Oncomelania hupensis, the intermediate snail host of Schistosoma japonicum, cannot be easily excluded from the integrated approach of schistosomiasis control in China. Application of chemical compounds, molluscicides, in snail habitats is a common method for snail control in addition to environmental modification. We conducted a systematic review and meta-analysis to assess the molluscicidal effects of the currently recommended 50% niclosamide ethanolamine salt wettable powder and a new 4% niclosamide ethanolamine salt powder developed by Chinese researchers. Literature was searched from three Chinese databases, i.e. Chinese Biomedical Database, VIP Database and Wanfang Database, on field mollusciciding trials of niclosamide in China . Molluscicidal effects on reduction of snail population of the 50% or 4% niclosamide formulations in field trial were evaluated 3 days, 7 days or 15 days post-application. Out of 90 publications, 20 papers were eventually selected for analysis. Publication bias and heterogeneity tests indicated that no publication bias existed but heterogeneity between studies was present. Meta-analysis in a random effect model showed that the snail mortality of 3, 7 and 15 days after spraying the 50% niclosamide ethanolamine salt wettable powder were 77% [95%CI: 0.68-0.86], 83% [95%CI: 0.77-0.89], and 88% [95%CI: 0.82-0.92], respectively. For the 4% niclosamide ethanolamine salt powder, the snail mortality after 3, 7 and 15 days were 81% [95%CI: 0.65-0.93], 90% [95%CI: 0.83-0.95] and 94% [95%CI: 0.91-0.97], respectively. Both are good enough to be used as molluscicides integrated with a schistosomiasis control programme. The 4% niclosamide ethanolamine salt powder can be applied in the field without water supply as the surrogate of the current widely used 50% niclosamide ethanolamine salt wettable powder. However, to consolidate the schistosomiasis control achievement gained, it is necessary to continuously perform mollusciciding more than twice annually in the field.The control efforts on  Oncomelania hupensis [O. hupensis habitats can be divided into three ecological types: (i) lake and marshland regions; (ii) hilly and mountainous regions; and (iii) plain regions with water networks [The transmission records of schistosomiasis japonica, a disease prevalent in the Yangtze River valley and southern part of China, can be traced back over 2,000 years . The dishupensis . Geograpnetworks ,5. The anetworks ,4,6. Soonetworks , which dnetworks ,9. The inetworks ,11. Snainetworks ,13. Favonetworks . In addinetworks ,16.Application of chemical compounds in snail habitats is a common method for elimination of intermediate host snails, in addition to environmental modification, such as cementing canals, re-adjusting irrigation systems, etc ,17. NiclMany similar studies have been carried out to evaluate the molluscicidal effect of different molluscicides in field trials in China. Due to the variation of environmental settings, a large variety of the molluscicidal effects on snail mortality (40-100%) no doubt impact the government decision makers to formulate the snail control strategy. Therefore, we conducted a systematic review and meta-analysis to assess the molluscicidal effect of the currently recommended 50% niclosamide ethanolamine salt wettable powder and of the new 4% niclosamide ethanolamine salt powder.A systematic search of the Chinese literature, including documents published from January 1, 1990 to April 1, 2010, was conducted to capture data on molluscicidal effect on snail mortality by niclosamide in field trials in China.Three major Chinese literature databases, namely Chinese Biomedical Database, VIP Database and Wanfang Database, were jointly searched for data pertaining to molluscicidal effect of the 50% niclosamide ethanolamine salt wet powder or the 4% niclosamide ethanolamine salt powder in field trials in China. Since the databases are domestic, the language of publication was Chinese. We used the terms "niclosamide", or "Luomiecide ", or "Qiangluocide " and "molluscicide", or "snail control". The abstracts of each screened publication were read carefully.2; (4) molluscicidal effects on mortality of O. hupensis were evaluated 3 days, 7 days or 15 days post drug application.Both inclusion criteria and exclusion criteria for searching published literature were settled in the first stage of the meta-analysis. The inclusion criteria of published literature were as follows: (1) the 50% niclosamide ethanolamine salt wettable powder or the 4% niclosamide ethanolamine salt powder was used; (2) molluscicidal experiments carried out in marshland; (3) the used dose of niclosamide in the field was 2 g/m2 was not standardized or used in a wrong approach; (2) no original data, such as number of observations and number of dead snails, were published in the paper; (3) review article, or books or conference abstracts; (4) editorials or letters to the editors without original data.The exclusion criteria of published literature were as follows: (1) application dosage of 2 g/mThe conclusion we draw may be distracted if we do not consider the bias that may exist from the literature review. Among all the biases, the publication bias is more difficult to control and adjust for. Identification of the bias is particularly important for the future adjustment. We applied "Metabias" function to test for funnel plot asymmetry based on a linear regression method, where the method "linreg" is the statistical test based on a weighted linear regression of the treatment effect on its standard error .Heterogeneity in meta-analysis refers to the variation in study outcomes between studies. The classical measure of heterogeneity is Cochran's Q. The I² statistic describes the percentage of variation across study that is due to heterogeneity rather than chance ,24, withR Package [All statistical analyses were done in  Package . All ratA total of 90 documents, with potential original data on molluscicidal effect by niclosamide in field trials in China, were identified. The flow diagram in Figure p value higher than 0.05. The linear regression figures showed all selected publications normally scattered around the regression line  plays an important role for the population dynamics. There is a general consensus among ecologists that small arthropods or invertebrate animals will follow the Gompertz logistic (GL) reproduction model, which means high turn-over rates occurred at low density [O. hupensis snails in the field, it is recommended to perform mollusciciding more than twice annually. While studies on the optimal times of annual mollusciciding to eliminate O. hupensis over certain years is still unknown and we need further investigations on the ecology of O. hupensis to understand its dynamics.Although this systematic review and meta-analysis identified that the molluscicidal effect of both formulations of niclosamide are good enough for the mollusciciding in a schistosomiasis control programme, both formulations cannot produce 100% snail mortality. Meta-analysis results showed that the snail mortality of the 50% and 4% formulations are 88% and 93%, respectively, 15 days after applying. It indicates the low density of  density ,49. TherThe authors declare that they have no competing interests.GJY and XNZ conceived and wrote the first version of the manuscript. GJY, WL, LPS, FW, KY and YXH revised the manuscript. XNZ and GJY finalized the manuscript. All of authors read and approved the final version of the manuscript.


Observational studies have demonstrated various correlations between hypertensive disorders of pregnancy and different weather parameters. We aim to study if a correlation exists between the incidence of eclampsia and pre-eclampsia and various weather parameters in the tropical coastal city of Mumbai which has the distinction of having relatively uniform meteorological variables all throughout the year, except for the monsoon season.We retrospectively analysed data from a large maternity centre in Mumbai, India over a period of 36 months from March 1993 to February 1996, recording the incidence of preeclampsia and eclampsia. Meteorological data was acquired from the regional meteorological centre recording the monthly average temperature, humidity, barometric pressure and rainfall during the study period. Study period was then divided into two climate conditions: monsoon season (June to August) and dry season September to May. The incidence of preeclampsia and eclampsia and the meteorological differences between the two seasons were compared.Over a 36-month period, a total of 29562 deliveries were recorded, of which 1238 patients developed preeclampsia (4.18%) and 34 developed eclampsia (0.11%). The incidence of preeclampsia did not differ between the monsoon and the dry season . The incidence of eclampsia was significantly higher in the monsoon . The monsoon was significantly cooler , more humid , and received higher rainfall  than the rest of the year. The median barometric pressure (1005 mb) during the monsoon season was significantly lower than the rest of the year .In the tropical climate of Mumbai, the incidence of eclampsia is significantly higher in monsoon, when the weather is cooler and humid with a lower barometric pressure than the rest of the year. This effect is not seen with preeclampsia. This strengthens the association of low temperature and high humidity with triggering of eclampsia. The aetiology of preeclampsia and eclampsia is not fully understood. Previous studies have shown a variable association of preeclampsia and eclampsia with the changing weather patterns of different seasons. These association studies often compared the incidence of preeclampsia and eclampsia against the climatic patterns seen in the three or four characteristic seasons in the study area. Studies coming from different parts of the world frequently give opposing results. There are two studies which demonstrate no relationship of meteorological factors on the incidence of eclampsia ,2. Most In our study, we intend to establish if any such association exists in the distinct climate of the tropical coastal city of Mumbai, India. Mumbai has a remarkably stable climate all throughout the year, except for the monsoon season, where the weather pattern is overwhelmed by heavy rainfall. We aim to assess the association of preeclampsia and eclampsia with various weather parameters in this unique climatic condition.The study population included all patients delivered in a large maternity centre in Mumbai. The project did not entail accessing any individual patient data or identifiable information and was reviewed by the hospital research committee. Prospectively maintained database recording all deliveries and associated complications was analysed retrospectively for a three-year period from March 1993 to February 1996 to identify the total number of deliveries, total number of preeclampsia cases and the total number of eclampsia cases. The data was then divided into two groups based on the two main seasons (June to August as monsoon season and September to May as the dry season). This division was based on the fact that the coastal city of Mumbai experiences a remarkably stable weather pattern all throughout the year except