polymorphism was significantly associated with cancer risk in the overall comparisons, compared with the wild homozygote. We also observed a significant association between the CYP2E1 DraI polymorphism and cancer risk for homozygote in the overall comparisons.In the present meta-analysis, we examined the association between two widely studied CYP2E1, our result showed a significantly high cancer risk for the c2 homozygote in Asian populations, with no such association being found among Caucasian population under any of the three genetic models. Ethnic differences may attribute to these different results, since the distributions of the less common c2 allele were different between various races, with a prevalence of ~25-50% and 5-10% among Asians and Caucasians, respectively [CYP2E1 c2 allele (5-10% against 25-50% for Asians).As for the PstI/RsaI polymorphism of ectively . The c2 CYP2E1 is presumed to confer susceptibility to HNSCC by metabolizing carcinogens but, unfortunately, few of the studies explored the interaction between the CYP2E1 genotype and smoking habits or alcohol consumption. This may be due to the low statistical power of the individual studies to detect such interactions; but all the studies which collected these data were utilized for this meta-analysis. The stratified meta-analyses of all the data on the PstI/RsaI genotype and smoking habits or alcohol consumption with respect to head and neck cancer risk produced no statistically significant results. These results suggest that when the environmental and genetic risk factors are both present, the combined effect on head and neck cancer seems to be no longer than additive of the separate effects. However, we cannot ignore the fact that owing to the low prevalence of c2 homozygotes in each study, even when data are pooled, the statistical power to detect an interaction remains low.In addition, the DraI polymorphism was associated with an increased cancer risk among Asians but not among Caucasians, suggesting an ethnic difference in terms of genetic and environmental factors . HoweverCYP2E1 is a phase I enzyme, which plays an essential role in the metabolic activation of low molecular weight compounds and pro-carcinogens such as N-nitrosamines, benzene, and halogenated hydrocarbons. There is strong evidence from in vitro studies suggesting that the rare allele of the RsaI/PstI polymorphisms in the CYP2E1 gene probably confers a higher risk of HNSCC susceptibility by increasing transcriptional and enzyme activity [CYP2E1 gene, in vitro expression studies indicate that it is associated with increased transcriptional activity [Currently, the mechanism whereby the rare allele of the RsaI/PstI and DraI polymorphisms increases the risk of head and neck cancer risk are still not clear. activity . Thus, tactivity . The AA CYP2E1 genotype and smoking habits/alcohol consumption are based on the small number of studies where such information is available. Nevertheless the number of subjects included in this part of the analysis comprised the largest sample of all. Third, the quality score of the individual studies included in our meta-analysis was assessed on the basis of trying to minimize the potential for selection bias, misclassification related to exposure, collection of data on potential confounders and method of statistical analysis. No validated quality assessment system currently exists, and it is evident that our quality scale has a subjective component.Some limitations should be considered when interpreting our results, in addition to those inherited from the meta-analysis. First, our results are based on unadjusted estimates, whereas adjustments for factors such as age and sex would produce a more precise analysis. The lack of this kind of information may cause serious confounding bias. Further large and well-designed studies need to be performed to further confirm all these results. Secondly, the subgroup meta-analyses dealing with interactions between the CYP2E1 and HNSCC. Our results indicate that the CYP2E1-PstI/RsaI and DraI polymorphisms may be a risk factor for head and neck cancer in Asian populations, and particularly for individual homozygotes for the unfavourable gene variant. However additional large studies are needed to validate our findings. Future studies should use standardized unbiased genotyping methods as well as homogeneous cancer patients and well-matched controls.In summary, this may be the first systematic and comprehensive meta-analysis of HNSCC: squamous cell carcinoma of head and neck; OR: odds ratio; CI: confidence interval; HWE: Hardy-Weinberg equilibrium.The authors declare that they have no competing interests.KFT organized all the research, performed data analysis and drafted the manuscript. YL collected the articles, co-worked in data analysis, as well as manuscript preparation. ZZ participated in the calculation and table generation, and helped to draft the manuscript. YMG and YYX participated in the data extraction and manuscript editing. GYF, LH and SYQ provided advice for preparing the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/575/prepubT-test and Funnel plot. Egger's test and Begger's funnel plot results to assess publication bias.Click here for file