during the monsoon season. Monthly averages of meteorological parameters  were acquired from the Regional Meteorological Centre, Mumbai for the study period.The incidence of preeclampsia and eclampsia and the meteorological parameters between the two seasons (June to August and rest of the year) were compared. Data was compared using Fisher's exact test, Chi-square test and Mann-Whitney test using GraphPad Prism version 4.03 for windows, GraphPad Software, San Diego California, USA.Over a 36-month period, a total of 29562 deliveries were recorded, of which 1238 patients developed preeclampsia (4.18%) and 34 developed eclampsia (0.11%). The incidence of preeclampsia (excluding patients progressing to eclampsia) did not differ between the monsoon and the dry season . The incidence of eclampsia was significantly higher in the monsoon  as compared to the dry season . By definition, monsoon received significantly higher monthly average rainfall when compared with the dry season i.e. rest of the year . The median maximum temperature in monsoon was 30.7°C and was significantly lower than during the rest of the year . The median relative humidity in monsoon was 85% and was significantly higher than during the rest of the year . The median barometric pressure) in monsoon was 1005 mb and was significantly lower than the rest of the year .During the rest of the year  the maximum temperature, morning barometric pressure, and the relative humidity remained very stable with a mean ± SD of 32.4 ± 1.1 C, 1012 ± 2.5 mb, and 71.3 ± 8 % respectively. Figures Available literature on the seasonal variation in the incidence of preeclampsia and eclampsia is much divided in its conclusion -13. PreeWe evaluated a large study population over a three-year period in a large maternity centre in Mumbai for the incidence of preeclampsia and eclampsia. Preeclampsia was defined by the presence of hypertension (systolic BP ≥ 140 mm Hg or diastolic BP ≥ 90 mm Hg in a women who were previously normotensive prior) and proteinuria after 20 weeks of gestation. Eclampsia was diagnosed by the presence of seizures in addition to preeclampsia, in the absence of any other obvious cause for seizures. All patients with the diagnosis of severe preeclampsia were closely monitored, treated with magnesium sulphate infusion and delivered as soon as possible. Mild preeclampsia was treated expectantly.Our study sample consisted of patients treated at a large maternity centre in Mumbai dealing with over 9500 deliveries a year. Our hospital treats only pre-registered patients, who have received antenatal care at our centre. All emergency un-booked referrals are taken by another state-run teaching hospital, which is next door to our centre. In effect our data is not biased by variation of referred cases as we deal with a large stable local population base. Also as our population base is local no significant delays in transporting patients to the hospital were anticipated.Our data shows a significant increase in the incidence of eclampsia in the monsoon season, with no such change in the incidence of preeclampsia. It is widely understood that preeclampsia and eclampsia are progressive manifestations of the same patho-physiological spectrum. Our study shows that the meteorological factors had no influence on the incidence of preeclampsia. In contrast, lower temperature, higher rainfall and humidity and lower barometric pressure were related to the triggering of seizures in patients primed with preeclampsia. This is similar to other studies associating eclampsia with lower temperatures -5 and inEvidence suggests that dehydration protects the brain from convulsions . The warIn contrast, over-hydration and hyponatremia is well known to be associated with triggering of seizures ,18. HypoIn our study, we have established a significant association of eclampsia to monsoon season, which may be explained by these effects.Contrary to the above explanation, there is some suggestion that plasma volume expands in summer when compared to winter months . It is nOur study has identified a pattern in the incidence of eclampsia, which probably is related to the climatic conditions in monsoon season. The exact mechanism by which meteorological parameters affect the patho-physiology of eclampsia is beyond the scope of our study. Further studies are needed to explore the exact mechanisms involved.There is a significant association between climatic factors and the occurrence of eclampsia, which is not seen in preeclampsia. Lower temperature, higher humidity and lower barometric pressure are linked to eclampsia. Exploring this association will help us to gain further insight into the pathophysiology of this condition.The author(s) declare that they have no competing interests.The pre-publication history for this paper can be accessed here:


The Ig Fc receptor family is an important link between the humoral and cellular immune systems. The association of a dimorphism in amino acid 131 (R/H) of the FcγRIIa with malaria severity, the R-allele being associated with a milder disease outcome, led to the investigation of the possible impact of this polymorphism in the interethnic difference in malaria susceptibility seen between the Fulani and Dogon in Mali.2-test.Plasma from individuals from Mali (164 Fulani and 164 Dogon) were analysed for malaria-reactive and total IgG subclass antibodies using ELISA, and the same individuals were also genotyped for the FcγRIIa R131H polymorphism using RFLP-PCR. Statistical analyses of the IgG subclass levels were done by unpaired t-test and ANOVA, and genotype differences were tested by χWhile the two ethnic groups showed a similar frequency of the FcγRIIa 131 R/H heterozygote genotype, 131R/R dominated over the 131 H/H genotype in the Dogon whereas the Fulani presented a similar frequency of the two homozygote genotypes. The two alleles were evenly distributed in the Fulani, while the Dogon were clearly biased towards the R-allele. The Fulani showed higher levels of anti-malarial IgG1, -2 and -3 antibodies, with a higher proportion of IgG2, than the Dogon. In the Fulani, H-allele carriers had higher anti-malarial IgG2 levels than R/R homozygotes, while in the Dogon, the R-allele carriers showed the higher IgG2 levels. For anti-malarial IgG3, the R-allele carriers in the Fulani had higher levels than the H/H homozygotes.Taken together, the results showed marked interethnic differences in FcγRIIa R131H genotypes. Furthermore, the results indicate that the FcγRIIa R131H genotype may influence the IgG subclass responses related to protection against malaria, and that IgG2 may be of importance in this context. Plasmodium falciparum malaria [P. falciparum antigens, have been shown to be related to protection [Malaria remains one of the most prevalent infectious diseases in the world today, with high morbidity and mortality, and no effective control strategies. A rational vaccine design is limited by the lack of knowledge of how the immune system clears the infection. Several studies relate high titers of anti-malarial IgG to protection from severe malaria, and seroepidemiological studies from different endemic areas have demonstrated an association between IgG antibodies of the cytophilic subclasses IgG1 and IgG3 with protection against  malaria . In otheotection , indicatThe Fcγ receptors on monocytes and other leukocytes are important structures in the immune defence against pathogens. The binding of antibodies to the Fc-receptors provokes important biological functions, e.g. antibody dependent cell-mediated cytotoxicity (ADCC) or inhibition (ADCI) and phagocytosis . In humaSeveral studies have demonstrated differences in susceptibility to malaria between different ethnic groups. In West Africa, the Fulani were shown to have a lower incidence of malaria than other sympatric groups, despite the same exposure and no differences in socio-cultural circumstances . StudiesIn an attempt to further define potential genetical and immunological factors contributing to these ethnic differences in malaria susceptibility, the FcγRIIa genotypes in sympatric individuals from Mali were analysed and related to both the total and the malaria reactive IgG subclass profiles.P. falciparum as the main parasite species. The entomological inoculation rate (EIR) is similar in both ethnic groups [The study area is located in the Mopti area about 850 km Northeast of Bamako, the capital of Mali. Four villages  were identified for the study. The Mantéourou and Naye are divided into two subdivisions, Mantéourou Peulh/Mantéourou Dogon and Naye Peulh/Naye Dogon-Dinsogou. The two subdivisions are separated by 300–500 meters and inhabited either by Fulani or Dogon. The other two villages, Binédama and Anakédié, are exclusively populated by either Fulani or Dogon, respectively, and are located approximately 1,000 meter apart. Malaria transmission is mesoendemic in the area, with c groups . In thisBlood was collected during the rainy season in September 2005 from 328 (164 Fulani and 164 Dogon) asymptomatic, randomly selected donors, aged 1–60 years. For PCR analyses, finger prick blood was collected on filter papers. For plasma samples, either finger prick blood was collected in capillaries or venous blood in vacutainer tubes, both with heparin as anti-coagulant. Spleen enlargement was established based on palpation and graded on the Hackett score (0–5). The proportion of individuals that had enlarged spleens was denoted spleen rate. Informed consent was obtained from all participants or their guardians and it was performed in two steps; first an orally community consent is obtained prior to the study, where the whole community is informed of the aim of the study. Second an individual consent is obtained before blood collection; an alphabetized volunteer sign the form and a non-alphabetized volunteer puts his fingerprint. The Ethical committee of the Faculty of Medicine and Pharmacy of Mali and the National Ethics Committee in Sweden approved the study.The capillaries were cut, and blood cells and plasma were separated by centrifugation. The plasmas were stored in -20°C.DNA was extracted from filter papers using Chelex-100, and then stored at -20°C. In brief, discs of the same size were cut out and incubated overnight in 1 ml of 0.5% saponin in phosphate-buffered saline (PBS) at 4°C, and were then washed 15–30 minutes in 1 ml PBS at 4°C. The discs were then boiled in 200 μl of 5% Chelex-100 in water for 15 minutes, and the DNA was collected in supernatants after centrifugation at 8,000 × g for 3 minutes.Plasmodium falciparum parasites (strain F32) were maintained in continuous cultures as described earlier [ earlier  and kept earlier . When th earlier .P. falciparum antigen in sodium carbonate buffer (pH 9.6) at the concentration 10 μg/ml. After blocking at 37°C with carbonate buffer containing 1% BSA (w/v) for 2 hours, the plates were washed with saline containing 0.05% Tween 20. Serum dilutions  were added to the plates and incubated 1 h at 37°C. To detect IgG2, IgG3 and IgG4, 50 μl biotin-conjugated mouse anti-human monoclonal antibodies  and alkaline phosphatase conjugated streptavidine  were used. Detection of IgG1 was done with a monoclonal mouse anti-human IgG1 antibody  and alkaline phosphatase