Studies assessing the relationships of anthropometry and testicular germ-cell tumour (TGCT) have reported heterogeneous findings.We undertook a systematic review and meta-analysis of the associations between adult height, weight, body mass index (BMI), and testicular cancer. Search strategies were conducted in PubMed, EMBASE, Scopus, and Web of Science on 26 May 2009. Studies that met our inclusion criteria were included in meta-analytic models using STATA 11.P<0.001) and weight  to be associated with TGCT. The meta-analysis of weight and TGCT produced a summary estimate, which indicated no association, although an analysis restricted studies to North American was suggestive of association .A total of 3255 references were retrieved, of which 14 met the inclusion criteria. Random effects meta-analysis found adult height (odds ratio (OR) per 5-cm increase 1.13, 95% confidence interval (CI) 1.07–1.19, This systematic review and meta-analysis has found evidence for a positive association of adult height and TGCT, and tentative evidence for an inverse association of BMI and TGCT. Testicular cancer is a relatively rare malignancy in the general population, but is the most commonly occurring cancer among young men in many countries . In addiAdult height is determined in early life by a combination of genetics, health and nutrition , EMBASE , Scopus , and Web of Science . Search strategies are available from the authors on request. Titles, abstracts, and keywords were assessed by two reviewers (CCL and MBC). Articles were compiled and managed using Endnote X3 software .To be included in the meta-analysis, studies had to meet the following criteria; (1) have at least 20 cases and 20 controls in the analysis; (2) recruited only incident cases to avoid survivorship bias; and (3) have a valid control group (no case-series or case-study designs were included). For the meta-analysis of adult weight, it was required to have the weight ascertained shortly before or around the time of diagnosis. For the meta-analysis of adult height, it was required to have the height ascertained at age 18 years or older. Both of these conditions had to be met for a study to be eligible for the meta-analysis of BMI. No restrictions were placed on year or language of publication. If a single study had reported results in more than one publication, that with the largest number of cases was included.Variables abstracted from studies included country of study population, institute or hospital, years of study, study design, patient selection criteria and source, mean age of study participants, mean age at diagnosis, race/ethnicity, data ascertainment method, and whether the study included all testicular tumours or only testicular germ cell tumours (TGST) (the most common type of testicular tumours).−2) was also examined as an ordinal variable using groupings of normal (BMI <25), overweight (BMI 25–29.9), and obese (BMI ⩾30). A random-effects meta-analysis was used according to the methods of I2 value and its 95% uncertainty intervals (UIs) were used to assess the consistency of the study-specific estimates  reviewed titles and abstracts of these articles and identified 189 articles of potential interest . The full-text of each article was retrieved and the manuscript was considered against the stipulated selection criteria for our review. Four additional original research articles were identified through review of citations of the full-text articles, for a total of 195 relevant articles. Any discrepancies regarding inclusion were discussed among all the three authors until consensus was reached. In total, 21 authors were contacted and requested for further information, of whom 19 replied and 8 provided additional information or unpublished data. In total, 14 articles met the selection criteria, provided the appropriate information or data to calculate OR, and were able to be included in at least two of the three  meta-analyses . We did Adult height and weight were used to calculate BMI of individuals of each study. Included studies used weight at TGCT diagnosis , at age P<0.001; I2 value, was 77% (95% UI 60–86%). The high heterogeneity was not explained by meta-regression of those variables specified a priori. The Egger and Begg's tests both indicated no small-study effects . A total of 12 studies contributed to the analysis of the association between adult weight and TGCT (P=0.508). The I2 value measuring between-study heterogeneity was 60% (95% UI: 24–79%). Egger and Begg's tests found no evidence for any small-study effects .In the meta-analysis of the association between adult height and TGCT, 13 studies were included which, in combination, produced a summary OR of 1.13 per 5 cm increase in height . A stratified forest plot graphically illustrates the differences in the relationship between adult weight and TGCT by continent (P=0.643) with an I2 value for between-study heterogeneity of 58%, whereas North-American studies produced a borderline statistically significant summary estimate of 1.01 per 1 kg increase in weight  with an I2 value of 0%. Meta-regressions of study design, country of study population, and data ascertainment method were all null.Meta-regression of adult weight by continent (North America against Europe) was of borderline statistical significance  indicating a moderate amount of between-study heterogeneity. The Egger and Begg's tests did not provide strong evidence for small-study effects .BMI was analysed both as a continuous and as a categorical metric in relation to TGCT. Thirteen studies were included in the continuous meta-analysis in which we found a tentative inverse relationship with TGCT with a summary risk estimate of 0.99 per one unit increase in BMI  and TGCT (P=0.011) with no heterogeneity indicated (I2=0%). Sensitivity analysis demonstrated that the study by I2=7%). When we compared the obese group with the referent group, BMI was not related to TGCT, with a summary risk estimate of 0.92 per unit BMI  with an I2 of 46% (95% UI: 0–100%). Again, sensitivity analysis showed that the study by P=0.068). The heterogeneity was lower after the omission of the I2=7%, 95% UI: 0–100%).In total, 11 studies contributed to the categorical meta-analysis of BMI (kg mand TGCT . For theVariables assessed using meta-regression included country of study population (grouped by continent), study design, and data ascertainment method. Meta-regression of variables such as mean age at diagnosis and mean age of study participants could not be conducted because they were not available for all included studies.In this systematic review and meta-analysis of body size in relation to TGCT, we found a positive association with height and an inverse association with BMI. The meta-analysis of weight and TGCT indicated no association. The association between height and testicular cancer has been examined by 17 studies, 13 of which were included in this meta-analysis. Although there was evidence of a moderate-to-high amount of heterogeneity among the 13 studies included, all but three reported a positive association  decreased significantly, from 48% to 7%. This study did differ somewhat in terms of study design when compared with the other studies included in the categorical BMI meta-analyses. It was a large, multi-centre case-control study in which cases were enroled both prospectively and retrospectively during a specified period. Controls were taken from the German National Health Survey; because TGCTs are more prevalent in younger men, only men aged 18–49 years were abstracted and chosen for these analyses. The authors could not calculate relative risks because the exact BMI of control individuals was not known; rather, BMI data was ascertained in categories of exposure , weight (continuous), and BMI  in relation to TGCT risk.a priori. The high levels of heterogeneity do not invalidate the findings presented herein, but they do warrant somewhat of a cautious interpretation. A fourth limitation is that weight was not measured in a uniform manner throughout the included studies. Rather, weight was measured at different points before or at the time of diagnosis, as specified in the individual study methods and summarised above. Weight can vary greatly throughout a person's life, and it may have more or less influence at different points in the disease process. Thus, to ensure meta-analytic validity and minimise heterogeneity, it is important that weight is collected at similar points in the disease process for each study included. The studies included here were limited to those in which weight was measured at or before diagnosis; post-diagnostic weight is more likely to be a reflection of treatment and stress associated with disease. Differences in the timing and method of weight measurement could have contributed to the heterogeneity of the weight meta-analyses, although a sensitivity analysis of the included studies demonstrated that no single study was dominant. One final limitation is the lack of prospective studies available for inclusion in this meta-analysis. There are very few cohort studies looking at the relationship between body size and TGCTs, and only one could be included in these meta-analyses (This analysis also has several limitations. It was not feasible to include every identified study in the meta-analysis, because three cohort studies (This study provides support for a positive association between height and TGCT, but little support for an association between weight and TGCT. Further investigation of the inverse relationship between BMI and TGCT may be warranted, for which the present findings lend only limited support.


Climate change will have major consequences for population dynamics and life histories of marine biota as it progresses in the twenty-first century. These impacts will differ in magnitude and direction for populations within individual marine species whose geographical ranges span large gradients in latitude and temperature. Here we use meta-analytical methods to investigate how recruitment (i.e. the number of new fish produced by spawners in a given year which subsequently grow and survive to become vulnerable to fishing gear) has reacted to temperature fluctuations, and in particular to extremes of temperature, in cod populations throughout the north Atlantic. Temperature has geographically explicit effects on cod recruitment. Impacts differ depending on whether populations are located in the upper (negative effects) or in the lower (positive effects) thermal range. The probabilities of successful year-classes in populations living in warm areas is on average 34 per cent higher in cold compared with warm seasons, whereas opposite patterns exist for populations living in cold areas. These results have implications for cod dynamics, distributions and phenologies under the influence of ocean warming, particularly related to not only changes in the mean temperature, but also its variability . Our second approach is based on standard correlational analyses of the long-term effects imposed by temperature trends on cod recruitment. This analysis considers, for the first time, to our knowledge, all 21 stocks and provides an update to current knowledge provided by earlier studies using subsets of available population data We are primarily interested in studying the response of cod recruitment (REC) to temperature variations owing to either the impact of thermal extreme events or trends. Because spawner stock biomass (SSB) might have linear and nonlinear effects on juvenile cod production rate , we use (b)T) during the cod spawning season, given that the early life-history stages are most sensitive to abiotic environmental variations T < 4°C while the warm group includes