conjugated goat anti-mouse Ig . The assay was developed with p-nitrophenyl phosphate disodium salt  as substrate and the optical densities were read at 405 nm in ELISA plate reader . The concentrations of anti-malarial antibodies, expressed as ELISA-units , were calculated from standard curves obtained in a sandwich ELISA with six dilutions of myeloma proteins of the IgG1-4 isotypes . No cross reactivity between the different subclass reagents were detected in this system.EIA/RIA plates  were coated overnight at 4°C with crude The total concentrations of IgG subclass 1–4 were determined with PeliClass human IgG subclass kit . In short, precoated micro titre plates where incubated with diluted sera  at 37°C for 1 h. For detection, an HRP-conjugated anti-human IgG antibody was used and the assay was developed with ABTS-substrate and read at 405 nm in ELISA plate reader .R131H FcγRIIa polymorphism (rs1801274) was investigated using restriction fragment length polymorphism (RFLP) analysis as described by Jiang et al [®  using a 10 min denaturation at 94°C followed by 35 cycles with 94°C for 30 sec, 56°C for 30 sec and 72°C for 45 sec. The final extension was at 72°C for 5 min. Five μl of the PCR product were used for the enzymatic digestion with 2.5 U Bsh12361  at 37°C over night. The resulting fragments  were analyzed on ethidium bromide stained 2.5% agarose gels.The ng et al . The PCRP. falciparum infection and to determine the number of merozoite surface protein (msp) 2 (FC27 and 3D7) clones per infection, a nested PCR was performed. Amplifications were done in a 10 μl reaction using 2.5 μl of DNA, iProof™ High-Fidelity Master Mix  and 500 nM of primer pairs corresponding to the outer conserved region of the polymorphic repetitive block 3 of msp2. DNA was denatured at 98°C for 1 min; then PCR was performed for 30 cycles of 98°C for 10 sec, 61°C for 20 sec and 72°C for 30 sec, with a final 5 min extension. One μl of the PCR product was re-amplified with primers specific for FC27 and IC/3D7 allelic types of msp2, using the following program: 30 sec at 98°C; 25 cycles of 98°C for 10 sec, 61°C for 20 sec and 72°C for 10 sec and a final extension for 5 min. The primer sequences were presented elsewhere [In order to detect an ongoing ®  using a 5 min denaturation at 95°C followed by 35 cycles with 95°C for 30 sec, 56°C for 30 sec and 72°C for 30 sec. The final extension was at 72°C for 5 min. The entire PCR product were used for the enzymatic digestion with 4 U DdeI (Invitrogen) at 37°C for 2 h. The resulting fragments were analyzed on ethidium bromide stained 2.5% agarose gels.To detect HbAS carriers a PCR procedure was used. In short, the PCR amplification was performed in 10 μl reactions using 2 μl of genomic DNA, 5 μl of 2× ready-to-use PCR Master Mix  and 0.25 μl of each primer ). The PCR was carried out in an Eppendorf Mastercycler2-test. Multiple regression analyses, with IgG subclasses as dependent variables and age, ethnicity, gender, parasite density and FcγRIIa R131H genotypes as independent variables, were performed to assess the influence on these variables on IgG subclass levels. Association analyses were performed in Unphased version 3.0.7.All the statistical analyses were performed in StatView version 5.0.1 if not stated otherwise. Differences in malariometric indexes between the two ethnic groups were tested with Mann-Whitney U-test. The observed genotype frequencies of the FcγRIIa R131H polymorphism in the two ethnic groups were tested for differences with χAs the antibody data did not show normal distribution, they were log-transformed, after which they all were normally distributed. Comparisons between the two ethnic groups were done using unpaired t-test. For analyses of genotype and IgG subclass levels, ANOVA was used. To determine differences between the populations, the Fisher's PLSD was used.2-test was used. Ethnic and genotype-based differences in parasite density, numbers of parasite clones and haemoglobin levels were analysed by Mann-Whitney U-test or Kruskal-Wallis test.To test for potential ethnic differences, as well as for genotype-based differences in spleen rate, χA p-value of 0.05 or less was considered to be significant, and p-values between 0.05 – 0.1 were considered to be marginally significant. No correction for the number of tests was done, so there is a risk of presenting some overestimated significances.There were no differences in age and gender between the two study populations Table . The parAll individuals of the two study groups were genotyped for the G494A single nucleotide polymorphism (R131H) in the FcγRIIa gene rs1801274). Both allele and genotype frequencies were found to be in Hardy-Weinberg equilibrium. While both ethnic groups showed a similar frequency of heterozygotes (131H/R), there was a statistically significant difference with regard to the frequency of homozygotes , while none of the other subclasses showed any difference between the two ethnic groups Figure .For the anti-malarial IgG antibody subclasses, the Fulani showed significantly higher levels of IgG1 p = 0.0003), IgG2 and IgG3 . For anti-malarial IgG4, both ethnic groups showed very low levels and no difference between the ethnic groups was seen (p = 0.2). Figure . The mos, IgG2 anThe patterns of the different IgG subclasses among the anti-malarial antibodies and total IgGs were compared between the two ethnic groups, analysing the ratios of IgG1/IgG2, IgG1/IgG3 and IgG2/IgG3 Table . IgG4 waP. falciparum-reactive IgG subclass levels, multiple regression analyses with the different IgG subclass antibody levels as dependent variables were performed. When adjusted for age, gender, ethnicity and parasite density, the FcγRIIa R131H genotype showed a weak but significant predictive effect on P. falciparum-reactive IgG3 levels (adjusted R2 = 0.324 and p = 0.027) with higher levels in individuals homozygous for the R-allele, and a non-significant effect on total IgG1 levels (adjusted R2 = 0.117 and p = 0.09), with higher IgG1 levels in HH carriers. When separating the two ethnic groups in the analyses, the FcγRIIa R131H genotype showed a tendency for a predictive effect on P. falciparum-reactive IgG2 levels in both Fulani and Dogon . Higher antibody levels where found in HH carriers in the Fulani, while RR carriers had higher levels in the Dogon,In order to see if the FcγRIIa R131H genotype could have an effect on the The total IgG1 levels in the Fulani were significantly but still weakly predicted by the FcγRIIa R131H genotype (p = 0.04).P. falciparum-reactive antibodies between the different FcγRIIa R131H genotypes were performed. Overall genotype differences regarding both IgG2 and IgG3 levels were seen for both ethnic groups, as well as a difference in IgG4 levels in the Fulani ethnic group . For the Fulani, the R/R individuals showed lower IgG2 concentrations than the H/R subjects (p = 0.05), but there was also a tendency for H/H individuals to present higher IgG2 levels than R/R genotype carriers. The concentrations of IgG3 were lower in the H/H individuals compared to the H/R heterozygotes (p = 0.01), but there was also a tendency for lower levels as compared to R/R subjects. For anti-malarial IgG4 concentrations, the H/H homozygotes had lower levels than H/R individuals (p = 0.04).In order to further decipher the possible influence of the FcγRIIa R131H genotypes on IgG subclass levels, comparisons of the levels of the different IgG subclasses among the up Table . For theAnalysis of the R-allele carriers in comparison with the H/H homozygotes revealed that there was only a difference regarding IgG3 in the Fulani Table , with R-In order to see if the FcγRIIa R131H genotypes affected haemoglobin levels, parasite density, number of circulating parasite clones, frequency of Hb AS and spleen rate, analyses of these parameters within each genotype were performed. No differences were found for parasite density, haemoglobin levels or spleen rate. The number of circulating parasite clones differed in the Fulani group, where individuals with the RR genotype had higher numbers of clones than HR and HH genotype individuals  .This study shows that two sympatric ethnic groups in Mali, the Fulani and the Dogon, exhibit differential frequencies in the expression of FcγRIIa R131H genotypes and allotypes. It furthermore, confirms and extends the previous findings regarding differences in anti-malarial responses between sympatric ethnic groups living in West Africa ,13. The The FcγRIIa R131H genotype results of this study contradict those from previous reports, where the R/R genotype has been associated with protection (reviewed by Braga ). Here iThe impact of the FcγRIIa R131H polymorphism on the clinical outcome of malaria has been shown in several studies ,23,27-29No differences between the two ethnic groups were shown when comparing total IgG subclasses, except for IgG4, were the Dogon showed slightly higher concentrations than the Fulani. This confirms the previous suggestion, that the relative resistance seen in the Fulani as compared to other sympatric ethnic groups appears to be pathogen related, and not a result of a generally more activated immune system in the Fulani .P. falciparum malaria [et al [Regarding anti-malarial IgG subclasses, the Fulani had higher levels of all subclasses, except for IgG4, which was very low in both ethnic groups. When calculating the percentage of anti-malarial IgG subclass antibodies within the total IgG concentrations, the Fulani showed a higher percentage of all IgG subclasses of anti-malarial antibodies, except for IgG4, where there were no differences between the two ethnic groups. For the ratios between IgG1:IgG2, IgG1:IgG3 and IgG2:IgG3, the Fulani had higher IgG1, IgG2 and IgG3 levels, while the Dogon had predominant IgG1 and IgG3 responses. Previous studies suggest that IgG1 and IgG3 are the most important IgG subclasses in the protection against malaria . HoweverCarriage of haemoglobin AS has been reported to influence the anti-malarial IgG2 concentrations in Gabonese children, which was independent of the FcγRIIa R131H genotype . PrelimiIn conclusion, the relative resistance to malaria seen in the Fulani as compared to the Dogon appears not to be a generally more reactive immune system, but rather a pathogen related phenomenon. Furthermore, the results indicate that the FcγRIIa 131H allele may be associated with a lower susceptibility to malaria in the Fulani group as compared to other sympatric ethnic groups, and also that the FcγRIIa R131H polymorphism may have an influence on the IgG subclass responses and thereby affecting the malaria susceptibility.The authors declare that they have no competing interests.EI contributed to the design of the study, performed the FcγRIIa genotyping and the ELISA, carried out the statistical analyses and drafted the manuscript. MV carried out the parasite genotyping. BM examined all study subjects in Mali and collected all clinical data. AL contributed to the FcγRIIa genotyping and the ELISA. NI performed the sickle cell analyses. AD and OKD were responsible for clinical data in Mali. KB designed the study. MTB and KB revised the manuscript and financed and supervised the research. All authors read and approved the final manuscript.