observations made at T > 6.5°C. Five stocks  do not have a sufficient (nine or more) number of observations in either temperature range and thus are not included in the groups.Temperature effects on cod recruitment have been shown to be stronger and opposite, at the lower and upper ends of the cod thermal distributional range . Thus, w(d)Random-effects meta-analyses were used to study the effects of (i) thermally extreme events  and (ii) temperature trends on recruitment success indices. Meta-analysis across cod stocks is possible through the encoding of the separate (stock-specific) responses on standardized, dimensionless metrics, the effect sizes, quantifying the degree and the direction of an effect, so that they can be subject to common statistical meta-analytic inference . The eff(i)T exceeds the stock-specific 75th percentile (T75%ile), while cold seasons (termed as ‘cool’ or ‘control’) are defined as those with T below the stock-specific 25th percentile (T25%ile). The choice of the exact percentiles is meant to derive an empirical basis for categorizing years when local cod biology was subjected to particularly unusual thermal conditions, which deviated substantially from the average, to which the life history and physiology of local populations are most likely adapted. Another advantage of using percentiles is that the two groups have equal numbers of observations. Our purpose is to compare (i) the probability of strong year-classes and (ii) the average year-class strength between seasons of extremely low and high temperature and to identify the most favourable conditions. Thus, we used the risk ratio (RR) and Hedges' g (HG) effect-sizes and also the Wilcoxon signed-rank test  for a given stock are characterized as those when (ii)z (FZ) correlation coefficient as the effect size . Special techniques and additional analyses were employed to address the possible influence of autocorrelation in the stocks time series, as explained in the electronic supplementary material.Meta-analyses evaluating impacts of thermal trends were based on the correlation between temperature and recruitment survival. We used the Fisher's 3.n = 7) and cold (n = 9) groups of stocks, using the SSB standardized measures of year-class strength . Significant results were mainly obtained for the warm stocks, while in the cold group the results were close to significant only for FZ meta-analysis using the Ricker model residuals. As explained in §2, concurrent thermal effects on both recruitment and SSB can obscure the results for the SSB standardized indices. Thus, for the cold group, we also analysed the effect sizes estimated for the two variables separately. Overall, our results show that temperature extremes and trends induce negative effects on the recruitment dynamics of the warm stocks, and vice versa for the cold stocks. The meta-analyses were checked for robustness to the exclusion of any stock by performing sensitivity analyses, and the results were generally satisfactory. We also performed cumulative meta-analyses, by progressively adding stocks with increasing ‘coldness’ or ‘warmness’ , to demonstrate how the effects become stronger when considering stocks closer to the extremes of cod thermal distribution.For each effect size, the meta-analyses were conducted separately for the warm ((a)T > T75th%ile) during the spawning season were shown to induce negative effects on recruitment dynamics of the warm stocks. Specifically, employing the RR . Similarly, when plotting the probabilities during warmer seasons versus the corresponding probabilities during the colder seasons, most of the points fall below the no-effect line, indicating that the probability of ‘successful’ events is higher during colder spawning seasons (Extreme high temperatures (g the RR  meta-ana seasons .a). The result, however, remains significant at p < 0.1 even if either of the stocks is excluded from the analysis. The cumulative of the Ricker residuals meta-analysis showed that the overall RR becomes significantly lower than 1 after including stocks with mean spring T > 7.5°C in the meta-analysis . The sensitivity and cumulative analyses for warm stocks recruitment survival were similar. The results remained significant (p < 0.1) when certain observations were excluded in order to eliminate autocorrelation  from those stocks (cod on Faroe Plateau and Icelandic cod) with substantial autocorrelation in the recruitment survival and Ricker residuals .The exclusion sensitivity plot of the Ricker residuals meta-analysis revealed that the result is in general robust and mostly sensitive to the Icelandic and Irish Sea cod a. The rea and p = 0.05 (but still remain significant at p = 0.1) only if Icelandic cod, which is among the stocks with higher sample sizes and weights in the analysis, was excluded (b). The cumulative meta-analysis showed that the pattern becomes evident after including stocks with mean T > 7.1°C . The corresponding overall Cohen's d . This result agrees with the HG meta-analysis, revealing that the average log(REC/SSB) is significantly lower at higher spring T conditions.The one-sided Wilcoxon signed-rank test  showed that, for the warm stocks, the median of the distribution of the log-transformed ratios between average recruitment survival during colder and warmer seasons is significantly negative b,c and figure 2c) shows that the result is robust to the exclusion of any stock. The pattern emerges after the stocks with average spring T > 7.1°C are included in the analysis .The across warm stocks correlation of both recruitment survival and Ricker residuals with spring temperature, corrected for autocorrelation at lag 1 , was found significantly negative (figure 3p < 0.05) were obtained for both the log-transformed recruitment and SSB (p = 0.10) positive correlation between the Ricker residuals and spring T (d). When either cod-3m or cod-4tvn, displaying negative FZ, is excluded from the analysis, the result becomes significant at p < 0.5 (d). The pattern becomes evident after stocks with average T < 4°C are included in the analysis .As in the previous meta-analyses for the cold stocks group, significant results  is an important factor affecting cod recruitment, reproductive success and spawner abundance, and we provided new evidence on the geographical patterns of these effects. Effects were prominent when considering either (i) the correlation between the temperature and population trends or (ii) the influence imposed by single events of exceptionally warm or cold seasons.T > 6.5°C; n = 7), compared with when spawning season temperature is relatively low. The pattern is opposite for the cold stocks , with both recruitment and SSB being positively affected in warmer seasons. Correlation between cod biomass and temperature has also been found in a previous meta-analytic study  and the lower (positive) thermal limits. In particular, high thermal events are associated with lower recruitment survival  for the warm stocks (ic study  and can ic study . Owing tThe main motives for investigating the sensitivity of cod recruitment to extreme temperature conditions were (i) the frequency of warm and cold seasons and/or years in some areas of the north Atlantic has changed in the last 120–140 years , while , impactsTherefore, part of our analyses focused on the effects of extreme temperature both across and within stocks. We compared cod dynamics for distinct cold and warm groups of stocks during (relatively) colder and warmer spawning seasons. The implemented methods, based on effect sizes quantifying and contrasting the magnitude and direction of extreme thermal events in natural populations, could be useful for the investigation of such effects in other single populations and/or, when combined with meta-analytic approaches, across a species range.Our results illustrating the consequences of extreme temperature on recruitment are consistent with recent studies investigating thermal habitats experienced by wild cod, based on data storage tagging experiments in the north-east Atlantic . Our deca priori used to designate the warm group of cod stocks. We believe therefore that the two studies  both indicate that temperatures during the spawning period exceeding 6.5–7°C may be detrimental for reproductive success. Similarly, we have also observed in a separate study using hierarchical Bayesian analyses of cod population dynamics that the maximum reproductive rate of cod in these same 21 populations also declines when spring temperatures exceed 5–6°C (Pan I) changes  during the cod spawning period (7°C) was close to the 75th percentile of spawning season temperature observed across the species range (6.5°C), using independent temperature datasets, and which we The predicted amplification of the extreme high thermal events and the reduction in the comparatively colder events  and poteNevertheless, even at favourable thermal conditions and reduced fishing, a variety of mechanisms and factors can prevent collapsed populations from recovering , includiThe presented multi-population level responses to extreme and mean temperature trends and the patterns in the spatial dependence of recruitment dynamics on temperature are difficult to resolve by single-stock analyses or by pooling all stock-recruitment data across stocks and treating the individual populations as one large undifferentiated population. These difficulties were mostly overcome by the use of meta-analyses, since patterns that are weak or not significant at local scales of single stocks, but which nevertheless are repeated in different geographical areas, or vary systematically across space, may generate significant and ecologically meaningful patterns at larger scales of organization e.g. . In partClimate change has been shown to have different levels of impact on different species, depending on their life-history traits and physiological tolerances . Thus, i


Sir,We thank the reader for his observations and the editor for having given us the opportunity to offer our explanation.The answer to the query that the genetic polymorphism studies should be performed on a population scale is – We agree with the observation; although desirable owing to the financial constraints involved, we could not undertake the study at a population level. However, the statistical relevance of the data gathered and its possible extrapolation to a larger group has been ensured by using appropriate statistical methods while interpreting the results.The answer to the query that race might have an implication on the results is – We again agree with the observation, the same having been reflected in earlier studies from different geographical locations including those from the Indian subcontinent, although with conflicting results. Our study was performed with the intention to gather data from patients of South Indian ethnicity in whom the prevalence of type 2 diabetes mellitus is high.–3The answer to the query that correlation between ACE polymorphism and diabetic retinopathy showed null correlation in different studies is – We restricted our study only to investigating the relationship of ACE gene polymorphism and diabetic nephropathy in type 2 diabetes mellitus patients. Hence, are unable to comment on the mentioned observation of the reader with regard to our study group.