F5 (G1691A), F2 (G20210A) and MTHFR , in 469 healthy blood donors from São Miguel Island (Azores). We also analysed the CYP2C9  and VKORC1 (G1639A) variants in fifty-eight individuals with predisposition to thrombosis (possessing at least one variation in F5 or F2 genes and one in MTHFR) to evaluate their warfarin drug response genetic profiles.The Azorean population presents the highest standardized mortality rate for cardiovascular diseases (CVD) when compared to mainland Portugal and other populations. Since thrombosis is a common cause of CVD, we assessed four polymorphisms in three thrombotic risk genes – 2, p < 0.001). The combined analysis of these variants identified twenty-two different genetic profiles . Complete homozygosity for all wild-type alleles (GG GG CC AA) was present in 11.7%, being GG GG CT AA (22.4%) the most frequent profile. The results also demonstrated that 12.4% (58 out of 469) of São Miguel islanders have increased genetic predisposition to thrombosis. Subsequently, we evaluated these individuals for their warfarin response genetic profiles. The data showed that seven out of fifty-eight individuals are poor metabolizers (two with CYP2C9*2/*2 and five with CYP2C9*2/*3 genotypes). VKORC1 polymorphism analysis identified twelve individuals (20.7%) with AA genotype, who probably will require lower doses of warfarin. The joint analysis of CYP2C9 and VKORC1 revealed that 79.3% (46 out of 58) of the individuals carry at least one polymorphism in these genes. Within these, twenty-five individuals (43.1%) need intermediate and/or low doses of warfarin, if treatment is started.Among the 469 individuals, the data showed that thrombotic risk allele frequencies – 1691A (4.9%), 20210A (1.8%), 677T (41.7%) and 1298C (24.8%) – were similar to other Caucasians, but significantly different from mainland Portuguese (χThe present study demonstrated, for the first time, that São Miguel, and possibly the Azores population, shows significant differences on allele frequencies of thrombotic risk factors when compared to mainland Portugal. This research constitutes a primary approach for future studies on CVD, as well as for the implementation of warfarin dosing protocols using the patient's genotypic information. F5; 1q23), resulting in resistance to activated protein C (APC). In the coagulation biochemical pathway, APC prevents blood clots from growing extensively large, by inactivating coagulation factor V (encoded by F5). The G1691A variation may increase the risk for thrombosis by 8 and 91 in heterozygous and homozygous states, respectively [et al. [F2; 11p11-q12) represents the second most important inherited thrombotic risk factor. A single base substitution (G20210A) in the F2 3'-untranslated region causes elevated plasma prothrombin levels. The same authors reported a 2.8-fold increase risk for venous thrombosis in heterozygous and homozygous variant individuals [MTHFR; 1p36.3), includes two variants – C677T and A1298C [et al. [et al. [Cardiovascular diseases (CVD) are complex disorders that include dysfunctional conditions of the heart, arteries and veins. Within these diseases, thrombosis is the third most common cardiovascular event worldwide . It can tion G202A in the CYP2C9 (Cytochrome P450 2C9) and VKORC1 (Vitamin K epoxide reductase complex subunit 1) genes account for approximately 50–60% of drug dosing variability [CYP2C9, the most frequent variants are: CYP2C9*1 , CYP2C9*2 (Arg144Cys) and CYP2C9*3 (Ile359Leu). These last two are associated with decreased metabolic efficiency of the CYP2C9 enzyme and increased risk of bleeding when administrated initial dosages of warfarin [VKORC1 gene results in decreased mRNA transcription and increased sensitivity to warfarin inhibition of hepatic synthesis of functional vitamin K-dependent coagulation factors [et al. [VKORC1 polymorphisms and classified individuals according to warfarin dose requirements into distinct groups: high (GG), intermediate (GA) and low (AA).The genetic characterization of the individual's potential risk for developing thrombosis and response to drugs is essential in clinical follow-ups, as well as for medical prescription. Warfarin is one of the most widely used anticoagulant drug, which requires a thorough risk-benefit analysis since the dose prescribed should avoid hemorrhagic complications and achieves suppression of thrombosis . The admiability ,14. Cytoiability . Althougwarfarin . A G>A v factors . Rieder  [et al.  studied F5, F2 and MTHFR – involved in blood clot formation. Moreover, in the individuals with predisposition to thrombosis, we evaluated the pharmacogenetic profile for warfarin, by characterizing CYP2C9 and VKORC1 polymorphisms. This information is important for the implementation of warfarin pre-prescription genotyping with the goal of performing individualized treatment.The Azores population presents the highest standardized mortality rate caused by CVD when compared to mainland Portugal   and otheF5 (G1691A), F2 (G20210A) and MTHFR . After evaluating the genetic variants in the aforementioned genes, we selected a subset of individuals who met one of the following criteria: 1. one variant in F5 and one in MTHFR ; 2. one variant in F2 and one in MTHFR  and 3. one variant in F5, F2 and MTHFR . These criteria were chosen to restrict the sample to individuals who would have a higher genetic risk for a thrombotic event, and hence fifty-eight individuals were analysed for CYP2C9 and VKORC1 polymorphisms.The population sample comprised a group of 469 São Miguel Island blood donors from the anonymized Azorean DNA bank located in the main Hospital of Azores archipelago, Portugal . São MigF5 and MTHFR polymorphisms . The genotyping protocol for the detection of the F5 polymorphism and MTHFR variants was adapted from Kumar et al. [et al. [ms Table  were evar et al.  and Skib [et al. , respectF2, CYP2C9 and VKORC1 , F2 (G20210A) and MTHFR  polymorphisms were assessed in 469 healthy individuals from São Miguel Island, Azores  was present at 9.8% . We did not detect 1691AA individuals. The results on the F2 analysis show an allele frequency of 1.8% for the 20210A variant. Although this polymorphism is also uncommon in other populations, one homozygous variant individual (0.2%) and fifteen heterozygous (3.2%) were observed in the present study. The frequency of the variant alleles in MTHFR (677T and 1298C) was 41.7 and 24.8%, respectively. Overall, 17.9% are 677TT , and 6.0% present the 1298CC genotype .Allele and genotype frequencies of F5, F2 and MTHFR (order in genotype profile) were observed in São Miguel's population . No heterozygous or homozygous profiles for all four variants were observed. Almawi et al. [F5 G1691A or F2 G20210A with MTHFR 677TT genotype, respectively, enhancing the risk for deep vein thrombosis (DVT). Based on this criterion, fifteen  and two individuals  have higher risk for DVT development  to 4.9% (São Miguel Island). This trend is not evident for F2 G20210A, where the values present a lower range, from 0.3% for African-American to 2.6% for Spanish. African populations showed the lowest frequency for MTHFR variants. Taken together, the results demonstrate that, in São Miguel, the frequencies for all polymorphisms were similar to those found in Caucasians.The comparison of allele frequencies of the thrombotic genetic risk factors – F5 or F2 genes and one in MTHFR, we studied CYP2C9 and VKORC1 gene polymorphisms. The allele frequencies for CYP2C9*2 and CYP2C9*3 were 15.5 and 10.3%, respectively (Table VKORC1 gene (G1639A), we identified twenty (34.5%) wild-type individuals, twenty-six (44.8%) heterozygous and twelve (20.7%) homozygous variant, corresponding to an allele frequency of 43.1% for 1639A  individuals that probably will require high doses of warfarin , and eight (13.8%) will need lower doses . The remaining seventeen individuals (29.3%) present one of the following genotypes: *1*1-AA→nine individuals, *1*2-GA→four, *1*3-GA→three and *2*3-GG→one, where all of them will possibly necessitate intermediate doses. In summary, twenty-five individuals (43.1%) will need intermediate and/or low doses of warfarin, if treatment is started.The majority of individuals with a genetic predisposition to thrombosis  carry at least one polymorphism in the genes involved in the metabolism of warfarin  are more frequent and combined with a genetic predisposition further enhance the risk of a thrombotic event. In the present study, the results obtained from 469 São Miguel healthy individuals demonstrate that approximately 5% have the uese, 1% , we did r et al. , this vaal, 2.5% .MTHFR C677T variant, which has a relatively high frequency throughout the world and is considered a low risk factor for vascular diseases [MTHFR 677T shows an allelic frequency of 41.7%, very similar to Europeans . Family-based investigation tends to have biased relative risk estimation, because oversampling of affected individuals is normally present. Therefore, the genetic characterization of disease variants in the general population is important to correct this potential bias. Consequently, the present research will aid the clinical follow-ups of patients with recurrent thrombotic events.The joint analysis of all variants demonstrated that 12.4% of the total sample (58 out of 469) has increased predisposition to thrombosis development. In addition, comparison of allele frequencies for all thrombotic risk factors between São Miguel and mainland Portugal revealed statistically significant differences  -, indicating that individuals with this genotype require the highest warfarin maintenance doses [In today's medicine, the association between pharmacogenetics and drug therapy represents an important tool to treat or control complex diseases. Drug treatment is based on the assumption that a particular dose will yield a predefined blood concentration in circulation and, thereby, establish the desired therapeutic effect . Recent d VKORC1 . Here, wce doses .CYP2C9*2 and CYP2C9*3 alleles demonstrate a close similarity with different European populations [CYP2C9*1/*2 and CYP2C9*1/*3) represent a middle range of metabolic activity – intermediate metabolizers, IMs [CYP2C9*2 and CYP2C9*3 variants have less activity and metabolize warfarin more slowly. Individuals with one or two of these variations – poor metabolizers (PMs) – have an enhanced response to warfarin, as well as an increased risk of bleeding when beginning and during treatment if not genotyped previously [CYP2C9*2/*2 and five with CYP2C9*2/*3 genotypes; Table CYP2C9 PMs may exhibit different pharmacokinetics than wild-type individuals if they are medicated with warfarin. Consequently, these individuals possibly will require non-conventional doses of drugs that need CYP2C9 enzyme for biotransformation [Considering São Miguel islanders with genetic predisposition to thrombosis, the data of ulations -44. Indiers, IMs ,44. The ormation .VKORC1, a target of warfarin inhibition, may explain 23% of this drug dosage variability [VKORC1 promoter, potentially resulting in decreased transcription of the gene product. In general, the results suggest that 65.5% of the fifty-eight individuals with predisposition to thrombosis will require intermediate and/or low doses of warfarin, if treatment is started. Personalized medicine relies on data based on the human genome and disease biology characterization for the development of preventive, diagnostic, and therapeutic strategies that target the underlying determinants of disease in populations with specific molecular profiles [Polymorphisms within iability . Rieder iability  divided profiles . For thiThe identification of hereditary risk factors, with appropriate clinical evaluation will allow the informed patient and physician to work together for effective management of thrombosis and prevention of subsequent thrombotic events. The present study demonstrated, for the first time, that São Miguel, and possibly the Azores population, shows significant differences on allele frequencies of thrombotic genetic risk factors when compared to mainland Portugal. This research constitutes a primary approach for future studies on CVD, as well as for the implementation of warfarin dosing protocols using the patient's genotypic information.CYP2C9: Cytochrome P450 2C9 gene; DVT: Deep vein thrombosis; EMs: Extensive metabolizers; F2: Prothrombin gene; F5: Factor V Leiden gene; IMs: Intermediate metabolizers; MTHFR: Methylenetetrahydrofolate reductase gene; OR: Odds Ratio; PCR-RFLP: Polymerase chain reaction – restriction fragment length polymorphism; PMs: Poor metabolizers; SNP: Single nucleotide polymorphism; tHcy: Total homocysteine; VKORC1: Vitamin K epoxide reductase complex subunit 1 gene; VTE: Venous thromboembolism.ADRs: Adverse drug reactions; APC: Activated protein C; CI: Confidence interval; CVD: Cardiovascular diseases; The authors declare that they have no competing interests.MTHFR variants and PRP participated in the analysis. LMV designed the study, provided scientific orientation and revised the manuscript. All authors read and approved the final manuscript.CCB and TP, contributed equally, by performing the experiments, statistical analysis and drafting the manuscript. RC genotyped


TGFBR1*6A (rs11466445) is a common polymorphic variant of the TGF-β receptor I gene and has been associated with tumour susceptibility. Nevertheless, the role of this polymorphism as a risk factor for colorectal cancer is controversial. The aim of this study was to assess the association between TGFBR1*6A and colorectal cancer, age, sex, tumour location and tumour stage in a Spanish population.TGF-β receptor type I is a mediator of growth inhibitory signals. TGFBR1 exon 1 and germline allele-specific expression were also conducted to obtain further information on the contribution of the TGFBR1*6A allele to CRC susceptibility.The case-control study involved 800 Spanish subjects: 400 sporadic colorectal cancer patients and 400 age-, sex-, and ethnic-matched controls. The odds ratio (OR) and 95% confidence interval (95% CI) for the TGFBR1*6A polymorphism were calculated using unconditional logistic regression adjusted for age and sex. Analysis of somatic mutations at the GCG repeat of TGFBR1*6A allele and CRC (p > 0.05). The OR was 1.147 (95% CI: 0.799–1.647) for carriers of the TGFBR1*6A allele and 0.878 (95% CI: 0.306–2.520) for homozygous TGFBR1*6A individuals compared with the reference. The frequency of the polymorphism was not affected by age, sex or tumour stage. The TGFBR1*6A allele was more prevalent among colon tumour patients than among rectal tumour patients. Tumour somatic mutations were found in only two of 69 cases (2.9%). Both cases involved a GCG deletion that changed genotype 9A/9A in normal DNA to genotype 9A/8A. Interestingly, these two tumours were positive for microsatellite instability, suggesting that these mutations originated because of a deficient DNA mismatch repair system.There was no statistically significant association between the 9A allele was detected in seven of the 14 heterozygous 9A/6A tumour cases. This could have been caused by linkage disequilibrium of the TGFBR1*6A allele with mutations that cause allele-specific expression, as was recently suggested.Allele-specific expression of the TGFBR1*6A allele does not confer an increased risk of colorectal cancer in the Spanish population.Our results suggest that the  APC, DNA mismatch repair genes, MYH, SMAD4 and BMPR1A/ALK3 [Colorectal cancer (CRC) is the main cause of cancer-related deaths in Spain. Although environmental and genetic factors associated with cancer have been described, current knowledge of the genetic basis of colorectal cancer is limited. About 5% of colorectal cancer cases are associated with germline mutations of genes such as SMAD proteins [TGFBR2 and SMAD4 may predispose to the development of hereditary non-polyposis colorectal cancer and juvenile polyposis, respectively [Low-penetrance cancer susceptibility alleles may increase the risk of cancer in a manner similar to that of pathogenic germline mutations in hereditary cancer genes. Transforming growth factor beta (TGF-β) is one of the most potent inhibitors of cell growth. TGF-β ligands interact with the type II receptor and then bind to the type I receptor, sending a signal to the nucleus through proteins . Inhibitectively .Recently, it has been suggested that a mouse model carrying a germline Tgfbr1 haploinsufficiency has constitutively reduced TGF-beta signalling that significantly enhances the development of colorectal cancer .TGFBR1*6A (rs11466445) is a common polymorphic variant of the TGF-β receptor I gene. It has been suggested that the TGFBR1*6A allele is a low-penetrance susceptibility factor for colorectal cancer [TGFBR1 is generated by deletion of three GCG triplets that code for three alanine molecules within a nine alanine (*9A) stretch sequence at exon 1. The TGFBR1*6A allele encodes a type I receptor with reduced growth-inhibitory signalling activity [TGFBR1*6A is a candidate tumour-susceptibility allele, is present in 13.7% of the general population and increases the risk of cancer by approximately 24% [TGFBR1*6A is responsible for a proportion of patients with hereditary non-polyposis colorectal cancer syndrome [et al. [TGFBR1*6A allele and prostate [et al. [TGFBR1 gene is dominantly inherited, segregates in families and occurs in sporadic CRC cases. This ASE confers a substantially increased risk of CRC . Moreover, the authors claim that the TGFBR1*6A allele is probably in linkage disequilibrium with one of the putative mutations that causes ASE, but TGFBR1*6A per se does not cause ASE. The causative germline changes have not been identified.Polymorphisms of various genes associated with the TGF-β pathway have been described as cancer risk factors. rs1146644 is a coml cancer . This vars1146644 is a comtely 24% . Moreovealanine *A stretchsyndrome , Skoglun [et al.  suggesteprostate  or bladdTGFBR1*9A/6A polymorphism, including mutational and ASE analyses, to clarify the effect of the TGFBR1*6A polymorphism on the risk of colorectal cancer in our population.Because of this controversy, we conducted a case-control study of the A total of 400 sporadic CRC cases and 400 controls from the Elche University Hospital and Castellon Provincial Hospital tissue banks were analysed. Written consent to be included in the tissue banks was obtained from each patient. Patients diagnosed with a familial cancer syndrome were excluded.This was a hospital-based case-control study. Controls with no personal history of cancer, selected with diagnoses considered unrelated to the exposures of interest, were selected from the same hospitals and matched with cases for age, sex and race/ethnicity. The study was approved by the ethical committees of the Elche University Hospital and the Castellon Provincial Hospital.The median age at diagnosis of CRC was 70 years (range 22–93 years) and that of the controls was 72 years (range 23–98 years). The sex distribution was 41.8% female (n = 167) and 58.2% male (n = 233) for colorectal cancer patients and 53.2% female (n = 213) and 46.8% male (n = 187) for the controls.Of the CRC cases, 24% had proximal colon tumours, 33.75% had distal colon tumours, 9.25% had sigma-rectum tumours and 25% had rectal tumours. In 8% of cases, the location of the tumour was unknown. Cases were classified according to tumour stage as low stage (stages I and II: 78.5% of cases) or high stage (stages III and IV: 21.5% of cases) . DNA isolation was performed using the EZ1 DNA Tissue kit and the EZ1 BioRobot  according to the manufacturers' instructions.DNA for the case-control study: Control DNA was obtained from peripheral blood samples (n = 400). DNA was isolated from the non-tumour colorectal tissue (n = 400) of cases after mechanical homogenization TGFBR1 exon 1 tumour mutation: Tumour tissue DNA was isolated after mechanical homogenization of colorectal tumour tissues (n = 69) as described previously.DNA for the EZ1 RNA Tissue kit and the EZ1 BioRobot .RNA extraction: RNA was isolated from normal colorectal tissue samples from 14 heterozygous 9A/6A CRC patients after mechanical homogenization using the Reverse Transcription Reagents, random primers  and 200–1000 ng of RNA.cDNA synthesis: A retrotranscription reaction was performed using CCACAGGCGGTGGCGGCGGGACCATG (5'-labelled with 6-FAM) for TGFBR1F and CGTCGCCCCCGGGAGCAGCGCCGC for TGFBR1R. PCR amplification was done using the Amplitaq Gold PCR Master Mix 2× in a total volume of 25 μl containing 1 M betaine (Sigma Aldrich). The PCR was run for 35 cycles at 94°C for 30 s and 72°C for 90 s followed by a 10 min final extension at 72°C. PCR products and size standards  were diluted in HiDi Formamide  and then resolved by capillary electrophoresis  using POP6 as polymer. GeneScan software was used for analysis of PCR fragments . Representative cases for each genotype were sequenced directly to confirm allele sizes (data not shown). A PCR product size of 119 bp corresponded to the most common allele, *9A, whereas a product size of 110 bp corresponded to the *6A allele.Genotyping of the TGFBR1 exon 1 polymorphism was conducted in the case-control study and used for the somatic mutation and ASE analyses. Genotype was assessed using PCR amplification and capillary electrophoresis. The PCR primers were et al. [A subset of colorectal tumour DNAs from 120 patients was screened for MSI status using five mononucleotide markers  and multiplex PCR as previously described by Buhard et al. .9A/6A CRC cases were included in the ASE analysis. TGFBR1 exon 1 amplicons generated from normal genomic DNA and corresponding normal cDNA were run in parallel. The ASE ratio was calculated by normalizing the ratio between the peak areas of the two alleles in cDNA with the same parameters in genomic DNA. A threshold ratio of a 33% difference was used to define ASE-positive cases [Fourteen heterozygous ve cases .TGFBR1*6A polymorphism using unconditional logistic regression adjusted for age and sex. We analysed for potential effect modification by age using an analysis stratified according to median age at diagnosis (≤ 70 years or >70 years). A χ2 test was used to evaluate differences in TGFBR1*6A carrier frequencies between the tumour and control groups and to analyse the association between the polymorphism and the clinical and pathological factors. A probability level of < 0.05 was considered significant.The Hardy-Weinberg equilibrium was checked among the control and case populations. In the case-control study, we estimated the odds ratio (OR) and 95% confidence interval (95% CI) for the TGFBR1*6A allele were 0.101 and 0.092 for cases and controls, respectively. Genotype distributions for cases and controls are presented in Table TGFBR1*6A allele and colorectal cancer incidence in our population (p = 0.517). The crude ORs were 1.147 (95% CI: 0.799–1.647) for carriers of the TGFBR1*6A allele and 0.878 (95% CI: 0.306–2.520) for TGFBR1*6A homozygous individuals compared with the reference (homozygous TGFBR1*9A).The genotype distribution in the case and control populations did not deviate significantly from that expected for a population in Hardy-Weinberg equilibrium (p = 0.295 for cases and p = 0.033 for controls). The allele frequencies for the TGFBR1*6A allele distribution between low and high stages (p = 0.66)  cancer patients than rectal cancer patients (p = 0.024) .First, we screened colorectal tumour DNA from 120 patients for MSI status. We found evidence of MSI in 14 cases . Two of 14 MSI tumours (14.29%) had a deletion of a TGFBR1*9A allele  [There is evidence that common variants of the TGF-β pathway alter TGF-β signalling and modify cancer risk. The gnancies . Recent 01–1.43)  or of bo01–1.43) .a priori that our study with 