Ultrasonography of temporal arteries is not commonly used in the approach of patients with suspected giant cell arteritis (GCA) in clinical practice. A meta-analysis of primary studies available through April 2004 concluded that ultrasonography could indeed be helpful in diagnosing GCA. We specifically re-examined the diagnostic value of the ultrasonography-derived halo sign, a dark hypoechoic circumferential thickening around the artery lumen, indicating vasculitic wall edema, in GCA.Original, prospective studies in patients with suspected GCA that examined ultrasonography findings of temporal arteries using the ACR 1990 classification criteria for GCA as reference standard, published through 2009, were identified. Only eight studies involving 575 patients, 204 of whom received the final diagnosis of GCA, fulfilled technical quality criteria for ultrasound. Weighted sensitivity and specificity estimates of the halo sign were assessed, their possible heterogeneity was investigated and pooled diagnostic odds ratio was determined.2) of both sensitivity and specificity of the halo sign, showed significant heterogeneity concerning the results between studies. Pooled diagnostic odds ratio, expressing how much greater the odds of having GCA are for patients with halo sign than for those without, was 34 . Diagnostic odds ratio was further increased to 65  when bilateral halo signs were present . In both cases, it was found that DOR was constant across studies.Unilateral halo sign achieved an overall sensitivity of 68%  and specificity of 91%  for GCA. The values of inconsistency coefficient (ITemporal artery edema demonstrated as halo sign should be always looked for in ultrasonography when GCA is suspected. Providing that currently accepted technical quality criteria are fulfilled, halo sign's sensitivity and specificity are comparable to those of autoantibodies used as diagnostic tests in rheumatology. Validation of revised GCA classification criteria which will include the halo sign may be warranted. According to the American College of Rheumatology (ACR) 1990 criteria for the classification of vasculitis, diagnosis of temporal or giant-cell arteritis (GCA), the most common form of systemic vasculitis in adults, is based on clinical grounds and the result of a temporal artery biopsy. The presence of three or more of the following five criteria, (1) age of 50 years and older, 2) new onset of localized headache, (3) temporal artery tenderness on palpation or decreased pulsation, (4) erythrocyte sedimentation rate of 50 mm/h and higher, (5) abnormal temporal artery biopsy, yields a sensitivity of 93.5% and a specificity of 91.2% for the diagnosis of GCA when compared to other vasculitides [ new onseIn 1997, Schmidt and colleagues were the first to examine the use of ultrasonography of the temporal arteries in the diagnosis of GCA . When apIn a more recent controlled study performed in our center , it was We conducted an extensive Medline/PubMed, EMBASE and Cochrane databases search for original primary studies published in any language through December 2009 that examined the sensitivity and specificity of the halo sign demonstrated by temporal artery ultrasonograply for GCA diagnosis versus the ACR 1990 criteria for the classification of vasculitis, used as a reference standard . The tem2). The I2 statistic is calculated from Q and can be interpreted as the percentage variability in study results attributable to between-study differences rather than chance [Sensitivity and specificity estimates of the halo sign together with their 95% confidence intervals were calculated for each study and for the total sample size of studies. Two meta-analytic methods of weighted independent estimation of overall sensitivity and specificity were applied to overcome biased estimation of such calculations; namely, the fixed effect model of Mantel-Haenzel weights each study by the inverse of its variance , thus isn chance . The degBecause the sensitivity and specificity are by their nature dependent on each other, a more appropriate analysis, which takes into account their independence, such as the summary receiver-operating characteristic (sROC) curve analysis was also applied. The sROC curve is well established as a method of summarizing the performance of a diagnostic test . The areSince an important extra source of variation in meta-analysis of diagnostic accuracy relates to the fact that different studies may have used, explicitly or implicitly, different thresholds to define positive and negative test results, Spearman correlation coefficient between the sensitivity and specificity was used (if the threshold effect exists an inverse correlation appears). A visual cue to the possible existence of this source of variation can be given by the plot the sensitivity and specificity on a ROC plane. If such a threshold effect exists, the points will show a curvilinear pattern. This hypothesis can be tested using Moses' regression model, both with unweighted and weighted, with inverse variance. If the slope (b) of the regression equation is not significantly different from zero, then we can assume that DOR is constant and that there is no heterogeneity between the studies.Of a total of 16 primary studies that examined the diagnostic value of the halo sign demonstrated by temporal artery ultrasonograply versus the ACR 1990 criteria for the classification of vasculitis, used as a reference standard ,6,16-28,As shown in Figure Notably, there was significant heterogeneity between the studies in terms of sensitivity , as well as in terms of specificity . As also shown by the sROC curves depicted in Figure As shown in Figure Finally, 4 studies, including a total of 380 patients provided data concerning the presence of bilateral halo in CDS ,17,21,24Our meta-analysis revealed a sensitivity of 68% and a specificity of 91% for the unilateral halo sign, as well as 43% and 100%, respectively, for the bilateral halo sign in temporal artery ultrasonography for GCA diagnosis, when the 1990 ACR criteria are used as the reference standard. Possible limitations of these criteria have been discussed in the literature , but revWe restricted our analysis to studies using the ACR criteria as reference standard. Those studies focusing only on comparison between ultrasonography and temporal artery biopsy findings -38 were A sensitivity of 68% and a specificity of 91% for the unilateral halo sign in temporal artery ultrasonography for GCA diagnosis, when the ACR criteria are used as the reference standard, are comparable to sensitivity and specificity estimates of other diagnostic tests that are widely used in rheumatology. For example, in a meta-analysis on the diagnostic value of autoantibody measurements in rheumatoid arthritis, the sensitivity and specificity of rheumatoid factor were 69% and 85%, respectively, whereas of anti-cyclic citrullinated peptide were 67% and 95% . Moreove2) of both sensitivity and specificity of the halo sign, showed significant heterogeneity concerning the results between studies, probably caused by a threshold effect. It is difficult to assume that this threshold effect is attributable only to ultrasonography findings per se. Other contributing factors may be differences in clinical diagnostic skills or biopsy method, and/or interpretation of histology results across studies. In addition, referral of patients for ultrasonography by physicians non-specialised to connective tissue diseases, i.e. internists, general practitioners, ophthalmologists, or neurologists may lead to a low pretest probability for positive GCA in some studies. Such factors may clearly result to variability between studies concerning the diagnostic thresholds.Interestingly, the values of inconsistency coefficient (IBecause of the trade-off nature of sensitivity and specificity, meta-analysis of diagnostic tests using these conventional expressions offers statistical challenges. Simple pooling of sensitivity and specificity may be not the most appropriate approach, as it ignores threshold differences. In addition, heterogeneity may lead to an underestimation of a test's performance. More recent evidence suggests that a more important index to define the accuracy of a given test result as a diagnostic tool is the pooled DOR . The DORThe results presented herein confirm that the halo sign in ultrasonography is useful in diagnosing GCA. A previously proposed algorithm  suggestsComparing to temporal artery biopsy, ultrasonography is very well tolerated, more accessible, less costly and a more rapid and easier to perform procedure, with high reproducibility. An additional important advantage of ultrasonography is that full-length examination of the superficial temporal and other cranial arteries is allowed. Experts in the field suggest that certain parameters should be fulfilled for a correct US examination of the temporal arteries; namely, high-quality color Doppler US equipment with a linear probe that exhibits a frequency of more than 8 MHz, adequate experience with vascular US, knowledge of the US image of a normal temporal artery, and standardized US machine adjustments . The colThe present meta-analysis, however, is by design unable to show a direct superiority of ultrasonography to microscopic examination of temporal artery biopsy, which might have some additional prognostic importance. As recently demonstrated in a study of 391 biopsy specimens from patients with GCA, the presence of giant cells was strongly associated with permanent visual loss, the most feared disease complication  On the oTo conclude, temporal artery edema demonstrated as halo sign in ultrasonography, providing that technical quality criteria are fulfilled , should GCA: giant-cell arteritis; ACR: American College of Rheumatology; DOR: diagnostic odds ratio; sROC: summary receiver operating characteristic; AUC: area under the ROC curve.The authors declare that they have no competing interests.A.A. participated in the design of the study, collected and analysed the data and helped to draft the manuscript. M. Kanakis participated in the design, collection and interpretation of data. M. Kyprianou performed the statistical analysis and participated in interpretation of data. P.P.S. conceived of the study and drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/11/44/prepub