400 sporadic CRC cases and 400 matched controls would enable us to detect an OR of 2.0 for 9A/6A heterozygous individuals (assuming a frequency of 0.15 in controls) and an OR of 3.0 for 6A/6A homozygous individuals (assuming a frequency of 0.0175 in controls) with 80% power .In association studies, contradictory conclusions may arise from population stratification or inappropriate sample size. We determined et al. [et al. [TGFBR1*6A carriers . The study did not provide information specifically about CRC or about sex matching between the groups. Zhang et al. (2005) suggested that the 12 case-control studies of Pasche [TGFBR1*6A and increased risk of cancer [Our results did not show a statistically significant association , which is in agreement with Skoglund et al. . To our f cancer . Similarf cancer -13 and iThere was little information on which to base the sample size for our study because information about this polymorphism in our population is limited and contradictory results were reported for another population. The results show that our study was under-powered for detecting weak associations between the TGFBR1*6A allele and CRC. The data indicate that a sample size of 10,000 cases and 10,000 controls is necessary to detect an OR of 1.15 with 80% power using a two-sided test with an alpha level of 5%.TGFBR1*6A allele. Previous case-control studies provide little information in this regard.In the present work, age and sex adjustment did have any effect on the risk conferred by the TGFBR1*6A allele between colon and rectal cancer cases. Moreover, when only colon cancer cases where considered, there was a difference of borderline significance for TGFBR1*6A carriers (p = 0.071) with an OR = 1.47 (95% CI: 0.979–2.203). No data are available regarding the risk of colon versus rectal cancer for the TGFBR1*6A allele because data from colorectal and colon cancer studies were pooled in the meta-analyses. Further studies with larger samples are needed to determine if the variant allele is associated specifically with colon cancer but not with rectal cancer. If confirmed, this may help explain the contradictory results in the literature.Interestingly, there was a significant difference in the frequency of the GCG repeat is a specific target for frameshift mutations in MSI colorectal tumours, and in the event, if somatic acquisition of TGFBR1*6A allele occurs in these MSI tumours, similar to metastatic CRC [GCG repeat at exon 1 of the TGFBR1 gene is a specific target for MSI tumours (p = 0.039), but the frequency of mutations is low  [TGFBR1*6A allele. Both mutated cases underwent the deletion of just one GCG triplet. No previous studies have documented the association between this polymorphism and MSI status. Unfortunately, our results did not enable us to clarify the role of the TGFBR1*6A allele in CRC risk.Because of the limitation that sample size exerts on association studies, we attempted to extract extra information from our data to elucidate the susceptibility effect of the minor allele of this polymorphism. First, we wanted to know if the atic CRC . The und 14.29%) . Neverthin vitro models shows that growth inhibitory signalling activity is low for the type I receptor coded by the TGFBR1*6A allele [TGFBR1*6A allele exists in the 6A/9A heterozygous individual, global functional reduction of the pathway would be evident and would increase the risk for CRC. Surprisingly, the results of the ASE study were opposite to what we had expected. The proportion of ASE positive cases was high (7/14), and there was predominant expression of the TGFBR1*9A allele in all seven ASE positive cases. The correct interpretation of our results became evident after the recent publication of the study of Valle et al. [TGFBR1 is a major contributor to genetic predisposition for CRC . They were unable to determine the causative mechanism of the ASE but the haplotype data suggested that ancestral mutations were implicated. One of the putative mutations that causes ASE is probably in linkage disequilibrium with the TGFBR1*6A allele but is not itself causative of ASE. Our results are in agreement with those of Valle et al. in that about 50% of 6A/9A heterozygous individuals have ASE and the 6A allele is expressed at a low level in all cases. The linkage disequilibrium between ASE and the TGFBR1*6A allele could explain the contradictory results in the literature on the association between this polymorphism and CRC.An additional strategy was used to address the role of the minor allele for this polymorphism in CRC. We tested whether gene expression level differed between the alleles of the polymorphism because previously reported data from A allele . We hypos high 7/, and theTGFBR1*6A allele and CRC, age, sex or tumour stage. The differences in haplotype frequencies between colon and rectal tumour patients should be confirmed by larger series. The somatic mutations that affected the GCG repeat in this study are a consequence of deficient MMR function. The ASE results suggest that there is linkage disequilibrium between the TGFBR1*6A allele and the mutations that cause ASE. Taken together, our results strongly suggest that the TGFBR1*6A allele does not confer an increased risk of colorectal cancer in the Spanish population.The case-control study did not show a statistical association between the The authors declare that they have no competing interests.AC participated in the design and coordination of the study, in the molecular genetic studies and helped to draft the manuscript. TM-B, AM-C, M-IC, CG and V-MB participated in the molecular genetic studies and helped to draft the manuscript. PM participated in the design of the study, performed the statistical analysis and helped to draft the manuscript. EO and RL participated in the biobanking of samples and helped to draft the manuscript. CG-P and AC participated in the design of the study and performed the statistical analysis. J-LS conceived the study, participated in its design and coordination 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/1471-2407/9/193/prepub


The aim of this study was to determine whether primary nocturnal enuresis (PNE) leads to alterations in glycosaminoglycan (GAG) excretion.Twenty subjects  with PNE were included in the study. Twenty-two healthy subjects were selected as a control group . Urinary GAG excretion was measured using a modified dimethylmethylene blue (DMD) assay of 24-hour urine.The mean urinary GAG content was 33.9 mg/g and 23.8 mg/g creatinine in patients with PNE and controls, respectively; patients with PNE thus had a higher mean GAG excretion than did age-matched controls (p < 0.05). The association between GAG level and enuresis frequency bordered on significance (p = 0.068).GAG excretion in patients with PNE was significantly higher than in normal children, suggesting that measurement of urinary GAG may be useful in evaluating physiopathological conditions of the bladder wall. Further studies are needed to confirm this finding. Nocturnal enuresis (NE) is defined as repeated, spontaneous voiding of urine during sleep in a child aged 5 years or older . AvailabRecent studies have focused on the importance of glycosaminoglycans  that line the transitional epithelium of the human bladder. GAGs play a structural role in the extracellular matrix, regulate ion permeability, and have anti-inflammatory functions on the bladder surface and wall -8. The aThe study included 20 children with PNE  and 22 healthy gender- and age-matched controls.5 CFU/mL), were all considered evidence of UTI [Exclusion criteria were conditions associated with large urine volume, abnormal neurological control, daytime wetting, and previous use of prescription medications for enuresis. Urinary symptoms were evaluated by taking of a history from each affected child and the parents, and by use of a questionnaire and bladder diary. All children underwent urinalysis. No child had any evidence of urinary tract infection (UTI). Exclusion of UTI was based both on urinalysis and absence of symptoms including dysuria, urination frequency or urgency, fever, or costolumbar or suprapubic pain. Pyuria, nitrite in the urine, leukocyte esterase activity (LEA) in the urine, or positive urine culture  after addition of dimethylmethylene blue  using bovine kidney heparan sulfate as a standard  . Under oAll values are presented as means ± SDs. Statistical analysis employed SPSS, version 10.0 . Statistically significant differences between parameters were established using Student's unpaired t test. Pearson correlations were used to determine the relationship between bedwetting frequency and urinary GAG level. A p value <0.05 was considered to indicate a significant difference.Of the 20 children currently bedwetting, 10 children had nocturnal enuresis every night, 4 at least three times a week, and 6 once or twice a week . We consider it likely that future work with larger numbers of patients would show a significant correlation between GAG level and enuresis frequency.e.g., interstitial cystitis or spina bifida) are probably derived from damaged bladder epithelium. Recently, Salvaggio and colleagues found that increased excretion of GAGs in spina bifida patients was an important parameter in evaluation of the physiopathological condition of the bladder wall [All cells apparently have GAGs on the surface, where the molecules constitute a matrix. Because of the negative charge of sulfate, GAGs repel each other, forming a type of cushion effect when cells interact. GAGs are very important in directing traffic at the surface, and constitute a necessary holding zone for many chemicals that must mutually associate at the cell surface. GAGs prevent small molecules from reaching the underlying tight junctions and cell membranes, and, hence, form a major permeability barrier . Severalder wall . The citInterstitial cystitis is often treated with exogenous GAGs such as heparin, chondroitin sulfate, hyaluronate, or semi-synthetic polysulfate. The mechanism of action is presumed to be coating of the bladder surface to replace chondroitin sulfate and heparin sulfate lost as a result of the disease.In the present study, GAG excretion was higher in patients with PNE than in healthy controls. Similarly, Ferrara and colleagues found that urinary GAG excretion was significantly elevated in patients with isolated NE compared to controls . They coCaione and associates investigated the effectiveness of a dextranomer/hyaluronic acid copolymer in treatment of urinary incontinence caused by sphincter incompetence in a total of 16 children and adolescents. At 6, 12, and 24 months of follow-up, patients reported improvement in daytime and nighttime dryness, respectively .Increased GAG excretion might result from either immaturity of bladder innervation, or chronic exfoliation of the bladder surface in response to infection. We suggest that quantitative and/or qualitative defects in the GAG layer might lead to reduction in functional bladder capacity.Although the association between bedwetting frequency and urinary GAG level only bordered on significance, analysis of a larger patient series might permit a stastically significant association between these variables to be demonstrated.Despite our small sample size, the between-group difference in urinary GAG values attained statistical significance, and we thus suggest that measurement of urinary GAG may be useful in evaluating physiopathological conditions of the bladder wall. Further studies are needed to confirm the validity of this observation.The authors declare that they have no competing interests.All authors have made substantial contributions to design of the work, in addition to analysis and interpretation of data; and have been involved in drafting the article and revising it critically for important intellectual content; and have given final approval of the version to be published.


Hypospadias is a congenital displacement of the urethral meatus in male newborns, being either an isolated defect at birth or a sign of sexual development disorders. The aim of this study was to assess the prevalence rate of hypospadias in different Districts of Italy, in order to make a comparison with other countries all over the world.We reviewed all the newborns file records (years 2001–2004) in 15 Italian Hospitals.th percentile) of any gestational age the prevalence rate of hypospadias was 6.25 per 1000 live births. Performing multivariate logistic regression analysis for different degrees of hypospadias according to severity, being born SGA remained the only risk factor for moderate-severe hypospadias (p = 0.00898) but not for mild forms (p > 0.1).We found an overall hypospadias prevalence rate of 3.066 ± 0.99 per 1000 live births . In newborns Small for Gestational Age . Pathogenesis of isolated hypospadias is multifactorial : however, the prevalence rate of hypospadias is higher in infants born small for gestational age than in newborns with normal birth weight. Hypospadias is a congenital displacement of the urethral meatus in male newborns, often associated to an incomplete development of the foreskin (prepuce) and abnormal penile curvature (recurvatum or hypospadias sine hypospadias). According to the exact localization of the meatus, hypospadias is named glandular or coronal (mild), penile (moderate), scrotal and perineal (severe). While most cases of mild forms are usually an isolated defect, the severe ones may be a symptom of a disorder of sexual differentiation . AccordiThe isolated malformation is likely caused by failed seam of the urethral folds at midline in the embryo. Patients need surgical repair, since they suffer from urological disorders and may have subfertility.So far only few studies reported the prevalence rate of hypospadias in Italy, but they all were based on birth defect registries or referred to particular districts in Sicily, where pollution level is actually high -6.We accurately assessed the prevalence rate and trends of hypospadias, including definition of different subtypes, in order to get exact data from several Italian districts for comparison with other countries all over the world.We reviewed all the newborns file records, years 2001–2004, in 15 Italian Hospitals, namely: Bologna, Chieti, Palermo, Parma, Pavia, Pisa, Torino , Carrara, Cecina, Livorno, Lucca, Massa, Nocera Inferiore, Pontedera, Versilia . As shown in figure The systematic inspection of all the neonates file records allowed us to avoid underreporting due to late or incomplete indications to local Registers of Congenital Malformations. By virtue of this method, we actually performed an accurate data collection. Unfortunately, we could not get the abortions file records: for this reason, we only calculated hypospadias rates among live births, instead of prevalence on all gestations.Diagnosis of hypospadias was made at birth, by Paediatricians expert of neonatal dysmorphology. Since we collected data from hospital file records, that is defined as a Population type-2 Study by Eurocat, we had to check all cases of neonates twice admitted to NICU for different reasons. Moreover, we looked for cases of neonates moving from a hospital to another into those districts where Hospitals are organized according to the Hub & Spoke Model .In order to allow comparison with previously reported studies, we defined glandular and coronal hypospadias (first-degree) as mild, while penile hypospadias (second-degree) as moderate and scrotal and perineal hypospadias (third-degree) were reported as severe ,8. We foTo the same purpose of comparison, hypospadias rates are finally presented as the number of cases per 1000 live births as well as in most studies already published.2 test for univariate analysis of categorical data, based on 2 × 2 cross-tabulations. We also performed Wilcoxon Rank Sum Test (Mann-Whitney) in order to assess risk factors separately. Finally, we used Multivariate Logistic Regression Analysis to show the likely relationship between hypospadias and pregnancy-related  or environmental factors .Hypospadias rates are presented as mean ± standard deviation. Statistics was performed using the software R Project for Statistical Computing. For comparison of prevalence rates, we used χThe study was approved by the local Ethical Committee of all the Hospitals included.Results are shown in table We reviewed a total of 78228 consecutive newborns file records. Male/female ratio was 0.892.We assessed 234 cases of hypospadias among 36890 live boys, giving an overall prevalence rate of 6.34 per 1000 male newborns (2.99 per 1000 live births). As shown in figure Within this sample population of newborns with hypospadias, 82.48% had mild hypospadias while 17.52% moderate-severe, without significant differences of severity during the study period. However, an increasing trend in prevalence of moderate-severe forms was observed in tertiary level perinatal centres.th percentile according to the normal tables for the Italian population [Adequate for Gestational Age) newborns (2.86 per 1000 live births) (p < 0.01).In newborns Small for Gestational Age  of any In preterm newborns  the prevalence rate of hypospadias was 6.34 per 1000 live births vs 2.53 per 1000 live births in neonates of g.a. ≥ 37 weeks (p < 0.01). Within the sample of preterm newborns, we found a prevalence rate of hypospadias of 19.08 per 1000 live births in SGA neonates vs 3.83 per 1000 live births among AGA neonates (p < 0.001).Finally, we calculated a prevalence rate of 5.28 per 1000 live births within SGA newborns born at term of pregnancy (g.a. ≥ 37 weeks), vs 2.58 per 1000 live births within the sample of AGA newborns at the same gestational age (p < 0.01).According to these data based on univariate analysis, both preterm birth and small birthweight for gestational age seem to be risk factors for hypospadias. However, performing Wilcoxon Rank Sum Test (Mann-Whitney), we found that preterm birth and small birthweight for gestational age are associated one to each-other (p = 0.0134). For this reason, we performed logistic regression, in order to evaluate the possible perinatal risk associated with hypospadias. Multivariate logistic regression analysis was performed for risk factors, including SGA, preterm birth and hospital of birth. Only SGA remained significant in the final model for hypospadias (p = 0.00482).Maternal age and multiple gestation were not related to hypospadias prevalence.We also performed multivariate logistic regression analysis for different degrees of hypospadias, according to severity. In the final model SGA remained the only risk factor for moderate-severe hypospadias (p = 0.00898), but not for mild forms (p > 0.1).In our sample the general prevalence of hypospadias results as high as the value observed in previous European and American studies (3–4 per 1000 live births) Table . The ratIn the 1980s European registers reported upward trends in hypospadias prevalence: the rate almost doubled in Wales, Sweden, Norway and Hungary . HoweverWe reported a steady trend of hypospadias in Italy in years 2001–2004 without any increase of severe forms.in vitro fertilization, pollution, medical techniques in neonates assistance, etc.). Effective recognition of endocrine disrupting properties of chemicals  that developing fetuses and children are exposed to is the first step to prevent environmental causes of disorders of sexual differentiation, including isolated disorders as hypospadias [Pathogenesis of isolated hypospadias is multifactorial, since genetic, endocrine and environmental factors have been implicated. These factors act in the early gestation disrupting the normal genitalia development in the male fetus, likely modifying androgen production or action . Becauseospadias .We included in the study tertiary, secondary and primary level perinatal centers, so that we got data from both nurseries for healthy newborns and NICU for neonates with any problems. The prevalence rate of hypospadias seems to be higher in infants born small for gestational age (SGA) than in newborns with normal birth weight . Our stuWe found differences among Italian hospitals that may be mainly related to the percentage of SGA and preterm newborns. For example, among tertiary level perinatal centres, we assessed a hypospadias prevalence rate of 4.87 per 1000 live births in Pisa, while 2.2 per 1000 live births in Chieti, considering a number of SGA of 9.5 per 1000 live births in Pisa and 3.3/1000 in Chieti. Similarly, among secondary and primary level perinatal centres, we showed a hypospadias prevalence rate of 3.83 per 1000 in Lucca (SGA: 43 per 1000) and 1.64 per 1000 in Nocera (SGA: 5.6 per 1000).Performing multivariate logistic regression analysis, we showed that SGA is a risk factor for all the different subtypes of hypospadias, even if the association is statistically significant only for severe ones. Because of this observation, we can speculate that mild and moderate hypospadias are likely due to environmental factors that disturb the pregnancy but not alter the whole growth of fetus. On the contrary, severe ones are probably correlate to genetics or important factors that are able to impair the general development of fetus-placental unit. In fact, few recently reported studies describe SGA newborns as a group at a major risk of severe birth defects.However, we can also speculate about pollution and about the effects of exogenous hormones influencing development. Recent studies, in fact, report that maternal intake of progestins during early pregnancy may be associated with an increased risk of hypospadias . ProgestThe authors declare that they have no competing interests.All the authors participated in the data collection. GP and SRT designed the study, carried out the data elaboration and coordinated the study.


One challenge in understanding complex diseases lies in revealing the interactions between susceptibility factors, such as genetic polymorphisms and environmental exposures. There is thus a need to examine such interactions explicitly. A corollary is the need for an accessible method of measuring both the size and the significance of interactions, which can be used by non-statisticians and with summarised, e.g. published data. The lack of such a readily available method has contributed to confusion in the field.SF) allows assessment of binary interactions in case-control studies. In this paper we describe its properties and its novel characteristics, e.g. in calculating the power to detect a synergistic effect and in its application to meta-analyses. We illustrate these functions with real examples in Alzheimer's disease, e.g. a meta-analysis of the potential interaction between a BACE1 polymorphism and APOE4: SF = 2.5, 95% confidence interval: 1.5–4.2; p = 0.0001.The synergy factor (Synergy factors are easy to use and clear to interpret. Calculations may be performed through the Excel programmes provided within this article. Unlike logistic regression analysis, the method can be applied to datasets of any size, however small. It can be applied to primary or summarised data, e.g. published data. It can be used with any type of susceptibility factor, provided the data are dichotomised. Novel features include power estimation and meta-analysis. The remarkable progress made in the understanding of single-cause diseases has not yet been matched in the study of complex conditions. One problem is that susceptibility factors, e.g. genetic and environmental, all contribute risk that is to varying extents contingent on the presence of other factors -4. ComplSF), derived from logistic regression models, which aims to address these needs.Yet, unravelling interactions has proved confusing (Discussion). There is a need for a readily accessible method of measuring their strength, available to non-statisticians and applicable to summarised data and to datasets of any size. Methods are also needed to calculate the power to detect an interaction and to perform meta-analyses of interactions from published data; these two functions have not so far been readily available. There is a particular need for an accessible method for referees; untested claims of synergy are regularly published. Here we present a statistic, the synergy factor  [SF), which depends on a multiplicative definition of the null hypothesis.In case-control studies, the parameter which is both estimable and interpretable as a relative risk is the odds ratio (OR) . In suchSF appears in Additional file SF) is equivalent to the interaction term defined by two binary factors in a logistic regression model. We test the hypothesis of no interaction, using a Normal approximation for the statistic ln(SF)/stderr(ln(SF)), where the standard error of ln(SF) is easily obtained via the delta method [SF to cope with empty cells and propose two bootstrap approximations and a Bayesian inferential procedure that can be used as alternatives to the Normal approximation. We also propose methodology to calculate the power of significance tests and to perform meta-analyses based on the SF.A full description of the methodology for significance tests based on the a method . This apx1 and x2, in the risk of a certain (binary) condition. Taking subjects with neither factor as reference, we first estimate the ORs for factor x1 alone (OR1), factor x2 alone (OR2) and both factors combined (OR12). The SF is then defined as: SF = OR12/(OR1 × OR2) and is the ratio of the observed OR for both factors combined, to the predicted OR assuming independent effects of each factor. Susceptibility factors may be associated with increased or reduced risk, i.e. risk or protective factors, respectively . In either case, interactions may be positive (synergy) or negative (antagonism). Thus, if SF > 1 (< 1), then there is a positive (negative) interaction between two risk factors. The opposite applies to protective factors.Let us assume we wish to estimate from a case-control study whether there is an interaction between any two (binary) factors, SF, construct a 4 × 2 table of the numbers of cases and controls in each of the 4 possible combinations of the two factors  as: Z = ln(SF)/stderr(ln(SF)) has asymptotically a standard normal distribution under the null hypothesis of no interaction.To obtain the statistical significance of a method  yields aAPOE4) and the GG genotype of the C/G polymorphism (rs638405) in exon 5 of the β-site APP-cleaving enzyme (BACE1) [BACE1 GG nor APOE4 as reference, the OR for BACE1 GG alone was 0.742 and that for APOE4 alone was 2.409. That gave a predicted OR of 1.788 (= 0.742 × 2.409) for the combination, compared with an observed OR of 4.934. Hence: SF = 2.76 (= 4.934/1.788), 95% confidence interval (CI): 1.25–6.09, ln(SF) = 1.015, stderr(ln(SF)) = 0.404, Z = 2.25 and p = 0.012. Thus the null hypothesis of no interaction was rejected and significant synergy was found. The observed joint effect of the two variants was nearly three times greater than the predicted joint effect.Let us take the potential interaction in risk of Alzheimer's disease (AD) between the ε4 allele of apolipoprotein E ( BACE1)  (Table 1  (Table SF), based on 10,000 simulated samples. This approximation does not depend on an asymptotic argument and gave p = 0.006. Figure SF) and the standard error: SF) with those of a standard normal distribution, shown as a straight line. Both graphs confirm the adequacy of the normal approximation, which we also tested formally using the Kolmogorov-Smirnov test (p = 0.66).Using the data of Table SF calculations may be performed using the Excel programme in Additional file The above example is of synergy between risk factors. Examples of antagonism and of protective factors are given in Additional file BACE1 GG and APOE4, respectively. The total sample size of that study [SF equals 1, the power is 0.05, which is the significance level used. In this example, 488 cases and 488 controls will be needed to have 80% power to detect an SF of 2. Power calculations may be performed on R, using a function available from MCB.Figure at study  was 524,APOE4 and the BACE1 exon 5 GG genotype in the risk of AD [BACE1 GG nor APOE4 as reference, the pooled SF obtained using the random effects model [p = 0.0001. The heterogeneity statistic based on three degrees of freedom was 1.88 (p = 0.76); the estimated random effects variance was 0. The results appear in Figure Table sk of AD -16. Thests model  was 2.51BACE1 studies , random effects model [The real examples in Tables es Table , the effts model  give furSFs ≥ 2, the main effects of the gene variants other than APOE4 were generally very weak. The ORs were ≤ 1.2 in 20 out of 36 cases and were only significant in 5 cases. Thus, preliminary screening for main effects will miss many, possibly most cases of epistasis.There is a common view that interactions, e.g. between genes (epistasis), should only be examined between risk factors that have already shown a significant main effect. But in many cases, such as most of the above, the association would be missed by the traditional single-factor approach -3. IndeeOR necessarily implies synergy. A single OR by itself says nothing about synergy; it is the relation between the three relevant ORs that matters. For instance, let us assume that two risk factors are associated with ORs of 3 and 5 alone and of 15 when combined. Although the combined value is impressive, there is no synergy: SF = 15/(3 × 5) = 1. Claims of synergy are frequently published on the basis of such invalid evidence. Indeed, we have noted at least 20 claims of interactions, in the field of AD genetics alone, that were published in leading journals in recent years, but which may be clearly refuted by SF analysis. There is thus a need for a readily accessible method of testing such claims.On the other hand, synergy can be too easily claimed. A common misconception is that a high combined SF analysis, being based on logistic regression analysis, is best used for assessing binary interactions [SF analysis may be performed twice, after stratification by the third factor, e.g. gender.We suggest that ractions . Variousractions ,20. Howeractions . Where aSF analysis should be the preferred method for stratification by covariates. Stratification can produce very small subsets, even of zero, which logistic regression analysis cannot handle. In contrast, SF analysis produces a realistic p value in each subgroup, if one adds 0.5 to each cell in any 4 × 2 table with at least one zero cell [Where the relevant data are available, logistic regression analysis is the appropriate method for adjusting for covariates, while ero cell ,22.SF analysis is simple to perform, through the Excel programmes in Additional files SF analysis was used to examine each of the 89 studies of interactions cited in that review. The method measures both the size and significance of a binary interaction, using either primary or summarised data. Unlike logistic regression analysis, it can be applied to datasets of any size, however small, even with zero cells (above). The method can be used with all types of susceptibility factors, both risk and protective, for instance, age, gender, diet, medication or genetic polymorphisms, provided the data are dichotomised, e.g. age ± 75 years. It can be applied both to synergistic and to antagonistic interactions. Novel features include power estimation (through an R function available from MCB) and meta-analysis, an increasingly important application (through the Excel programme in Additional file The authors declare that they have no competing interests.MCB and DJL conceived the method. MCB devised the various statistical applications and worked out the necessary proofs. ADS advised on applications, e.g. in AD genetics. OC and DJL selected real examples from the AD literature and tested the method on those examples. MCB and DJL wrote the initial drafts of the manuscript and all authors read those drafts and contributed to the revisions. All authors approved the final manuscript. OC submitted the article.Methods. text + 8 references.Click here for fileExamples of various types of interactions. text + 3 tables + 3 references.Click here for fileCortina-Borja 2009, SF calculator. Excel programme.Click here for fileCortina-Borja 2009, SF calculator, meta-analysis. Excel programme with macro.Click here for file


MBL2) variants with between 12 to 25% allele frequency in Caucasian and other populations, result in markedly reduced expression of functional protein. Prospective epidemiologic studies, including a nested, case-control study from the present population, have demonstrated the ability of MBL2 genotypes to predict complications of atherosclerosis,. The genetic control of MBL2 expression is complex and genetic background effects in specific populations are largely unknown.Mannose-binding lectin (MBL) forms an integral part of the innate immune system. Persistent, subclinical infections and chronic inflammatory states are hypothesized to contribute to the pathogenesis of atherosclerosis. MBL gene  and promoter (H and Y) alleles that have been seen in other populations. In addition, MBL levels were found to be positively associated with male gender and hemoglobin A1c levels, but inversely related to triglyceride levels. Correlation was not found between MBL and other markers of inflammation.MBL2 expression to clinical and environmental factors, including inflammatory markers.New data is presented concerning the effects of known genetic variants on MBL levels in an American Indian population, as well as the relationship of  MBL2), between 12 to 25% allele frequencies in Caucasian and other populations [lower levels of circulating MBL [MBL2 genotypes and coronary artery disease (CAD) was previously confirmed in the American Indian, Strong Heart Study (SHS) cohort  [high levels of MBL may be involved in complications related to cardiovascular disease (CVD). Thus, the role of MBL in cardiovascular disease appears to be a double-edged sword, due to hitherto only partially resolved mechanisms [Early innate human defenses against microbial invaders include mannose-binding lectin (MBL), a serum protein, which acts as a microbial opsonin and initiates activation of the so-called lectin complement activation pathway. Deficiencies in innate immunity have been hypothesized to allow chronic inflammatory states that may contribute to the pathogenesis of atherosclerosis . Common ting MBL  and are ting MBL  and cardting MBL , but notting MBL . An indeariates) . On the chanisms .Although considerable effort has focused on the genotypic determination of MBL levels, our understanding of the role of various promoters and structural genetic variations remains incomplete. New promoter variants continue to be identified ,15 and gMBL is also known to increase during an acute-phase reaction . SamplesThe previously mentioned SHS case/control study matched cases and controls on age, gender and recruitment center (Arizona (AZ), Dakotas (DK) and Oklahoma (OK)). From this group a subset was chosen to include 186 of the controls without regard to genotype and an additional 51 cases limited to those known to be homozygous for high expressing "HYA" alleles.MBL2 genotypes and other covariates were determined from both of these groups. American Diabetes Association criteria [Ascertainment of fatal and nonfatal cardiovascular events occurring between examinations was accomplished by medical record review and/or yearly participant contact . Cases wcriteria  were useDetermination of MBL levels was done at Rigshositalet in Copenhagen, Denmark, using a double sandwich ELISA method as previously described .MBL2 gene was assessed for the presence of the B (G54D), C (G57E) or D (R52C) structural variations, and two promoter polymorphisms, one a G/C transition at -550 bp  [The alleles)  and the alleles) . Qualityalleles) . GenotypMBL2 similar to previous reports [MBL2 analysis [MBL2 (LOW_1) was assumed from the presence of an O_O or LA_O genotype. The LOW_2 category consisted of either O_O, LA_O or LA_LA genotypes. These risk categories were compared with reference groups ALL_1 or ALL_2, consisting of all genotypes not included in either LOW_1 or LOW_2 respectively; or the HIGH category (either HA_HA or HA_LA). There is ample documentation of the biologic effect of these various genotypes on basal MBL levels [Composite categories of genotypes predicted to result in reduced expression of  reports , primarianalysis  were alsL levels , althougMBL2 expression using SPSS version 10.1.0 software. The distributions of all subgroups in table The Chi square statistic was used to evaluate proportions between groups. Multivariate regression models and comparison of means (t test of independent samples) were used to understand the contribution of the various factors to Approval consistent with the Helsinki Declaration was obtained from all relevant Institutional Review Boards and tribes, and all participants gave informed consent.Table There were no significant differences in the prevalence of O (15.3%) and H (67.2%) alleles in this control sample, compared with an earlier SHS report .These findings confirm low levels of MBL among individuals with composite genotypic groups  , as wellAmong controls only, comparing means of composite genotypes showed highly significant differences  between either the HYA_HYA or HIGH groups and any of the YA_O, HA_O, LA_O, O_O, LOW1, or LOW2 groups. Figure Only cases with HYA_HYA genotype were included to allow adequate numbers to compare with controls of identical genotype. If the mean MBL levels for case and control groups of identical genotype are compared , then a crude p value of 0.812 is obtained.MBL2 within the control group. A backward selection criterion of F ≥ 0.1 was used and results are summarized in table Multivariate regression analysis was used to investigate the interplay of various demographic, environmental, genetic factors on expression of MBL2 gene has been implicated in important pathophysiologic processes including susceptibility to infections [MBL2 expression and as recently as 2006, new variants have been identified [MBL2 expression and function will continue to be uncovered. The worldwide high population prevalence of these variants suggests an apparently ambivalent role of MBL2 in a number of pathogenetic and homeostatic processes [Genetic variation in the fections -26, chrofections ,6,8,27, fections . Furtherfections ,30. It ientified ,31. It ientified ,33, varientified , and epirocesses -37.MBL2 variants in native populations of North America [MBL2, as in the present study. Since MBL levels are higher in those with diabetes  [In this report we have generally confirmed the previously reported influence of the three main structural variants and the two most widely investigated promoter variants (H/L and X/Y). This is of importance due to the demonstrated association between CVD and these variants in this particular, non-Caucasian population. Other studies have reported on the prevalence of  America ; but havdataset) , a corredataset) . Others dataset) ; but no Strengths of this study include confirmation of the presumed link between genotype and expressed protein, increasing confidence in a possibly causal association between MBL and cardiovascular disease in this population. These results also extend the previously identified genotype/phenotype correlations to another population with a unique genetic background. A weakness is the previous matching structure of the sample and the need to compare HYA/HYA case genotypes with the same control genotypes. There were an insufficient number of previously matched pairs with this genotype to allow a matched analysis, thus the aggregate comparison between these two groups needs to be interpreted with caution.MBL2 expression may improve our ability to understand and disrupt the pathogenetic mechanisms involved in cardiovascular disease.The findings from our study corroborate previous research that has demonstrated the important effects of genetic variants and the association of metabolic abberations with MBL levels. Further understanding of The authors declare that they have no competing interests.All authors have read and approved the final manuscript. LB: Originated and conducted the majority of the analysis, drafted the manuscript and is the corresponding author. RF and SD: Supervised and conducted the genotyping. KN, JM, EL, BH, JU and VP: Provided recommendations on analysis and assisted in editing the manuscript. YZ: Provided provided critical statistical supplort and validation. PG: Supervised laboratory determination of MBL levels, as well as providing major contributions in developing the analytic strategy and key editorial recommendations.The pre-publication history for this paper can be accessed here: